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Background C‐reactive protein (CRP) and paraoxonase 1 (PON1) might increase and decrease in canine leishmaniasis (CanL), , and both can rapidly normalize after therapy. Recently, supplementation of domperidone with conventional therapy , increasing the activity of cells involved in acute phase responses in vitro. This combined therapy has been recommended to treat mild forms of CanL; however, no studies have investigated the effects of domperidone supplementation on early CRP or PON1 changes in dogs with CanL. Objectives The aim of this study was to evaluate whether domperidone, added to conventional treatments, modifies CRP concentration and PON1 activity kinetics in CanL dogs responsive to conventional therapy. Methods Serum CRP concentrations and PON1 activities were measured in dogs with mild CanL before (t‐0) and 3 (t‐1), 7 (t‐2), 14 (t‐3), and 21 (t‐4) days after treatment with N‐methylglucamine antimoniate and allopurinol alone (n = 18) or combined with domperidone (n = 18). Results C‐reactive protein concentrations increased at t‐1 in the domperidone group, especially when the CRP concentration at t‐0 was normal. However, the concentrations normalized at t‐4 in 18/18 dogs compared with 14/18 dogs not receiving domperidone. The median PON1 activity decreased at t‐1 in the domperidone group, and this decrease was more significant in dogs with normal PON1 activity at t‐0. Conclusions Based on these results, transient increases in CRP concentrations or decreases in PON1 activities after domperidone administration should not be erroneously interpreted as signs of a worsening disease process.

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

Paraoxonase 1 (PON1) is an inflammation marker associated with lipid oxidation and is used as a diagnostic marker in people. There is no information about the suitable substrate and analytic performance in cats, or its biological behavior compared with other inflammation markers. Our aims were to validate a paraoxon-based method to measure PON1 activity in feline serum, to assess stability of PON1 under different storage conditions and the impact of interfering elements, to determine a reference interval (RI) for healthy cats, and to correlate PON1 activity with 2 major acute-phase proteins. Intra- and inter-assay precision, accuracy, and RI were assessed using fresh serum. The same specimens were stored at room temperature, refrigerated, or frozen, and retested at defined intervals. Hemolysis, lipemia, and icterus were simulated to study interferences. PON1 results were compared to serum amyloid A (SAA) and alpha-1-acid glycoprotein (AGP) results. Analytical validation yielded precise and accurate results. PON1 activity is stable for up to 24 h at room temperature and up to 48 h at 4°C. Freezing at −20°C results in an increase after 72 h, with return to baseline values after 1 wk, that again increases after 6 mo. Only hyperlipemia interfered with PON1 activity. The RI based on 71 healthy cats was 58–154 U/L. PON1 activity was negatively correlated with AGP, but not with SAA. Serum PON1 activity can be measured accurately in cats, and it acts as a negative acute-phase protein.

To document changes in urinary biomarker concentration and conventional diagnostic tests of acute kidney injury (AKI) following hypotension and fluid resuscitation in anaesthetized dogs.Experimental, repeated measures, prospective study.A group of six male adult Greyhound dogs.Following general anaesthesia, severe hypotension was induced by phlebotomy, maintaining mean arterial blood pressure (MAP)40 mmHg for 60 minutes, followed by resuscitation with intravenous gelatine solution to maintain MAP60 mmHg for 3 hours. Following euthanasia, renal tissue was examined by light microscopy (LM) and transmission electron microscopy (TEM). Urinary and serum concentrations of neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CysC), and gamma-glutamyl transpeptidase (GGT), serum creatinine and urine output were measured at baseline and hourly until euthanasia. Data are presented as mean and 95% confidence interval and analysed using repeated measures analysis of variance with Dunnett's adjustment, p0.05.Structural damage to proximal renal tubular cells was evident on LM and TEM. Urinary biomarker concentrations were significantly elevated from baseline, peaking 2 hours after haemorrhage at 19.8 (15.1-25.9) ng mLUrinary NGAL, CysC and GGT concentrations, and UPC were consistently elevated within 1 hour of severe hypotension, suggesting that proximal renal tubules are damaged in the earliest stage of ischaemia-reperfusion AKI. Measurement of urinary biomarkers may allow early diagnosis of AKI in anaesthetized dogs. Urinary GGT concentration and UPC are particularly useful as they can be measured on standard biochemistry analysers.

Background To date, little information is available about the effect of preanalytical factors on the urinary protein-to-creatinine (UPC) ratio in cats. Objectives We aimed to evaluate the effect of a commercially available cat litter, creatinine measurements at three different dilutions of urine, and different storage conditions on the UPC ratio in cats. Methods Feline urine specimens were prospectively collected. Twenty-two whole-urine specimens were placed uncovered and in contact with cat litter for 1 hour; 25 urine supernatants were diluted 1:10, 1:20, and 1:100 for creatinine measurements. The correlation, difference, agreement, and concordance in classifying specimens according to International Renal Interest Society staging were determined. Storage effects on UPC ratios were assessed in specimens stored for 6 hours at +20℃ (n = 20), 1 week at +4℃ (n = 20), and 3 months at −20℃ (n = 25). Specimens were also subjected to four freeze-thaw cycles (n = 20). Results were compared, and clinical significance was assessed by comparing each UPC ratio to the inter-assay range of the baseline value. Results Exposure to cat litter did not affect UPC ratios. A positive proportional bias was found in the 1:100 dilution compared with the 1:20 dilution; however, concordance was high for all comparisons. At +20, +4℃, and after four repeated freeze-thaw cycles, UPC ratios were stable. Compared with baseline values, UPC ratios decreased (P < .01) after 8 and 12 weeks at −20℃. However, all UPC ratios were within the inter-assay variability of the baseline value. Conclusions Exposure to cat litter did not affect UPC ratios, but further studies are necessary to evaluate other potential variables. The effects of the dilutions and storage conditions were clinically acceptable, although the 1:20 and 1:100 dilutions were not perfectly comparable.

Paraoxonase-1 (PON-1) activity is a new inflammatory and oxidative marker. Technical effects and biological factors could affect the accuracy of PON-1 activity measurement. We investigated the effects of storage at different temperatures, repeated freeze–thaw cycles, interferences from hemolytic, lipemic, and icteric samples, and seasonal effects on PON-1 activity in horses. We evaluated 2 substrates with an automated spectrophotometer. Ten equine serum samples were stored under different conditions. Although storage at room (21°C) or refrigeration (4°C) temperature induced a statistically significant decrease ( p < 0.05) in PON-1 activity, this is not diagnostically relevant. PON-1 activity in frozen samples (−20°C) was stable for short-term storage; diagnostically significant ( p < 0.01) fluctuations were observed after 1 mo. Four repeated freeze–thaw cycles were assessed, and all cycles affected PON-1 activity ( p < 0.01); however, this was diagnostically significant only after the 4th cycle. Hemolysis induced an overestimation of PON-1 activity; lipemia and hyperbilirubinemia did not change PON-1 activity. Thirty-four horses were sampled monthly for 1 y, and PON-1 activity was higher in autumn ( p < 0.05) and winter ( p < 0.05) than in spring and summer.

Several protein biomarkers have been shown to be useful for the early diagnosis of acute kidney injury (AKI) in animals and people. Multiplex assays for measurement of a panel of renal biomarkers in canine samples have recently become available. This study compared the use of two such assays, versus previously validated ELISAs, to measure five biomarkers in canine samples during ischaemia-reperfusion (IR) AKI. Blood and urine was collected from six male anaesthetised greyhounds that underwent 1-h of renal ischaemia (severe hypotension induced by acute haemorrhage) and 2-h of reperfusion (intravenous fluid resuscitation). Histology confirmed presence of acute tubular injury at 2 h of reperfusion. Concentrations of clusterin, cystatin C, kidney-injury molecule 1 (KIM-1), monocyte chemoattractant protein 1, and neutrophil gelatinase-associated lipocalin (NGAL) at baseline and following IR, measured by two different multiplex assays and previously-validated single analyte immunoassays, were compared. Only NGAL was significantly elevated following IR with all assays investigated. Whether concentrations of the other four biomarkers were significantly increased following IR depended on the assay used. Concentrations of cystatin C and KIM-1 measured with the multiplex assays were of a vast magnitude lower than those measured with the corresponding single analyte ELISAs. We conclude that further validation is required before these assays can reliably be used to measure AKI biomarkers in canine samples.

Novel urinary biomarkers are increasingly utilized for the diagnosis of acute kidney injury (AKI) in dogs. Magnetic-bead based immunoassays for the simultaneous measurement of multiple biomarkers represent a potentially efficient and cost effective tool for investigators; however there is limited data to support their reliable use in dogs. Analytical validation of a commercial multiplex assay for the measurement of five AKI biomarkers: clusterin, cystatin C, kidney-injury molecule 1 (KIM-1), monocyte chemoattractant protein 1 and neutrophil gelatinase-associated lipocalin (NGAL) in canine urine was performed. The effect of pre-analytical factors including potential interfering substances and sample storage methods were investigated. Urine from 110 healthy dogs was used to determine reference intervals for each biomarker measured, according to American Society of Veterinary Clinical Pathology guidelines. Additionally, urine from 21 dogs with pyuria was used to evaluate the impact of pyuria on biomarker concentration. The assay performed with acceptable accuracy and precision for the measurement of NGAL only. Clinically relevant urine concentrations of bilirubin, haemoglobin, and synthetic colloid solutions led to interference (mean percentage difference > +/− 15% compared to control) with measurement of all or some of the biomarkers. All biomarkers were stable in urine stored at 20–22 °C for 2 h, 4 °C for 12 h, or -20 °C for 6 months. Reference intervals could not be established for KIM-1 due to unacceptable measurement imprecision (intra- and inter assay coefficient of variation 45% and 20% respectively). Urine NGAL concentration was significantly elevated in pyuria (P

Interest is growing in measurement of novel biomarkers for the diagnosis of acute kidney injury. Multiplex assays may provide a rapid and cost-effective way of measurement; however, sparse information is published regarding their use in dogs. We aimed to validate a commercial magnetic bead–based assay for 5 biomarkers: clusterin (Clus), cystatin C (CysC), kidney injury molecule 1 (KIM-1), monocyte chemoattractant protein 1 (MCP-1), and neutrophil gelatinase–associated lipocalin (NGAL). Intra- and inter-assay imprecision, linearity under dilution (LUD), spike recovery (S-R), and hemoglobin interference were evaluated using serum from healthy and diseased dogs. Additionally, the effect of sample type (serum vs. plasma) was investigated. All values for Clus and MCP-1 were outside the assay’s measurable range. Intra- and inter-assay precision were acceptable for NGAL (CVs 8.8% and 13.2%, respectively). Regression analysis of LUD and S-R indicated good linearity for CysC and NGAL. Hemolysis did not affect measurement of any biomarker. Measured concentrations of CysC ( p = 0.018) and NGAL ( p = 0.015) were significantly lower in sodium citrate plasma compared to serum. We conclude that this magnetic bead–based assay is precise and accurate for NGAL measurement in canine serum. Inappropriate standards for MCP-1 and Clus, and poor accuracy for KIM-1 measurement, suggest that this assay cannot reliably quantify those biomarkers in canine blood. Measurements of CysC in canine blood using this assay must be interpreted with caution given inter-assay imprecision.

Hepcidins are an evolutionarily conserved class of liver-expressed peptide, from which the twenty-five amino acid hormone, hepcidin-25 (herein hepcidin), has gained significant notoriety as the master regulator of iron homeostasis in mammals. Hepcidin maintains iron homeostasis by controlling the dietary absorption of iron and the mechanisms of recycling cellular iron stores. With the physiological significance of this hormone well established, it has emerged as an informative biomarker. In a comparison of the genome, transcriptome and peptidome of Canis lupis familiaris, we reveal the size of the hepcidin peptide in the canine, previous reports of which were contradictory to the evolutionary conservation predicted by genome annotation. Here, measurement of the peptide by mass spectrometry, following isolation from greyhound blood serum, revealed an amino acid sequence and peptide mass, differing from all accounts to date, yet demonstrating perfect sequence identity to that of the greater Canidae lineage of the Carnivora. Importantly, in the greyhound, the measured hepcidin peptide showed a similar temporal pattern to total serum iron, consistent with our understanding of hepcidin regulating iron homeostasis, in agreement with human diagnostics, and providing added translational evidence of the measured peptide being the iron regulatory hormone of the Canidae.

Background: High molecular weight (MW) synthetic colloids interfere with refractometry. The effect of low MW synthetic colloids on refractometry is unknown. Objectives: To investigate the effect of 6% hydroxyethyl starch 130/0.4 (HES) and 4% succinylated gelatin (GELO) on the refractometric measurement of total plasma protein (TPP) and colloid osmotic pressure (COP) in canine plasma. Methods: Heparinized plasma from 10 dogs was diluted with 0.9% saline (NaCl), HES, and GELO in ratios of plasma:fluid, 9:1, 8:2, 7:3, 6:4, and 5:5. The biuret TPP (TPPb), refractometric TPP (TPPr), and COP assays were performed for all dilutions. Agreement (bias, 95% limits of agreement (LOA)) was tested between the TPPb (NaCl) and the TPPr of the NaCl, HES, and GELO plasma dilutions, and between the TPPb (NaCl) and the TPPb of the HES and GELO plasma dilutions. Results: A small bias (0.06 g/dL, 95% LOA −0.51 to 0.39 g/dL) existed between TPPb (NaCl) and TPPr (NaCl). A large bias was found between TPPb (NaCl) and TPPr (HES) (1.38 g/dL, 95% LOA 0.30 to 3.10 g/dL), and TPPr (GELO) (1.18 g/dL, 95% LOA 0.53 to 2.88 g/dL). A small bias was found between TPPb (NaCl) and TPPb (HES) (0.08 g/dL, 95% LOA −2.04 to 2.20 g/dL), and a large bias was found between TPPb (NaCl) and TPPb (GELO) (1.00 g/dL, 95% LOA −0.59 to 2.60 g/dL). Increasing overestimation of TPPb (NaCl) by TPPr (HES and GELO) and TPPb (GELO) occurred with increasing colloid volumes. Conclusions: HES and GELO interfered with TPPr, and GELO also interfered with TPPb, in vitro. These variables should be interpreted with caution for dogs that have received these synthetic colloids.

Feline infectious peritonitis (FIP) is characterised by the presence of systemic inflammation accompanied by oxidative stress. Paraoxonase-1 (PON-1) is a negative acute phase reactant produced by the liver. A paraoxon-based method has been validated to measure PON-1 activity in feline serum. The aim of this study was to investigate the usefulness of PON-1 activity as a biomarker to discriminate FIP from other diseases with similar clinical signs. Of 159 cats enrolled, 71 were healthy, 34 had FIP and 54 had another disease but presented with clinical signs that could be consistent with FIP. PON-1 activity was lower (P

The cannabinoid receptor type 2 (CB2) is the peripheral receptor for cannabinoids, involved in the homeostatic control of several physiological functions. Male mitotic germ cells express a high level of CB2, whose activation promotes their differentiation in both in vitro and in vivo experiments, controlling the correct progression of spermatogenesis. However, it remains elusive if CB2 activation in spermatogonia could affect reproductive success in terms of fertility and healthy pregnancy outcomes. In this study, we explored the effects of male CB2 activation on sperm number and quality and its influence on next generation health. We show that exposure of male mice to JWH-133, a selective CB2 agonist, decreased sperm count, impaired placental development and reduced offspring growth. These defects were associated with altered DNA methylation/hydroxymethylation levels at imprinted genes in sperm and conserved in placenta. Our findings reveal that paternal selective activation of CB2 alters the sperm epigenome and compromises offspring growth. This study demonstrates, for the first time, a new role of CB2 signaling in male gametes in causing epigenetic alterations that can be transmitted to the next generation by sperm, highlighting potential risks induced by recreational cannabinoid exposure.

Background Synthetic colloid solutions, administered by rapid infusion to volume‐depleted dogs, might be present in high concentrations in subsequent urine samples. The potential for these solutions to affect the performance of ELISA measurements due to sample matrix effects when studying kidney injury biomarkers requires investigation. Objective We aimed to investigate two different synthetic colloid solutions, 4% succinylated bovine gelatin (GEL) and 6% hydroxyethyl starch (HES), for potential interferences with a commercially available canine neutrophil gelatinase‐associated lipocalin (NGAL) ELISA. Methods Assay interference was assessed by measuring the linearity of NGAL concentrations measured using a canine NGAL ELISA after serial dilution of a canine pooled urine sample with an assay diluent, GEL, or HES. Results NGAL recovery from urine specimens containing up to 75% HES and up to 62.5% GEL was within acceptable limits (80%‐120%). NGAL recovery from the urine specimen containing 75% GEL was poor (76%). Linear regression analysis demonstrated excellent linearity under dilution when a canine urine sample was diluted with the assay diluent, GEL, or HES. Conclusions The presence of large amounts (>62.5%) of GEL in canine urine samples could cause negative interference in the performance of the NGAL ELISA investigated.

Background The ΔWBC (the ratio between DIFF and BASO counts of the Sysmex XT‐2000iV), hereafter defined as ΔTNC (total nucleated cells), is high in effusions due to feline infectious peritonitis (FIP), as cells are entrapped in fibrin clots formed in the BASO reagent. Similar clots form in the Rivalta's test, a method with high diagnostic accuracy for FIP. Objectives The objective of this study was to determine the diagnostic accuracy for FIP and the optimal cutoff of ΔTNC. Methods After a retrospective search of our database, DIFF and BASO counts, and the ΔTNC from cats with and without FIP were compared to each other. Sensitivity, specificity, and positive and negative likelihood ratios (LR+, LR−) were calculated. A ROC curve was designed to determine the cutoff for best sensitivity and specificity. Results Effusions from 20 FIP and 31 non‐FIP cats were analyzed. The ΔTNC was higher (P 2.5 had 100% specificity. Conclusions The ΔTNC has a high diagnostic accuracy for FIP‐related effusions by providing an estimate of precipitable proteins, as the Rivalta's test, in addition to the cell count. As fibrin clots result in false lower BASO counts, the ΔTNC is preferable to the WBC count generated by the BASO channel alone in suspected FIP effusions.

Quantification of proteinuria is a fundamental step in staging dogs with chronic kidney disease and in monitoring the course of disease or the efficacy of anti-proteinuric treatments. Analytical precision and accuracy of the proteinuria assessment could be affected by several factors such as biological variability, different operators and quality control materials. The aim of this study was to assess whether inter-laboratory variability could affect the urinary protein to creatinine (UPC) ratio and whether this variability may affect patient classification according to the International Renal Interest Society (IRIS) sub-staging system. The same urine samples were analysed in three different laboratories using different instruments and different reagent brands. The results of the three laboratories were highly correlated to each other although urinary protein (UP), urinary creatinine (UC) and the UPC ratio of one laboratory were found to be significantly higher than those of the other two. No significant differences between the other two laboratories were recorded. The concordance in classifying dogs according to the IRIS guidelines was good if all three proteinuria categories were analysed separately or if borderline proteinuric (BP) dogs were included in the proteinuric group, and very good if BP dogs were merged into the non-proteinuric group. The inter-laboratory variability in UPC ratio measurement was not so great as to impede the identification of proteinuric dogs, but may influence the estimation of the magnitude of proteinuria.

Background: Proteinuria quantification with the urinary protein-to-creatinine (UPC) ratio is part of the diagnostic process in feline patients suspected of chronic kidney disease (CKD). In affected cats, monitoring and substaging of the UPC according to the International Renal Interest Society (IRIS) guidelines is also necessary for appropriate patient management. No information is available about the possible effects of analytical variability on urinary proteins (UPs) and the UPC ratio in cats. Objectives: This study aimed to determine whether imprecision and method-dependent differences due to the two dye-binding methods, pyrogallol red-molybdate (PRM) and Coomassie brilliant blue (CBB), could affect IRIS substaging. Methods: Urine samples were collected from proteinuric and nonproteinuric cats. Intra-assay and inter-assay repeatability were assessed with both the PRM and CBB methods. Urinary supernatants (n = 120) were tested using both methods. Agreement between the methods and concordance with sample classification according to IRIS guidelines were determined. Results: On average, the PRM method yielded a higher CV (UP 8.4 ± 5.2%; UPC 9.5 ± 4.8%) than the CBB method (UP 5.6 ± 2.6%; UPC 7.2 ± 2.6%), but similar rates of misclassification were found in samples with UPC ratios close to the IRIS cut-off. Although the two methods were correlated, the CBB method tended to yield UPs and UPC ratios that were significantly higher (P < 0.0001) than that of the PRM method. The Passing-Bablok test also found constant and proportional errors between the PRM and CBB methods. Concordance in substaging samples according to IRIS was good (k coefficient = 0.62). Conclusions: The two methods were precise, but the higher UPC ratios obtained with the CBB methods might affect the interpretation using the IRIS guidelines and clinical decisions.

Background Pro-active case detection (Pro-ACD), in the form of voluntary screening and treatment (VSAT) following community mobilisation about ‘asymptomatic malaria’, is currently being evaluated as a tool for Plasmodium falciparum elimination in Preah Vihear Province, Cambodia. Methods A qualitative study was conducted to explore community understanding, perceptions, expectations and acceptability of the Pro-ACD intervention in order to identify aspects that could be improved in future Pro-ACD activities. This was ancillary to a three-round VSAT campaign, carried out in three villages between December 2015 and March 2016. Qualitative data collection began shortly after the end of the three rounds of screening. Purposive sampling was used to select participants. Nine focus group discussions with participants (n = 46) and non-participants (n = 40) in the Pro-ACD screening were conducted, in addition to in-depth interviews with key village figures (n = 9). Results Health promotion messages were well delivered and received, but it was difficult for many villagers to understand the messages around ‘asymptomatic malaria’. Overall, villagers and village leaders had a positive opinion about the VSAT intervention. Acceptability was high, as a direct consequence of favourable perceptions towards the screening activity: the Pro-ACD intervention was seen by the local population as an effective, inexpensive, reliable and readily available tool to protect individuals and the community from the insurgence of malaria. Physical absence and lack of time (both linked to work-related activities) were the main reasons for non-participation. Conclusions Although VSAT was generally well perceived and accepted, the ‘time factor’ related to the need to satisfy essential daily subsistence requirements played a significant role in determining participation in the screening. More well-adapted and meaningful Pro-ACD approaches could be implemented by improving the timing of the testing activites, and strengthening community participation and engagement to increase acceptability.

A striped dolphin (Stenella coeruleoalba) calf stranded alive because of a Salter-Harris fracture type 1 of a caudal vertebra and remained in a provisional rehabilitation facility for 3 days where the fracture stabilization was attempted, but he died the day after bandaging. Serum and urine samples were collected during hospitalization (days 1, 2 and 3 serum and day 2 urine). Serum analysis showed increased urea, alanine transaminase, aspartate transaminase, and serum amyloid A values, while creatinine was below the lower limit. Urine analysis showed urinary protein-to-creatinine ratio of 5.3 with glomerular proteinuria. Postmortem analyses demonstrated a severe rhabdomyolysis and myoglobinuric nephrosis, suggestive of capture myopathy syndrome. We report, for the first time, the clinico-pathological changes during this condition in a striped dolphin.

Highlights • Holy Birman cats (HB) seem to be predisposed to feline infectious peritonitis (FIP). • In this study, indicators of innate and specific immunity were assessed in HB cats. • CD4/CD8, IFN-γ, IL12/IL4 were higher and IL-4 was lower in HB than in other breeds. • The rate of shedders, but not of seropositivity, was lower in HB than in other cats. • Th1 immunity seems to be stronger in HB. This may reduce the rate of shedders., The aim of this study was to assess whether Holy Birman cats (HB) have a peculiar immune profile and a higher rate of infection by feline coronaviruses (FCoV). Leucocyte and lymphocyte subsets, antibody titers, α1-acid glycoprotein (AGP), globulin fractions, IL-4, IL-12 and IFN-γ in blood and fecal FCoV excretion were determined in HB (n = 75) and in cats from other breeds (n = 94). Significantly higher CD4/CD8 ratio, IFN-γ concentration and IL12/IL4 ratio and significantly lower IL-4 concentration and proportion of shedders were found in HB than in other breeds. No other differences were found. In conclusion, this study did not provide evidence of peculiar immune profiles in HB, except for a prevalent Th1 profile, that may explain why in our caseload the rate of shedders was lower in HB than in other breeds.

This study was designed to determine if the activity of paraoxonase (PON1), an antioxidant enzyme that works as a negative acute phase reactant, is a better predictor for the clinical recovery of leishmaniotic dogs receiving standard treatments compared with inflammatory markers such as C reactive protein (CRP) and electrophoretic fractions. For this purpose we tested 20 healthy dogs (controls) and 39 leishmaniotic dogs classified as sick (group A, n=23) or severely sick (group B, n=16) and tested at admission and after 3, 7, 14, 21, 28, 35 and 42 days. At admission, CRP and electrophoresis were altered in both groups, while PON1 activity was abnormal only in group B. There were no differences related to the outcome (mortality, complications or time of recovery). PON1 activity normalized in about 2 weeks in dogs that had abnormal values at admission and a final positive outcome; CRP normalized in 4-6 weeks and electrophoretic fractions were still altered after 6 weeks. The results show that, at admission, inflammatory markers did not predict the outcome of leishmaniasis. PON1 activity decreased only in some dogs with systemic inflammation but not in those with mild leishmaniasis: when decreased, PON1 normalized earlier than other markers in dogs that responded to treatment. This finding most likely depends on the rapid decrease in oxidative phenomena. PON1 activity should therefore be tested on admission: if low values are recorded, severe inflammation may be suspected and PON1 measurement may be repeated during treatment to early identify responsive dogs.

This study was aimed at determining the serum concentration of homocysteine (Hcy) and big endothelin-1 (big ET-1, the precursor of endothelin) in dogs with chronic kidney disease (CKD) with and without hypertension, proteinuria and inflammation, in order to explore their role as biomarkers of hypertension associated with CKD. Hcy and big ET-1 were measured using an enzyme-linked immunosorbent assay and an enzymatic cyclic reaction, respectively, in dogs with CKD staged, as proposed by the International Renal Interest Society (IRIS), using serum creatinine, urinary protein to creatinine (UPC) ratio and systolic blood pressure, and classified as affected or not by inflammation based on the serum concentration of C-reactive protein (CRP). Serum Hcy was significantly higher in dogs of IRIS stages II, III and IV compared with controls and in proteinuric compared with non-proteinuric dogs. No differences relating to the degree of hypertension or to the CRP concentration were found. Serum big ET-1 significantly increased in dogs of IRIS stage IV compared with controls, in proteinuric compared with non-proteinuric dogs, in dogs with severe hypertension compared with those without hypertension, and in dogs with increased CRP compared to those with normal CRP concentrations. Hcy only correlated with serum creatinine but big ET-1 significantly correlated with serum creatinine, UPC ratio, systolic blood pressure, and increased CRP. In conclusion, both Hcy and big ET-1 increase in dogs with CKD. Although further research is needed, big ET-1, but not Hcy, may also be considered as a biomarker of hypertension.

Many feline breeds have been generated from a small number of ancestors. Thus, breed-specific peculiarities can be expected, which could include haematological and biochemical measurements. Despite this, there are only a few reports on breed-specific reference intervals (RI). This information is essential in routine practice where results from individual patients are usually compared with an RI. The aim was to compare haematological and biochemical data from clinically healthy Abyssinian, Holy Birman, Norwegian Forest and Siberian cats with published RIs to assess whether the published RIs are acceptable in these breeds. Comparison with established RIs using guidelines from the National Committee for Clinical Laboratory Standards and the American Society of Veterinary Clinical Pathology, revealed a number of breed-related clinicopathological differences. New RIs were established, but in most cases the new RIs overlapped with published RIs, and the use of the breed-specific data would minimally affect the clinical interpretation of laboratory results. Important differences that could result in misinterpretation of laboratory results were as follows: microcytosis and high α2-globulin concentrations in Abyssinian cats; high serum creatinine, α2-globulin and glucose concentrations in Holy Birman cats; high serum alkaline phosphatase activity and calcium and phosphate concentration in Norwegian Forest cats; low β2-globulin and γ-globulin concentrations in Norwegian Forest and Siberian cats. Breed-specific RIs should be used for these analytes. In addition, care should be taken in interpreting clinicopathological data in purebred cats for which specific RIs have not been established.

High density lipoproteins (HDLs) are pivotal in innate immunity and decrease in serum during inflammation. Several studies have been done about lipoprotein changes in transition cows but little is known about their changes in newborn calves. The aim of this study is to provide information about HDLs in newborn calves, by defining the possible age-related changes in healthy calves compared with adults and by assessing the possible differences in calves with inflammation. Lipoprotein electrophoretic separation (reported as percentages) and colorimetric measurement of HDL (HDL-C) were performed on healthy cows and calves in order to identify possible differences in the lipoprotein profile due to the age. Then, age-matched calves with inflammatory conditions were also evaluated. Results showed that in calves HDL% and VLDL% were lower (mean values ± SD: 77.6% ± 8.6% and 2.6% ± 2.5%, respectively) and LDL% was higher (19.7% ± 7.4%) than in adults (89.0% ± 3.9%; 5.2 ± 2.1% and 5.8% ± 3.1%, respectively). Sick calves revealed a decrease of both HDL% (mean values ± SD: 61.0% ± 22.1%) and HDL-C (22.8 ± 11.6 mg/dL) and an increase of VLDL% (12.1% ± 13.1%) compared with controls (77.6% ± 8.6%; 41.5 ± 11.2 mg/dL and 2.6% ± 2.5%, respectively). Paraoxonase-1 activity, influenced by inflammation and oxidation, was measured, and it appeared correlated with HDL% and HDL-C in sick calves. In conclusion, this study revealed that HDLs concentration in healthy calves is lower than in adults, and further decreases in calves with inflammation, likely due to oxidation.

Meiosis is a crucial process for the production of functional gametes. However, the biological significance of many genes expressed during the meiotic phase remains poorly understood, mainly because of the lethal phenotypes of the knockout mice. Functional analysis of such genes using the conditional knockout approach is hindered by the lack of suitable Cre transgenic lines. We describe here the generation of transgenic mice expressing Cre recombinase under the control of the meiotic Spo11 gene. Using LacZ-R26loxP and EYFP-R26loxP reporter mice, we show the specific expression and activity of Cre during meiosis in males and females. Spo11Cre mice were then crossed with floxed Nbs1 and JAM-C mice to produce conditional knockouts. A strong reduction of Nbs1 and JAM-C protein levels was found in the testis. Although Nbs1-deleted mice developed minor gonadal abnormalities, JAM-C-knockout mice showed a spermiogenetic arrest, as previously described for the null mice. These results provide strong evidence that Spo11Cre transgenic mice represent a powerful tool for deleting genes of interest specifically in meiotic and/or in postmeiotic germ cells.

Background: The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. Objectives: The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. Methods: EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various breeds, both sexes, and 2–6 years of age. Samples were stored for up to 12 hours at 4 °C and then analyzed on the ADVIA 120 and the Hemat 8. A 100-cell to 200-cell differential leukocyte count was performed by 3 independent observers on May-Grunwald-Giemsa-stained smears. Intra-assay precision of the ADVIA 120 was determined by analyzing 5 replicates each of 10 of the blood samples. Results: Results from the ADVIA were significantly higher than those from the impedance counter for RBC count, total WBC count, hemoglobin concentration, red cell distribution width, MCH, and MCHC, and significantly lower for HCT and platelet count. Significantly higher neutrophil and basophil counts and significantly lower lymphocyte counts were obtained with the ADVIA 120 compared with manual counts. Based on Passing–Bablok regression analysis, RBC and platelet counts were in good agreement between the 2 analyzers; a constant and proportional bias was present for other values. Coefficients of variation for erythrocyte parameters on the ADVIA were

Early detection of inflammation in neonatal calves allows early intervention, which may reduce mortality. Paraoxonase-1 (PON1) is a negative acute phase protein in humans. The aims of this study were to investigate age-related variability in serum PON1 activity and its clinical usefulness in neonatal calves. In healthy calves (n=9), PON1 activity increased with age from 2 to 21 days of age. There was no significant increase in PON1 activity in healthy calves from days 21 to 120 (n=15), but PON1 activity was significantly higher in adult cattle (n=45). In sick calves, serum PON1 was significantly lower in calves7 days of age with diarrhoea (n=8) and in calves28- to 120-days-old with respiratory disease (n=8) in comparison with age matched controls (n=20 and n=15, respectively). These results support the role of PON1 as a negative acute phase protein in cattle.

In order to assess whether the concentration of high density lipoproteins (HDLs) changes in leishmaniotic dogs before and after treatment, HDL cholesterol (HDL-Chol and HDL%), C reactive protein (CRP) and activity of the antioxidant enzyme paraoxonase (PON-1) were measured in sera from 10 controls and 10 leishmaniotic dogs. Seven of these latter were sampled also 3, 7, 14, 21 and 28 days after treatment with antimonials and allopurinol. HDL-chol, and PON-1 were low in leishmaniotic dogs at admission and increased after treatment. HDL-chol and HDL% correlated positively with PON-1 and negatively with CRP suggesting that HDLs decrease through an oxidative mechanism. Therefore, HDLs may be used to monitor the magnitude of oxidation associated with inflammation in leishmaniotic dogs.

Background: Information about laboratory reference intervals (RIs) of European Hedgehog (Erinaceus europaeus) hospitalized at rehabilitation centers is scarce. Objective: The purpose of this study was to establish hematologic and biochemical RIs for rehabilitated hedgehogs before the release into the wild, and to assess whether sex and management of the center influence laboratory results. Methods: Blood was collected from 50 hedgehogs at 3 centers. Thirty-eight animals were included in the study based on normal body weight, absence of clinical signs of disease, Bunnell index > 0.80, and absence of hibernation during overwintering. CBCs were performed using an automated laser cell counter followed by morphologic analysis of blood smears. Clinical biochemistry was performed using an automated spectrophotometer. RIs were determined as recommended by the ASVCP guidelines. Results: Hematology profiles revealed a prevalence of lymphocytes, a constant presence of nucleated RBCs, Howell-Jolly bodies and basophils, and bilobed nuclei in neutrophils and eosinophils. Biochemistry profiles were characterized by higher creatinine and urea concentrations, and higher ALP and GGT activities compared with other domestic species. The sex did not influence the results. Conversely, numbers of eosinophils, activated and large granular lymphocytes, and concentrations of total protein, glucose and cholesterol were different among the centers, likely due to different management practices (eg, antiparasitic treatments, environmental exposure to microorganisms, diet). Conclusion: The RIs established in this study can be used to monitor the health status of hedgehogs in rehabilitation centers. As management practices appeared to influence some variables, it is recommended to standardize the management protocols to minimize their influence on laboratory data.

Paraoxonase 1 (PON1) is a negative acute phase protein bound to high density lipoproteins (HDL) and during the acute phase response (APR) protects HDL from peroxidation. The aim of this study was to assess the relationship between PON1 and HDL in canine babesiosis, a disease characterized by oxidative damages and by an APR. PON1, HDL and C-reactive protein (CRP), were measured in blood collected from 15 controls and 29 dogs with babesiosis sampled at admission, and on days 1 and 7 after treatment. At admission, PON1 and HDL were significantly lower in affected dogs. HDL concentration increased at day 1 while PON1 increased and CRP decreased at day 7. This suggests that the decrease of PON1 at admission is in part due to an increased consumption, the decreased HDL may depend on lipid peroxidation and its rapid increase after treatment may depend on the antioxidant activity of PON1.

The aim of this study was to use normal immunocompetent mice to set up a model for endometriosis which allowed to study the dynamic aspects involved in initiation and progression of the disease. Thirty mice were surgically transplanted with autologous endometrium and at 3 weeks showed evidence of endometriosis. Diagnosis of endometriotic lesions was histologically confirmed. Visual inspection using a caliper revealed that, after an initial decrease in size (from 33.44+/-2.33 mm2 to 24.24+/-2.37 mm2 (p0.01)) detected at 3 weeks after transplantation, there was a significant increase of lesion area from 21.30+/-3.15 mm2 to 43.93+/-6.29 mm2 (p0.05) in the following 4 weeks. When we compared these results to those obtained in mice which underwent bilateral annessiectomy, we observed that, when bilateral annessiectomy was performed simultaneously to endometrial transplantation, lesion surfaces were similar between mice which were or were not subjected to bilateral ovariectomy. On the other hand, when bilateral annessiectomy was performed at second laparotomy and then evaluated after 4 weeks, differently from what observed in control mice, surface values decreased from 21.24+/-2.29 mm2 to 10.58+/-3.40 mm2 (p0.01). Finally, progression of lesions in estrogen supplemented mice seems less evident than in control mice since only a slight but not significant increase in size (from 21.32+/-3.32 mm2 to 26.18+/-6.98 mm2, p=0.32) was detected. The results presented herein demonstrate that surgically induced endometrial implants in mice are dynamic lesions and that implantation and progression of endometriosis represent different stages in the ethiopathogenesis of the disease. Moreover, we showed that progression, but not implantation, of ectopic endometrium is dependent upon the functionally and structurally integrity of the ovaries. This is a model of endometriosis established in normal immunocompetent mice, and, consequently, may represent a reliable tool for testing new immunological therapeutical approaches and studying the role of different genes using transgenic mice.

CHRONIC monocytic leukaemia (CMoL) is characterised by the proliferation of mature monocytes that predominate in the peripheral blood ([Monteith and Cole 1995][1]). CMoL is frequently indolent, with a persistent monocytosis that is unresponsive to therapeutic agents (Messick 2000). In human beings

The effects of constant rate infusion (CRI) of lidocaine on sevoflurane (SEVO) requirements, autonomic responses to noxious stimulation, and postoperative pain relief were evaluated in dogs undergoing opioid-based balanced anesthesia. Twenty-four dogs scheduled for elective ovariectomy were randomly assigned to one of four groups: BC, receiving buprenorphine without lidocaine; FC, receiving fentanyl without lidocaine; BL, receiving buprenorphine and lidocaine; FL, receiving fentanyl and lidocaine. Dogs were anesthetized with intravenous (IV) diazepam and ketamine and anesthesia maintained with SEVO in oxygen/air. Lidocaine (2mg/kg plus 50 μg/kg/min) or saline were infused in groups BL/FL and BC/FC, respectively. After initiation of lidocaine or saline CRI IV buprenorphine (0.02 mg/kg) or fentanyl (4 μg/kg plus 8 μg/kg/h CRI) were administered IV in BC/BL and FC/FL, respectively. Respiratory and hemodynamic variables, drug plasma concentrations, and end-tidal SEVO concentrations (E'SEVO) were measured. Behaviors and pain scores were subjectively assessed 1 and 2h post-extubation. Lidocaine CRI produced median drug plasma concentrations

In humans, the glycosylation pattern of serum and of membrane glycoproteins is associated with invasiveness of tumors: specifically, α2,6-sialylation and α2,3-sialylation are associated with metastasizing and nonmetastasizing tumors, respectively. In turn, the type of sialylation depends on the activity of α2,6 or α2,3 sialyltransferase (ST) enzymes. Because of the high prevalence of metastasizing tumors with biological behavior similar to the human counterpart, female dogs with metastasizing neoplasms could provide a good animal model for investigating the potential roles of sialic acid (Sia) and ST enzymes in the pathogenesis of metastatic tumors. The aims of this study were (1) to validate a solid-phase method based on lectin staining of serum and tissue homogenates to investigate sialylation and ST activity and (2) to compare the results obtained with this method and with lectin staining and to collect preliminary information on sialylation and ST activity in dogs with ( n = 8) and without ( n = 8) mammary tumors. The data recorded in healthy dogs revealed that serum and tissue glycoproteins are prevalently characterized by a α2,6 sialylation, but ST-α2,3 seems to be the most active enzyme in both samples. Sia-α2,3 and ST-α2,3 activity decreases in serum and tissues of dogs with tumors, especially in a dog with metastasis, suggesting that the equilibrium between ST-α2,6 and ST-α2,3 activity shifts toward the former, as reported in humans.

To determine the temporal sequence of pregnancy-induced changes in insulin action and secretion, awake midpregnant (11-12 days) and late pregnant (19-20 days) rats underwent a two-step euglycemic hyperinsulinemic or a hyperglycemic clamp study after a 24-h fast. During euglycemia, insulin-stimulated increments in glucose uptake and clearance in midpregnant rats were reduced by 60-70% at the lower dose (insulin approximately 360 pM) and by 20-30% at the higher dose (insulin approximately 1,750 pM; P < 0.01 vs. virgin controls). Insulin action was also diminished in late pregnant rats. However, the magnitude of resistance did not increase. Insulin-mediated suppression of glucose production was only minimally impaired in midpregnancy. In contrast, glucose production was virtually unchanged in late pregnancy, even at the highest insulin dose. During hyperglycemia, insulin responses in late pregnancy were markedly increased 5-fold above controls and 2.5-fold above midpregnant rats (P < 0.05). We conclude that rat pregnancy is characterized by the early appearance of peripheral insulin resistance. As pregnancy progresses toward term, marked hepatic insulin resistance and insulin hypersecretion develop, whereas peripheral insulin resistance demonstrates negligible changes. These data imply that insulin hypersecretion during late pregnancy is most closely linked to hepatic insulin resistance, at least in 24-h-fasted animals.

A 6-year-old intact female Labrador Retriever had diabetes mellitus, which had been difficult to control with insulin. The dog also had a solid ductal mammary carcinoma with very rapid growth, which was temporally related to onset of hypoglycemia. Eight months after initial diagnosis of diabetes, the dog had a hypoglycemic crisis. Insulin administration was stopped and serum glucose concentration returned to normal. Three months after discontinuing insulin, another hypoglycemic crisis occurred. During subsequent months, serum glucose concentrations remained at life-threatening levels (1.64-2.12 mmol/L, reference interval 4.44-6.66 mmol/L) simultaneously with an increase in the size of the mammary tumor, which reached a diameter of about 16 cm. At the time of surgery for removal of the tumor serum glucose concentration was 2.20 mmol/L and was then monitored every 3 hours after excision of the tumor. The glucose concentration continued to rise and reached 9.99 mmol/L 12 hours after the removal of the mammary tumor. Immunohistochemical staining demonstrated expression of insulin growth factor-2 by tumor cells, which apparently had caused the hypoglycemia during tumor growth even in a diabetic dog. Hyperglycemia associated with diabetes was pronounced after excision of the tumor and had been masked by the paraneoplastic effect of the tumor.

Interferon gamma (IFN-g) plays an important role in cell mediated responses against mutated feline coronavirus strains (FCoV) involved in the pathogenesis of feline infectious peritonitis (FIP). The aim of this study was to establish a combined in silico and in vitro approach to assess feline leukocyte production of IFN-g in response to selected peptides of the nucleocapside protein (N) of FCoVs. To this aim, we designed, through a bioinformatic approach, 8 potentially immunogenic peptides from the protein N corresponding to sequences of residues 14, 182, 198 detected only in FCoVs from FIP cats (virulent strains), only in FCoVs from healthy cats (avirulent strains) and both in FIP and in healthy cats (mixed strains). The peptides or a sham solution were incubated with whole blood from 16 cats (7 healthy and 9 with chronic diseases other than FIP) and IFN-g concentration was measured on plasma using an ELISA system. RT-PCR expression of IFN-g mRNA was also evaluated after incubation of the peptides or a sham solution with whole blood from 4 clinically healthy cats. The mean plasma concentration of IFN-g in samples incubated with peptides decreased and the expression of IFN-gmRNA did not change compared with the sham solution, except for some cats with chronic diseases (which probably have a ‘‘pre-activated’’ immune response). These cats responded to ‘‘avirulent’’ or ‘‘mixed’’ peptides by increasing the concentration of IFN-g and the expression of IFN-g mRNA. The combined approach employed in this study allowed us to identify potentially immunogenic peptides of FCoV N protein that can modulate the production of IFN-g especially in cats with a ‘‘pre-activated’’ cell mediated response.

Classification of leukemias requires specialized diagnostic techniques. Automated preliminary indicators of neoplastic cells in blood would expedite selection of appropriate tests.The objective of this study was to assess the capacity of the Sysmex XT-2000iV hematology analyzer to identify neoplastic cells in canine blood samples.Blood samples (n=160) were grouped into 5 categories: acute leukemia (n=30), chronic leukemia (n=15), neoplasia without blood involvement (n=41), non-neoplastic reactive conditions (n=31), and healthy dogs (n=43). WBC counts, WBC flags, scattergrams, percentages of cells with high fluorescence intensity, and percentages of cells in the lysis-resistant region were evaluated alone or in combination to establish a "leukemic flag." Sensitivity, specificity, negative (LR-) and positive (LR+) likelihood ratios, and the number of false-negative (FN) and false-positive (FP) results were calculated, and receiver operating characteristic curves were designed for numerical values.Among single measurements and parameters, only the evaluation of scattergrams minimized FN and FP results (sensitivity 100%, specificity 94.8%, LR+ 19.17, and LR- 0.00), although their interpretation was subjective. The more objective approach based on the generation of a "leukemic flag" had a sensitivity of 100%, specificity of 87.0%, LR- of 0.00, and LR+ of 7.67.Using a novel gating strategy the Sysmex XT-2000iV may be used effectively to screen canine blood for hematopoietic neoplasia.

Leishmania infantum interferes with the oxidative metabolism of phagocytes. In order to assess whether derivatives of reactive oxygen metabolites (d-ROMs) decrease due to infection or increase due to inflammation, d-ROMs were measured in serum collected from control dogs (Group 1; n = 12), from dogs seropositive for Leishmania either symptomatic (Group 2; n = 27) or not (Group 3; n = 14), and from dogs with other diseases (Group 4; n = 16). The concentrations of d-ROMs in the four groups, expressed in Carratelli Units (U CARR) were, respectively, 75.4 ± 39.5 (median, 81.6), 108.2 ± 96.3 (73.4), 73.5 ± 62.2 (62.0), 127.7 ± 97.3 (94.3). There were no significant differences between groups, but dogs with values higher than the reference interval were found, mostly in Groups 2 and 4 (which had serum C-reactive protein levels consistent with inflammation), whilst low values were occasionally found in Groups 2 and 3. Inflammation may mask decreases in d-ROMs induced by Leishmania infection.

The aims of this study were to validate a colorimetric method to measure total sialic acid (TSA) in feline serum and to investigate the serum concentration of TSA in clinically healthy cats seronegative (n = 9) and seropositive (n = 48) for feline coronavirus (FCoV), and in cats affected by feline infectious peritonitis (FIP, n = 28), tumors (n = 20), or inflammation (n = 16). The correlation between TSA and alpha(1)-acid glycoprotein (AGP) was also investigated. The method employed in this study is precise and accurate at TSA levels (in mg/L) commonly encountered in feline serum. No significant differences between seropositive (385.6 +/- 192.2 mg/L) and seronegative (433.5 +/- 179.0 mg/L) cats were detectable, suggesting that the simple infection by FCoVs does not influence TSA levels. Compared with seropositive controls, the concentration of TSA was higher in cats with FIP (556.7 +/- 268.3 mg/L, P = 0.003), tumors (522.5 +/- 294.4 mg/L, P = 0.028), and inflammation (546.8 +/- 208.3 mg/L, P = 0.018). The discriminating power of TSA for FIP is moderate (area under the ROC curve = 0.65) and the likelihood ratio is higher than 3.0 only at high TSA levels. Consequently, TSA could support a diagnosis of FIP only at extremely high serum concentration (800 mg/L) or when the pre-test probability of FIP is high. No correlations were found between the TSA and AGP concentrations in cats with FIP, suggesting that sialylated proteins other than AGP are present. Both the antibody titre and the degree of AGP sialylation were negatively correlated with TSA levels, suggesting that increased TSA may contribute to reduce the burden of FCoVs.

Intensive insulin treatment during diabetic pregnancy is complicated by maternal hypoglycemia. To investigate whether pregnancy may contribute as an independent hypoglycemia risk factor, awake pregnant rats that were near term underwent stepped insulin hypoglycemic (3.4 and 2.3 mM) clamp studies in the fasted and nonfasted states. In the fasted state, the glucagon response to hypoglycemia was completely suppressed in the pregnant rats (P0.01). Epinephrine, but not norepinephrine, was also diminished by approximately 70-75% at both hypoglycemic steps, and more exogenous glucose was needed to maintain hypoglycemia during pregnancy. To avoid the potential confounding effect of increased ketone levels (beta-hydroxybutyrate was approximately 170% higher in the pregnant rats), experiments were repeated in the nonfasting state when ketosis was eliminated in both groups. The nonfasted pregnant rats continued to show near complete suppression of the glucagon response, even at glucose levels of 2.3 mM. In contrast, a brisk response occurred in nonpregnant controls when glucose fell to 3.4 mM. Although epinephrine levels in the pregnant rats were also markedly suppressed during the milder hypoglycemic stimulus, they approached values seen in nonpregnant controls when glucose was lowered further to 2.3 mM. We concluded that in the rat, pregnancy markedly suppresses glucagon responses to hypoglycemia. The release of epinephrine, but not norepinephrine, is also blunted, especially during mild hypoglycemia. These findings suggest that pregnancy may impair glucose counterregulation by inhibiting glucagon and epinephrine release during hypoglycemia.

Preimplantation embryos up to the 8-cell stage of development use lactate and pyruvate but not glucose or Krebs cycle intermediates to support growth, development, and cleavage. The dominant effect of dichloroacetic acid (DCA) is the irreversible stimulation of pyruvate dehydrogenase (PDH) activity, thus accelerating the oxidative metabolism of pyruvate and lactate. To test the hypothesis that early induction of oxidative metabolism in 2-cell murine embryos accelerates preimplantation embryo cleavage rates, female B6C3F1 mice at 6 to 8 weeks of age were superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and mated. All 2-cell stage embryos were randomly assigned to culture media with or without 130 micrograms/mL DCA. The developmental stage of all embryos was then noted every 24 hours for a total of 72 hours. Chi-square analysis and the method of average rank sum were used to compare the distribution of embryos at each observation point. At 24 hours, DCA-exposed embryos had achieved an advanced stage of growth and development relative to controls (average rank sum, P = .026; chi-square distribution, P = .047). Subsequently, at 48 and 72 hours, neither the average rank sum nor the chi-square distribution was different. Our data suggest that DCA accelerates early growth and development of murine embryos before implantation, possibly through the early induction of oxidative metabolism.

OBJECTIVE To assess sensitivity and specificity of manual and automated measurements of reticulocyte percentage, number, and production index for classification of anemia in dogs. DESIGN Retrospective case series SAMPLE 174 blood smears from client-owned dogs with anemia collected between 1993 and 2013 for which reticulocyte parameters were determined manually (nonregenerative anemia, 22; preregenerative anemia, 23; regenerative anemia, 28) or with an automated laser-based counter (nonregenerative anemia, 66; preregenerative anemia, 17; regenerative anemia, 18). PROCEDURES Diagnostic performance was evaluated with receiver operating characteristic (ROC) curves by considering preregenerative anemia as nonregenerative or regenerative. Sensitivity, specificity, and positive likelihood ratio were calculated by use of cutoffs determined from ROC curves or published reference limits. RESULTS Considering preregenerative anemia as non regenerative, areas under the curve (AUCs) for reticulocyte percentage, number, and production index were 97%, 93%, and 91% for manual counting and 93%, 90%, and 93% for automated counting. Sensitivity, specificity, and positive likelihood ratio were 82% to 86%, 82% to 87%, and 4.6 to 6.4, respectively. Considering preregenerative anemia as regenerative, AUCs were 77%, 82%, and 80% for manual counting and 81%, 82%, and 92% for automated counting. Sensitivity, specificity, and positive likelihood ratio were 72% to 74%, 76 to 87%, and 2.7 to 6.2, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Whereas all reticulocyte parameters identified regeneration in anemic dogs, the performance of specific parameters was dependent on the method used. Findings suggested that lower cutoffs than published reference limits are preferred for reticulocyte number and production index and higher cutoffs are preferred for reticulocyte percentage. Reticulocyte production index may be useful when the pretest probability of regeneration is moderate.

In humans, homocysteine (Hcy) is employed to monitor renal, cardiovascular, and other diseases and their complications. The aim of the current study was to define the analytical performances of an enzymatic method not yet validated in dogs for measuring homocysteine, and to assess the possible clinical usefulness of Hcy measurement. Using conventional approaches, the analytical performances were investigated by assessing, imprecision, inaccuracy, and interference of hemoglobin, triglycerides, and bilirubin. The possible clinical usefulness of Hcy determination was assessed by comparing the results of healthy dogs ( n = 8) with those of dogs with heart disease ( n = 10), inflammation ( n = 6), gastrointestinal disorders ( n = 7), neoplasia ( n = 8), renal failure ( n = 4), trauma ( n = 7), and other miscellaneous diseases ( n = 6). Preliminary evaluation of this enzymatic method showed it to be precise at Hcy levels close to or higher than the values in dogs with renal or cardiac disorders that had the highest Hcy levels. By contrast, at low Hcy levels, which were recorded basically in control dogs, the method suffers from high imprecision. The sample of choice is serum. The use of icteric samples should be avoided, while hemoglobin and lipids have only a minor effect on Hcy measurement. In conclusion, the enzymatic method employed in the current study provides useful information in dogs and could be used to monitor cardiac and renal disorders, in which Hcy concentrations are elevated.