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Background: Chihuahua represents an increasingly widespread breed predisposed to cardiac disease. N-terminal pro-B-type natriuretic peptide (NT-proBNP) might be a useful point-of-care biomarker for dogs suspected of having heart disease, but breed differences have been reported. The urinary aldosterone-to-creatinine ratio (UAldo: C) appears to be a good indicator of renin-angiotensin-aldosterone system activity in dogs, but Chihuahuas showed significantly higher UAldo: C than other breeds. The objective of this study was to assess preliminary breed-specific reference intervals for NT-proBNP and UAldo: C in healthy Chihuahuas and evaluate sex differences in these parameters., Results: Forty-three healthy Chihuahuas dogs were enrolled. The median NT-proBNP was 347 (125-515) pmol/L, and the median UAldo: C was 2.59 (1.57-4.61) µg/g. The NT-proBNP reference interval was 125 (90% CI 125-125) - 2121.4 (90% CI 941.6-2248) pmol/L. 91% of the Chihuahuas were below the nonbreed-specific cut-off (900 pmol/L). The UAldo: C reference interval was 0.6 (90% CI 0.5-0.9) - 16.8 (90% CI 10.9-27.4) µg/g. No significant sex differences in NT-proBNP or UAldo: C were found., Conclusions: The median value, interindividual coefficient of variation and reference interval of NT-proBNP were in line with those reported for other small breeds. In contrast to previous studies, no sex differences in NT-proBNP were detected. As previously suggested, Chihuahuas seem to be characterized by higher values of UAldo: C than other breeds., (© 2024. The Author(s).)

The urinary normetanephrine to creatinine ratio (uNMN/Cr) and urinary metanephrine to creatinine ratio (uMN/Cr) are commonly evaluated for the diagnosis of pheochromocytomas (PCC) in dogs. This study aimed to establish reference intervals for uNMN/Cr and uMN/Cr in 56 healthy dogs in Japan and to investigate the effect of urine collection methods on these measurements in 15 non-PCC dogs. The upper limits of reference intervals for uNMN/Cr and uMN/Cr were 124.4 nmol/mmol (90% confidence interval [CI] 107.7-137.0) and 121.1 nmol/mmol (90% CI 102.8-145.1), respectively. Both uNMN/Cr and uMN/Cr were significantly higher when urine was collected in the hospital compared to at home. Several factors, including the method of urine collection, should be considered when utilizing these reference intervals.

Objectives: To evaluate the prevalence of bacterial presence in free-catch urine samples preceded by either a standardised prepped ("clean-catch") protocol versus unprepped (non-cleaned) voiding., Materials and Methods: The study was a single-centre prospective single-blinded randomised controlled trial. Urine samples were obtained from 100 client-owned dogs presenting for routine evaluation. Dogs were randomly assigned to either the prepped group (preputial or peri-vulvar area cleaned with sterile saline before collection) or the unprepped group (no preliminary cleansing) stratified by sex. Urinalysis and urine culture (blood and MacConkey agar) were performed on all samples. Significant bacterial presence on urine culture was defined as >10 4  colony forming units (CFU)/mL., Results: There were no statistically significant associations between prepped versus unprepped collection method or sex with a urinalysis positive for bacteriuria. However, on culture, significant bacterial growth was almost five times more likely to be associated with males relative to females (odds ratio 4.59, 95% confidence interval 1.61 to 13.10). The probability of finding a positive culture was not statistically associated with prep method (odds ratio 1.43, 95% confidence interval 0.50 to 4.08)., Clinical Significance: For the majority of dogs without clinical signs of urinary tract infection, free-catch urine collection does not result in significant bacteriuria found on analysis or culture. The presence of bacteria found in free-catch samples may be secondary to sample contamination or subclinical bacteriuria. Sample contamination or subclinical bacteriuria may be more prevalent in male dogs., (© 2024 British Small Animal Veterinary Association.)

Advanced glycation end products (AGEs), formed via the Maillard reaction (MR) during processing of foods, have been implicated in inflammatory and degenerative diseases in human beings. Cellular damage is primarily caused by AGE binding with the receptor for AGEs (RAGE) on cell membranes. An isoform of RAGE, soluble RAGE (sRAGE), acts as a decoy receptor binding circulating AGEs preventing cellular activation. Pet food manufacturing involves processing methods similar to human food processing that may increase dietary AGEs (dAGEs). We hypothesized that diet, plasma and urine AGEs, and serum sRAGE concentrations would differ between thermally processed diets. This study examined the association of four differently processed diets: ultra-processed canned wet food (WF); ultra-processed dry food (DF); moderately processed air-dried food (ADF) and minimally processed mildly cooked food (MF) on total plasma levels of the AGEs, carboxymethyllysine (CML), carboxyethyllysine (CEL), methylglyoxal hydroimidazolone-1, glyoxal hydroimidazolone-1, argpyrimidine, urine CML, CEL and lysinoalanine, and serum sRAGE concentration. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used to measure AGEs. sRAGE concentration was measured using a commercial canine-specific enzyme-linked immunosorbent assay kit. Total dAGEs (mg/100 kcal as fed) were higher in WF than in other diets. Plasma total AGEs (nM/50 μL) were significantly higher with WF, with no difference found between DF, ADF, and MF; however, ADF was significantly higher than MF. Urine CML (nmol AGEs/mmol creatinine) was significantly higher with DF than with WF and MF. There were no significant differences in total urine AGEs or serum sRAGE concentration between diets. In conclusion, different methods of processing pet foods are associated with varied quantities of AGEs influencing total plasma AGE concentration in healthy dogs. Serum sRAGE concentration did not vary across diets but differences in total AGE/sRAGE ratio were observed between MF and WF and, ADF and DF., (© 2024 The Authors. Journal of Animal Physiology and Animal Nutrition published by Wiley‐VCH GmbH.)

Background: Furosemide, a diuretic that acts on the loop of Henle, is commonly used to treat congestive heart failure in veterinary medicine. Some owners have difficulty in administering oral tablet medication to animal patients, which leads to noncompliance, especially during long-term administration. Oral disintegrating film (ODF) has the advantages of easy administration via a non-invasive route, rapid dissolution, and low suffocating risk. The objective of this study was to research the pharmacokinetic (PK) profiles and diuretic effect of furosemide after intravenous (IV), orally uncoated tablet (OUT), and newly developed ODF administration in healthy beagle dogs. In this study, a furosemide-loaded ODF (FS-ODF) formulation was developed and five beagle dogs were administered a single dose (2 mg/kg) of furosemide via each route using a cross-over design., Results: The most suitable film-forming agent was sodium alginate; thus, this was used to develop an ODF for easy drug administration. No significant differences were detected in the PK profiles between OUT and FS-ODF. In the blood profiles, the concentration of total protein was significantly increased compared to the baseline (0 h), whereas no significant difference was detected in the concentration of creatinine and hematocrit compared to the baseline. FS-ODF resulted in a similar hourly urinary output to OUT during the initial 2 h after administration. The urine specific gravity was significantly decreased compared to the baseline in each group. The peak times of urine electrolyte (sodium and chloride) excretion per hour were 1 h (IV), 2 h (OUT), and 2 h (FS-ODF)., Conclusions: These results suggest that the PK/PD of furosemide after administration of newly developed FS-ODF are similar to those of OUT in healthy dogs. Therefore, the ODF formulation has the benefits of ease and convenience, which would be helpful to owners of companion animals, such as small dogs (< 10 kg), for the management of congestive heart failure., (© 2021. The Author(s).)

The urinary microbiota is the collection of microbes present in urine that may play a role in host health. Studies of urine microbiota have traditionally relied upon culturing methods aimed at identifying pathogens. However, recent culture-free sequencing studies of the urine microbiota have determined that a diverse array of microbes is present in health and disease. To study these microbes and their potential role in diseases like bladder cancer or interstitial cystitis, consistent extraction and detection of bacterial DNA from urine is critical. However, urine is a low biomass substrate, requiring sensitive methods to capture DNA and making the risk of contamination high. To address this challenge, we collected urine samples from ten healthy dogs and extracted DNA from each sample using five different commercially available extraction methods. Extraction methods were compared based on total and bacterial DNA concentrations and bacterial community composition and diversity assessed through 16S rRNA gene sequencing. Significant differences in the urinary microbiota were observed by dog and sex but not extraction method. The Bacteremia Kit yielded the highest total DNA concentrations (Kruskal-Wallis, p = 0.165, not significant) and the highest bacterial DNA concentrations (Kruskal-Wallis, p = 0.044). Bacteremia also extracted bacterial DNA from the greatest number of samples. Taken together, these results suggest that the Bacteremia kit is an effective option for studying the urine microbiota. This work lays the foundation to study the urine microbiome in a wide range of urogenital diseases in dogs and other species., Competing Interests: The authors have declared that no competing interests exist.

Electrophoresis of urine to evaluate protein fractions in dogs with proteinuria to differentiate glomerular from tubular damage has increased in recent years; however, capillary electrophoresis (CE) of urine has not been reported in a study of > 40 healthy animals, to our knowledge. We aimed to establish reference intervals (RIs) for the urine protein fractions obtained by CE of urine from healthy dogs. We obtained urine samples from 123 clinically healthy dogs of both sexes between December 2016 and April 2019; urine was frozen until CE was performed. The electrophoretic patterns obtained were divided into 5 protein fractions, and RIs were established in percentages and absolute values using nonparametric methods. RIs were obtained for the fractions (F) as follows: 5.5 to 56.2% for F1, 3.2 to 16.5% for F2, 3.5 to 16.2% for F3, 17.8 to 69.8% for F4, and 5.1 to 23.9% for F5. These RIs obtained by CE might be useful clinically as a basis for comparison with pathologic samples. Age was a statistically significant factor for F2 ( p  = 0.01) and F3 ( p  = 0.02), and sex was a statistically significant factor for F1 ( p  = 0.03).

Oxytocin (OT) promotes pro-sociality, bonding, and cooperation in a variety of species. Measuring oxytocin metabolite (OTM) concentrations in urine or saliva provides intriguing opportunities to study human and animal behaviour with minimal disturbance. However, a thorough validation of analytical methods and an assessment of the physiological significance of these measures are essential. We conducted an analytical validation of a commercial Enzyme Immunoassay (EIA; Arbor OT assay kit) to measure OTM concentrations in dog, wolf, and human urine samples. To test the assay's ability to detect changes in OTM concentrations, we administered oxytocin intranasally to 14 dogs. Assay performance with regard to parallelism was acceptable. Assay accuracy and extraction efficiency for dog and wolf samples were comparable to a previously validated assay (Enzo OT assay kit) but variation was smaller for human samples. Binding sensitivity and antibody specificity were better in the Arbor assay. Average OTM concentrations were more than twice as high as in comparable samples measured with the Enzo assay, highlighting a lack of comparability of absolute values between different assays. Changes in OTM concentrations after intranasal treatment were detected reliably. The Arbor assay met requirements of a "fit-for-purpose" validation with improvement of several parameters compared to the Enzo assay.

Refractometry is utilized routinely to evaluate canine urine specific gravity (USG) in veterinary clinical settings. We aimed to determine if the magnitude of interobserver reliability when assessing canine USG via refractometry could impact clinical judgment. USG was determined in 38 dogs by 3 registered veterinary technicians (RVTs) using both an optical analog refractometer and a digital refractometer. Summary statistics were reported, interobserver reliability was assessed via intraclass correlation coefficient (ICC) analysis through a 2-way mixed-effects model, and agreement between RVT pairs was compared through Bland-Altman plots. The median analog refractometer USG measurement was 1.018 (range: 1.004-1.040) and for the digital refractometer was 1.0176 (1.0035-1.0357). The analog refractometer average measure ICC was 0.995 (95% CI: 0.992, 0.997; p  < 0.001). The digital refractometer average measure ICC was 0.999 (95% CI: 0.999, 1.000; p  < 0.001). Strong agreement between all pairs of RVTs was seen via Bland-Altman plots for both analog and digital refractometers, with 95% CIs spanning no more than 0.002 in either the positive or negative direction for all pairings. The interobserver variability in canine USG measurements by RVTs was trivial and did not impact clinical judgment and decision-making.

Cimicoxib is a selective COX-2 inhibitor (coxib) registered for the treatment of pain and inflammation in dogs. Pharmacokinetics of some coxibs have been described in dogs and cats. In cats, total body clearance values are lower and terminal half-lives of the coxibs are longer than those in dogs. The aim of this work was to evaluate if this is also the case for cimicoxib. For this purpose, blood pharmacokinetics and urinary excretion after IV administration were compared between these species. The in vitro metabolism of cimicoxib was also evaluated using canine and feline microsomes. In canine and feline microsomes, the formation rate of demethyl-cimicoxib, a phase 1 metabolite, was decreased in presence of quinidine, a specific human cytochrome P450 (CYP)2D6 inhibitor. IC 50 values were 1.6 μM and 0.056 μM with canine and feline microsomes, respectively. As quinidine was about 30 times more potent in feline microsomes, the affinity of cimicoxib to the enzyme was considered to be about 30 times lower than that in canine microsomes. Total body clearance (Cl B ) of cimicoxib, was 0.50 L/h kg in dogs and 0.14 L/h kg in cats (P < 0.01) and terminal half-life, T ½λz , was 1.92 and 5.25 h, respectively (P < 0.01). Dose eliminated in urine was 12.2% in dogs and 3.12% in cats (P < 0.01). Conjugated demethyl-cimicoxib represented 93.7% of this amount in dogs and 67.5% in cats. Thus cimicoxib, like other veterinary coxibs, was eliminated more slowly in cats. Both CYP2D15 (the canine ortholog of CYP2D6) and UDP-glucuronyltransferase enzyme systems have reduced ability to produce metabolites of cimicoxib in cats., (Copyright © 2021 Vetoquinol SA. Published by Elsevier Ltd.. All rights reserved.)

The stability of canine urine samples is essential when the samples cannot be analyzed immediately. The objective of this study was to investigate the stability of canine urine samples at room temperature and under refrigerated conditions. Samples from 20 dogs were collected, divided, and stored at 4°C and 20°C. The samples were examined up to 48 h after collection for specific gravity, pH, protein, bilirubin, glucose, ketones, and sediment and at 4 h and 24 h for bacterial growth. Specific gravity and all chemistry parameters were stable for a minimum of 48 h in 90% of samples. The sediment was stable, apart from crystals. The bacterial growth of 3 bacterial species tested in vitro , as well as the clinical samples, was mostly constant over 24 h at the refrigerated temperature. In urine samples stored at room temperature, the total number of aerobic growing bacteria was increasing. The results of our study showed that routinely measured parameters were stable in unpreserved urine for a minimum of 4 h and up to 48 h in most cases. If it is not possible to culture urine immediately, it is recommended that urine samples be stored at 4°C for a period of up to 24 h., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Urolithiasis is highly prevalent in dogs and cats, with struvite and calcium oxalate being most commonly diagnosed. Some commercial diets aimed at reducing the risk of urolithiasis are based on inclusion of sodium chloride (NaCl) in an attempt to dilute the urine and the risk of crystallization, but more information on the effect of differing levels of sodium inclusion is needed. The objective of this study was to compare the short-term effect of four diets differing only in NaCl content (base diet with 0.3% sodium and diets with added NaCl to achieve 0.7, 1.0 and 1.3% sodium as fed) on urinary ion concentrations and relative supersaturation (RSS) of struvite and calcium oxalate in dogs and cats. In both species, there was a significant increase in water intake and urine volume as dietary NaCl increased. Urine sodium concentration increased with increasing dietary NaCl. The highest sodium diet increased urinary calcium excretion in dogs only, while decreasing urinary calcium concentration. Calcium oxalate RSS and struvite RSS both significantly decreased, with the lowest RSS values reported on the highest sodium diet in both dogs and cats (p < .001). These results suggest that an increase in dietary NaCl decreases RSS values in both dogs and cats. Despite an increase in urinary calcium excretion in dogs, urinary calcium concentration and calcium oxalate RSS were lower on high sodium diets due to urine dilution. Long-term studies are needed to confirm the relationship between RSS and stone occurrence and recurrence., (© 2020 The Royal Canin. Journal of Animal Physiology and Animal Nutrition published by Blackwell Verlag GmbH.)

Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite's RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 μm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 μg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 μg/mL, and oxypurinol at 10-200 μg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Serum creatinine concentrations are higher in Greyhounds when compared with nonsighthound breeds. Greyhounds might also have higher urine creatinine concentrations compared with other breeds, which could affect urine protein-to-creatinine ratio (UPC) references., Objectives: We aimed to determine the UPC reference intervals (RIs) in healthy nonracing Greyhounds and compare this with UPC values in a group of healthy nonsighthounds and with the current International Renal Interest Society (IRIS) guidelines., Methods: The study used an observational cross-sectional design, involving clinically healthy, nonracing Greyhounds (n = 98) and nonsighthound dogs of similar weight, age, and sex (n = 24). Packed cell volumes, total solids, urine protein concentrations, serum and urine creatinine concentrations, urine specific gravity (USG) measurements, and UPCs were determined. Linear regression was used to compare urine creatinine and urine protein concentrations, relative to the USG measurements, between Greyhound and nonsighthound groups. Greyhound UPC RIs were determined using nonparametric methods and compared with UPC values in nonsighthounds and current IRIS guidelines., Results: Mean urine creatinine concentrations, adjusted for USGs, were approximately 22% higher in Greyhounds compared with nonsighthounds (P = .002). Mean urine protein concentration (P = .46) and UPC (P = .1) were not significantly different between Greyhounds and nonsighthounds. The upper limit of the Greyhound UPC RI was 0.20 or 0.42, depending on whether strict or moderate exclusion criteria were applied, respectively., Conclusions: Greyhounds have higher urine creatinine concentrations than nonsighthounds. Although the suggested RI for UPCs in Greyhounds is slightly lower than the cut-offs recommended in generic canine IRIS guidelines, this difference is not likely to be clinically significant., (© 2020 American Society for Veterinary Clinical Pathology.)

Nonadrenal diseases (NAD), including congestive heart failure (CHF), can affect the conversion of cortisone to cortisol favoring the production of cortisol's urinary downstream metabolites 5α/5β-tetrahydrocortisol (THF) relative to tetrahydrocortisone (THE). We hypothesized that healthy dogs would have lower urinary levels of cortisol, cortisone, THF, and THE than dogs with hypercortisolism (HC) or CHF, and the latter would have higher urinary levels of THF and lower THE than dogs with HC. Four, 9, and 8 dogs with HC, CHF, and normal health, respectively, were included in a pilot prospective cross-sectional study. A single morning voided urine sample was analyzed for urinary cortisol metabolites by liquid chromatography-mass spectrometry. The percentages of conjugated urinary metabolites were significantly higher in dogs with CHF than in healthy dogs ( p  = 0.001), and not different in HC dogs ( p  = 0.07). Log-transformed urine cortisol metabolites-to-creatinine ratios in healthy dogs were significantly lower than the 2 other groups ( p  < 0.001). The urinary free THE:THF ratio was significantly higher ( p  < 0.001) than the urinary total and conjugated THE:THF ratios. Health status did not affect the total, conjugated, and free THE:THF ratios ( p  = 0.61). Additional studies are needed to investigate differences in cortisol metabolites between dogs with HC and NAD to accurately discriminate between the groups.

Urine specific gravity (USG), which is usually measured by refractometry, is an important indicator of renal concentrating ability. Few studies have evaluated refractometers with separate scales for canine and feline urine. Variables such as protein content or storage time may influence the USG. We compared the effects of measuring USG with a refractometer with single or separate scales for canine and feline urine, investigated inter- and intra-observer variability, and measured agreement between whole urine and supernatant. We evaluated the correlation between USG and osmolality, the influence of urinary protein on USG and osmolality, and the impact of storage time up to 6 mo. We examined 252 canine and 126 feline samples. Bland-Altman analysis revealed higher USG values of the single-scale refractometer than the dual-scale refractometer, with a mean difference (bias) of < 0.001 for canine and 0.003 for feline specimens. Inter- and intra-observer variability were acceptable. Good agreement was shown between USG of whole urine and supernatant. Correlations between USG and osmolality were excellent (0.98-0.99, p  < 0.001). Proteinuria up to 1 g/L had no major impact on USG or osmolality. Storage time had no significant effect on USG. The difference between the refractometers is clinically irrelevant, and the use of a refractometer with separate feline and canine scales is unnecessary. Whole urine and supernatant stored up to 6 mo can both be used for USG measurement. The influence of proteinuria <1 g/L on USG and osmolality is negligible.

The accuracy of urine analyzers used for dogs and cats has remained uncertain. This study examines the agreement between results of urine analysis obtained using two devices marketed for animals and for humans and the results of quantitative biochemical analysis. The degrees of concordance for bilirubin and ketones in the same category were ~80%, but for pH these were only ~60% in dogs and cats. Degrees of concordance for protein and the UP/C ratio clearly differed between the devices for animals and humans. We found that values for bilirubin and ketones obtained using urine analyzers may be reliable, but pH is unlikely to be accurate enough to be clinically useful for dogs and cats.

Background: Refractometry is often used in clinical veterinary medicine to estimate urine concentration. Variability among commonly-used refractometers has not been critically evaluated., Objective: This study aimed to evaluate the variability of urine specific gravity (USG) among four refractometers and compare results of USG measurements with those of urine osmolality (U osm ), the gold standard for determining urine concentrations., Methods: USG was determined in 100 dogs using three optical refractometers, the American Optical Abbe refractometer 10450, Reichert TS 400 refractometer, and Heska Veterinary refractometer 2737-E02, and one digital refractometer, the Misco Palm Abbe Digital refractometer #PA203. Results were compared between each pair of refractometers and between each refractometer and urine osmolality determined by freezing point depression using a Multi-Osmette 2430E osmometer. Results were analyzed with Bland-Altman plots and Passing-Bablok regression analysis., Results: The Reichert, Heska, and Misco refractometers provide USG measurements that can be used interchangeably based on small differences, consistently less than 0.002, between the USG measurements. The AO refractometer measured USG values with much larger differences, which were deemed clinically significant by the study parameters. None of the refractometers were able to accurately predict U osm or vice versa within a clinically acceptable range., Conclusions: Varying degrees of differences were seen in the USG measurements among the different refractometers. These differences were refractometer-dependent, and the results from one instrument could affect clinical decisions., (© 2019 American Society for Veterinary Clinical Pathology.)

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., (© 2019 American Society for Veterinary Clinical Pathology.)

Assessing changes in oxytocin (OT) levels in response to a variety of social stimuli has become of major interest in the field of behavioral endocrinology. OT is involved in the regulation of various aspects of social behavior such as tolerance, and the formation and maintenance of social bonds but also the regulation of stress. All of these aspects have been identified as potential targets of selection during the domestication process. Therefore, comparing the role of the oxytocinergic system in various aspects of dog and wolf social behavior, might help to understand whether this system was involved in the domestication process. Studies assessing OT levels in dogs and wolves have used invasively collected plasma and serum samples and non-invasively collected urine samples. However, when using an assay system on a new species a careful and complete validation of the method is of crucial importance, and to date no proper validation, to assess urinary OT levels in dogs and wolves, has been reported. We therefore conducted an analytical validation of an Enzyme Immunoassay (EIA) for the measurement of OT in urine of dogs and wolves, using a commercially available EIA. Stability tests revealed that OT levels degrade over time when stored at 4 °C, but are little affected by repeated thawing. In addition, our results indicate that the variance in OT levels is slightly lower when phosphoric acid is added following collection to prevent OT degradation. Long term storage tests revealed that urinary OT levels are least variable when stored as extracts in ethanol at -20 °C, rather than as unextracted urine samples. Validation results were acceptable with regard to parallelism, but values for accuracy and extraction efficiency were not meeting the standard criteria usually applied to steroid EIAs, especially when assessed for the lower range of the assay. The results of this study highlight the importance of an analytical assay validation, since even if validation parameters are not optimal, if published, they allow readers to estimate the relevance of studies using the validated method., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Urine specific gravity (USG) is an integral part of the urinalysis and a key component of many clinical decisions, and fluctuations in USG have the potential to impact case management., Objectives: To determine the intraindividual variability of first morning USG results in healthy dogs., Animals: One hundred three healthy client-owned dogs., Methods: Dogs were deemed healthy based on clinical history and physical examination findings. Repeated USG measurements were performed over the course of 2 weeks. Three urine samples were collected each week for a total of 6 samples per dog. Sample collection was distributed evenly throughout the week. Urine samples were acquired immediately upon waking and before any ingestion of liquids, food, or exertion of physical activity in the dogs. All measurements were made using the same Misco digital refractometer., Results: Intraindividual USG was variable over the course of the study. The mean difference between the minimum and maximum USG for each dog was 0.015 (SD, 0.007). The within-week difference between the minimum and maximum USG was less than over the complete 2-week study (0.009 [SD 0.006] for week 1 and 0.010 [SD 0.007] for week 2). The mean coefficient of variance across all 6 time points was 15.4% (SD 8.97%)., Conclusions and Clinical Importance: Clinically important variation occurs in USG in healthy animals and might impact clinical decision-making when diagnostic cutoff points are utilized. Clinicians should be aware of inherent variability in this clinical variable when analyzing results., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Medication control in greyhound racing requires information from administration studies that measure drug levels in the urine as well as plasma, with time points that extend into the terminal phase of excretion. To characterize the plasma and the urinary pharmacokinetics of flunixin and enable regulatory advice for greyhound racing in respect of both medication and residue control limits, flunixin meglumine was administered intravenously on one occasion to six different greyhounds at the label dose of 1 mg/kg and the levels of flunixin were measured in plasma for up to 96 hr and in urine for up to 120 hr. Using the standard methodology for medication control, the irrelevant plasma concentration was determined as 1 ng/ml and the irrelevant urine concentration was determined as 30 ng/ml. This information can be used by regulators to determine a screening limit, detection time and a residue limit. The greyhounds with the highest average urine pH had far greater flunixin exposure compared with the greyhounds that had the lowest. This is entirely consistent with the extent of ionization predicted by the Henderson-Hasselbalch equation. This variability in the urine pharmacokinetics reduces with time, and at 72 hr postadministration, in the terminal phase, the variability in urine and plasma flunixin concentrations are similar and should not affect medication control., (© 2019 John Wiley & Sons Ltd.)

Objective: To quantify the magnitude and duration of changes in urine chondroitin sulfate concentration (uCS) as a result of oral administration of a chondroitin sulfate-containing supplement in dogs., Animals: 8 healthy privately owned dogs., Procedures: A urine sample was collected from each dog via cystocentesis on day 1; free-catch midstream urine samples were collected once daily on days 2 through 5. Pretreatment uCS was established from those samples. Each dog then received a chondroitin sulfate-containing supplement (20 to 30 mg/kg, PO, q 12 h) for 8 days (on days 7 through 14). Urine samples were collected on days 8 through 12 and day 15. For each sample, uCS was quantified by liquid chromatography-tandem mass spectrometry. Variable urine concentration was accounted for by dividing the uCS by urine creatinine concentration (uCrea) to determine the uCS:uCrea ratio. Pretreatment uCS:uCrea ratios were compared with treatment uCS:uCrea ratios to calculate the fold change in uCS after supplement administration., Results: Among the study dogs, oral administration of the chondroitin sulfate-containing supplement resulted in a 1.9-fold increase in the median uCS:uCrea ratio. Data obtained on days 8 through 12 and day 15 indicated that the daily increase in uCS remained consistent and was not additive., Conclusions and Clinical Relevance: Results indicated that oral administration of supplemental chondroitin sulfate to dogs modestly increased uCS within 24 hours; however, subsequent supplement administration did not have an additive effect. A potential therapeutic benefit of persistently increased uCS in preventing recurrent urinary tract infections in dogs warrants investigation.

Background: Small RNA sequencing (RNA-seq) of biofluids is challenging due to the relative scarcity of microRNAs (miRNAs), limited sample volumes, and the lack of a gold standard isolation method. Additionally, few comparisons exist for the RNA isolation and sequencing methods of biofluids., Objectives: We aimed to compare the performance of six commercial RNA isolation kits and two library preparation methods for small RNA-seq using canine serum and urine., Methods: Serum and urine were collected from seven dogs with protein-losing nephropathy, and the samples were pooled. Total RNA from serum (2 mL) and urine (10 mL) was isolated in triplicate using three methods each for serum (Zymo Direct-zol, mirVana PARIS, miRCURY Biofluids) and urine (Qiagen exoRNeasy, Norgen Urine Exosome, miRCURY Exosome). For each sample type, the two kits yielding the highest RNA concentration were selected, and small RNA-seq was performed using TruSeq and NEXTflex library preparations. Data were analyzed by CPSS 2.0 and DESeq2., Results: For serum, Zymo Direct-zol combined with NEXTflex was the only combination that enabled successful library preparation, while for urine, Qiagen exoRNeasy combined with NEXTflex outperformed other combinations for detecting miRNAs. The total number of miRNAs detected in serum and urine was 198 and up to 115, respectively. miRNA expression in serum was distinct from urine. Furthermore, the library preparation method introduced a higher variation of urine results than the RNA isolation method., Conclusions: Different isolation and library preparation methods show significant differences in miRNA results that could affect biomarker discovery. Small RNA-seq provides an unbiased, global assessment to compare these methods in canine biofluids., (© 2019 American Society for Veterinary Clinical Pathology.)

Background: Microscopic evaluation of urine is inconsistently performed in veterinary clinics. The IDEXX SediVue Dx® Urine Sediment Analyzer (SediVue) recently was introduced for automated analysis of canine and feline urine and may facilitate performance of urinalyses in practice., Objective: Compare the performance of the SediVue with manual microscopy for detecting clinically relevant numbers of cells and 2 crystal types., Samples: Five-hundred thirty urine samples (82% canine, 18% feline)., Methods: For SediVue analysis (software versions [SW] 1.0.0.0 and 1.0.1.3), uncentrifuged urine was pipetted into a cartridge. Images were captured and processed using a convolutional neural network algorithm. For manual microscopy, urine was centrifuged to obtain sediment. To determine sensitivity and specificity of the SediVue compared with manual microscopy, thresholds were set at ≥5/high power field (hpf) for red blood cells (RBC) and white blood cells (WBC) and ≥1/hpf for squamous epithelial cells (sqEPI), non-squamous epithelial cells (nsEPI), struvite crystals (STR), and calcium oxalate dihydrate crystals (CaOx Di)., Results: The sensitivity of the SediVue (SW1.0.1.3) was 85%-90% for the detection of RBC, WBC, and STR; 75% for CaOx Di; 71% for nsEPI; and 33% for sqEPI. Specificity was 99% for sqEPI and CaOx Di; 87%-90% for RBC, WBC, and nsEPI; and 84% for STR. Compared to SW1.0.0.0, SW1.0.1.3 had increased sensitivity but decreased specificity. Performance was similar for canine versus feline and fresh versus stored urine samples., Conclusions and Clinical Importance: The SediVue exhibits good agreement with manual microscopy for the detection of most formed elements evaluated, but improvement is needed for epithelial cells., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Proteinuria is a marker of chronic kidney disease in dogs and a risk factor for increased morbidity and death. Predictive models using the results of readily available screening tests could foster early recognition., Objective: To determine whether urine specific gravity (USG) and semiquantitative category of dipstick protein can be used to predict urinary protein-to-creatinine ratio (UP : C) and to examine the effect of urine culture results on UP : C in dogs., Animals: Three hundred ninety-four dogs (482 visits) presented to a university Community Practice Clinic or Veterinary Teaching Hospital between January 2011 and November 2015., Methods: Retrospective study. Medical records were searched to identify dogs for which urinalysis, UP : C measurement, and urine culture testing were performed during a single hospital visit. Urine specific gravity, UP : C, dipstick protein concentration, and findings of urine sediment analysis and urine culture were recorded. Regression or Spearman correlation analysis was used to test for relationships between UP : C and USG within dipstick categories and between UP : C and bacterial colony-forming units per milliliter, respectively. Cohen's kappa test was used to evaluate agreement between urine culture and UP : C testing., Results: There were significant (P < .05) weak negative correlations (R 2 range, 0.14-0.37) between UP : C and USG for all nonnegative urine protein dipstick categories. The presence of a positive urine culture did not agree with the presence of abnormal UP : C (κ = -0.06)., Conclusions and Clinical Importance: Within dipstick protein categories, UP : C cannot be accurately predicted from USG. Repeating UP : C measurement after resolution of urinary tract infection is advisable., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

The aim of this study was to compare fecal microbiome, plasma, fecal and urine metabolomes, and serum biochemistry of adult companion dogs according to body condition scores. Blood, serum/plasma, urine, and fecal samples were collected from 66 clinically healthy, adult companion dogs of either normal weight (NW), overweight (OW), or obese dogs (OB). analyses included fecal microbiome analyses via 16S ribosomal RNA gene amplicon; sequencing, nontargeted plasma, fecal, and urine metabolomics using liquid chromatography/gas chromatography-mass; spectrometry, and serum biochemistry for each dog. Few significant differences in serum biochemistry and fecal microbiome Operational Taxonomic Unit (OTU) were found between weight groups and there was high OTU variation between individual dogs. NW dogs had higher relative abundance of the genus Eubacterium (log-fold change 4.3, adjusted P value = .003) and lower relative abundance of the family Bifidobacteriaceae (log-fold change -3.6, adjusted P value = .02) compared to OB dogs. The microbiome of NW dogs had higher OTU richness compared with OB dogs. Metabolome analysis showed 185 plasma, 37 fecal, and 45 urine metabolites that significantly differed between NW and OW or OB dogs. There were notable significant differences in relative abundance of several plasma phospholipid moieties and fecal volatile fatty acids between weight phenotypes. The combinations of host and gut microbiota and metabolic shifts suggest a pattern that could help detection of early metabolic changes in overweight dogs before the development of obesity related disease. The results of this study support the need for continued investigation into sensitive measures of metabolic aberrancies in overweight dogs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Centrifugation is the primary method used to perform urine sediment analyses, but evaluation of other methods is required to validate centrifugation., Objectives: Non-urine materials were used to examine the repeatability (precision) and effectiveness (recovery) of four sediment methodologies on red blood cell (RBC) and white blood cell (WBC) counts., Methods: Four urine sediment methods were compared using commercially available quality control material (QCM) and fresh canine RBCs in a diluent. Treatments included (a) 5 mL centrifugation at 390g for 5 minutes; (b) 1.5 mL centrifugation at 3900g for 45 seconds; (c) 60 µL of neat (unspun urine) in a microtiter well; and (d) 30 µL of neat on a slide with a coverslip. A within-run precision using QCM was followed by a one-run comparison test performed with a suspension of canine erythrocytes. RBC morphology was also examined., Results: All results are listed in order of Methods A-D. Percent coefficients of variation (%CVs) for WBCs were 23.2%, 33.7%, 15.0%, and 27.2%. Red blood cells %CVs were 34.3%, 29.2%, 16.2%, and 24.4%. Average WBC counts in ten fields of view (FOV) ± 1 SD were 26.4 ± 6.1, 14.2 ± 4.8, 32.8 ± 4.9, and 1.6 ± 0.4. Average RBC counts in 10 fields of view (FOV) ± 1 SD were 45.3 ± 15.5, 23.9 ± 7.0, 38.4 ± 6.2, and 2.6 ± 0.6. The one-run comparison test reports average RBC counts per FOV at 55.2, 23.4, 92.8, and 13.8. The percentages of abnormal RBCs were 92.2%, 74.8%, 7.0%, and 55.1%., Conclusions: Method C had the best reproducibility, a lower frequency of cell morphology abnormalities, and similar cellular counts to those of Methods A and B., (© 2018 American Society for Veterinary Clinical Pathology.)

The role of epigenetic alterations during cancer has gained increasing attention and has resulted in a paradigm shift in our understanding of mechanisms leading to cancer susceptibility. Sulforaphane (SFN) is a naturally occurring isothiocyanate derived from the precursor glucosinolate, glucoraphanin (GFN), which is found in cruciferous vegetables such as broccoli. Sulforaphane has been shown to suppress tumour growth by several mechanisms including inhibiting histone deacetylases. The objective of this study was to provide a detailed analysis of sulforaphane absorption following a single oral dose of a broccoli sprout supplement in normal dogs. A single dose of broccoli sprout supplement (with active myrosinase) was orally administered to 10 healthy adult dogs. Blood and urine samples were collected prior to dosing, and at various time points post-dosing. Plasma total SFN metabolite levels peaked at 4 h post-consumption and were cleared by 24 h post-consumption. Urinary SFN metabolites peaked at 4 h post-consumption, and remained detectable at 24 and 48 h post-supplement consumption. A trend for decrease in histone deacetylase activity was observed at 1 h post-consumption and a significant decrease was observed at 24 h post-consumption. The data presented herein indicate that oral SFN is absorbed in dogs, SFN metabolites are detectable in plasma and urine post-dosing, and SFN and its metabolites have some effect on histone deacetylase activity following a single dose., (© 2018 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Urine dipstick results may vary between operators/methods. The magnitude of variation across the veterinary field is currently unknown. The aim of this study was to compare the precision of urine dipstick results between standard direct visual and automated reading methods when performed by several operators. Urine samples were pooled and divided into three aliquots: one plain, one with glucose and one with serum. Final year students, veterinary surgeons and veterinary nurses, blinded to each sample, were then asked to perform dipstick analysis with direct visualisation and an automated analyser, and their technique was observed. A subsequent session was undertaken with samples which had pH titrated to achieve an acidic, neutral or alkaline value. Sixty-four veterinary students, 20 veterinary surgeons and seven veterinary nurses performed the first (n=61) or second (n=30) part of the study. Precision was greater using the automated reader. The most common observed technique errors were: lack of sample mixing, for both visual and automated methods, and not timing readings as per manufacturer instructions when performing visual analysis. This study suggests that in an environment with multiple operators, as is the case in veterinary teaching or large private hospitals, automated urine dipstick reading improves precision of results., Competing Interests: Competing interests: None declared., (© British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Introduction: This study examined whether the angiotensin II receptor blocker telmisartan had inhibitory effects on drug-induced renin-angiotensin-aldosterone system (RAAS) activation in normal dogs., Animals: Five healthy laboratory beagles were used in this study., Methods: Each dog received amlodipine (0.5 mg/kg, q12h, PO) alone for 14 days. Starting on the next day, animals received both amlodipine and telmisartan (1.0 mg/kg, q24h, PO) for 84 days. Systolic blood pressure, heart rate, plasma biochemical variables (blood urea nitrogen, creatinine, and electrolytes), plasma renin activity, and 24-h urinary aldosterone elimination (U-Aldo) were measured before amlodipine administration; at day 0; and at days 1, 7, 14, 28, 56, and 84 of telmisartan treatment., Results: Telmisartan was associated with significant decreases in systolic blood pressure on day 56 (p=0.046), whereas heart rate did not significantly change during this treatment (p=0.061). Plasma renin activity was significantly increased on days 1, 7, 28, 56, and 84 during telmisartan administration (all p=0.04). No change in median U-Aldo was detected following telmisartan administration (p=0.241). When U-Aldo was evaluated in individual animals, two dogs displayed evidence of aldosterone breakthrough., Conclusions: Telmisartan administration did not suppress RAAS activation. The appearance of aldosterone breakthrough supports the incomplete blockade of RAAS activation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Markers of kidney dysfunction and damage have potential to detect chronic kidney disease (CKD) in early stages. However, data on long-term variation of these markers in healthy dogs is lacking and is crucial for the interpretation of results., Hypothesis/objectives: To determine temporal variations of serum cystatin C (sCysC) and urinary retinol-binding protein (uRBP), neutrophil gelatinase-associated lipocalin (uNGAL), immunoglobulin G (uIgG), and C-reactive protein (uCRP) in healthy dogs., Animals: Eight clinically healthy adult Beagles were evaluated., Methods: Longitudinal observational study. Serum cystatin C was determined by particle-enhanced nephelometric immunoassay. Urinary retinol-binding protein, uNGAL, uIgG and uCRP were determined by ELISA and concentrations were indexed to urinary creatinine. Within- and between-dog variance components (VC) and within-dog coefficients of variation (CV) were determined from blood and urine collected at eight time points over 1.5 years., Results: Urinary C-reactive protein (uCRP) concentrations were consistently below the detection limit (5.28 ng/mL). Mean ± within-dog standard deviation for sCysC, uRBP/c, uNGAL/c and uIgG/c was 0.15 ± 0.01 mg/L, 0.09 ± 0.03 mg/g, 2.32 ± 2.03 μg/g and 12.47 ± 10.98 mg/g, respectively. Within-dog CV for sCysC, uRBP/c, uNGAL/c and uIgG/c was 8.1%, 33.7%, 87.2% and 88.1%, respectively., Conclusions and Clinical Importance: Serum cystatin C, uRBP/c, uNGAL/c and uIgG/c exhibit a wide range of long-term within-dog variability. Researchers and veterinarians might need to take this into account when interpreting their results. To assess their diagnostic and predictive ability, future studies need to establish reference ranges for healthy dogs and dogs with CKD., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Urine protein loss is common in dogs with chronic kidney disease (CKD). Currently available noninvasive means of evaluating CKD in dogs cannot accurately predict the severity of glomerular and tubulointerstitial damage. Electrophoretic analysis of urine proteins can indicate the compromised renal compartment (glomerular vs tubular), but extensive evaluation of protein banding pattern associations with histologic damage severity has not been performed in dogs., Objectives: We aimed to evaluate electrophoretic banding patterns as indicators of the presence and severity of glomerular and tubulointerstitial damage in dogs with naturally occurring, predominantly proteinuric CKD., Methods: We performed a retrospective study using urine and renal tissue from 207 dogs with CKD. Urine protein banding patterns were correlated with histologic severity of renal damage. Sensitivity and specificity of banding patterns for the detection of glomerular and tubulointerstitial damage were determined., Results: Banding patterns were 97% sensitive and 100% specific for the detection of glomerular damage and 90% sensitive and 100% specific for the detection of tubulointerstitial damage. Correlations between composite banding patterns and the severity of renal damage were strong, while glomerular banding patterns correlated moderately with glomerular damage severity, and tubular gel scores correlated weakly to moderately with the severity of tubulointerstitial damage., Conclusions and Clinical Importance: Urine protein banding patterns are useful for the detection of glomerular and tubulointerstitial damage in dogs with proteinuric CKD., (© 2018 The Authors. Veterinary Clinical Pathology Published by John Wiley & Sons Ltd.)

Background: Urine pH is an integral part of a complete urinalysis, and is commonly measured in veterinary practice using semiquantitative reagent strips., Objective: The aim of this study was to compare the urine pH of dogs and sheep, using visual interpretation of dipstick reactions, and using a pH-meter as the reference method. Agreement between the 2 methods was also assessed. An additional objective was to compare the urine pH before and after centrifugation., Methods: A total of 50 voided urine samples from sheep and 52 from dogs were collected into sterile containers. For pH measurements, 2 methods were used, a pH-meter and urine dipstick reagent pads. Measurements were performed using urine samples before (whole urine) and after centrifugation (urine supernatant). For comparison of the 2 methods, Passing and Bablok regression analysis and Bland-Altman plots were used., Results: The equation created to assess agreement between the 2 methods in dogs showed a constant bias at -0.14 and a positive proportional bias at 0.98. From a clinical standpoint, total bias was below and above the maximum acceptable bias in sheep and dogs, respectively. Clinically acceptable bias was also found using centrifuged urine samples in sheep, but the urine pH values before and after centrifugation were nearly identical in dogs., Conclusion: Urine dipstick reagent pads and pH-meters can be used interchangeably to determine urine pH in sheep without needing centrifugation. In contrast, pH-meters provide more accurate pH measurements than urine dipstick pads in canine urine, which is not improved by centrifugation., (© 2018 American Society for Veterinary Clinical Pathology.)

Background: The dipstick is a first-line and inexpensive test that can exclude the presence of proteinuria in dogs. However, no information is available about the analytical variability of canine urine dipstick analysis., Objectives: The aim of this study was to assess the analytical variability in 2 dipsticks and the inter-operator variability in dipstick interpretation., Methods: Canine urine supernatants (n = 174) were analyzed with 2 commercially available dipsticks. Two observers evaluated each result blinded to the other observer and to the results of the other dipstick. Intra- and inter-assay variability was assessed in 5 samples (corresponding to the 5 different semi-quantitative results) tested 10 consecutive times over 5 consecutive days. The agreement between observers and between dipsticks was evaluated with Cohen's k test., Results: Intra-assay repeatability was good (≤3/10 errors), whereas inter-assay variability was higher (from 1/5 to 4/5 discordant results). The concordance between the operators (k = 0.68 and 0.79 for the 2 dipsticks) and that of the dipsticks (k = 0.66 and 0.74 for the 2 operators) was good. However, 1 observer and 1 dipstick overestimated the results compared with the second observer or dipstick. In any case, discordant results accounted for a single unit of the semi-quantitative scale., Conclusions: As for any other method, analytic variability may affect the semi-quantitation of urinary proteins when using the dipstick method. Subjective interpretation of the pad and, to a lesser extent, intrinsic staining properties of the pads could affect the results. Further studies are warranted to evaluate the effect of this variability on clinical decisions., (© 2018 American Society for Veterinary Clinical Pathology.)

Background: Lower urinary tract infections are common in dogs, and Escherichia coli is the most common bacterial pathogen isolated. The literature has conflicting evidence regarding the inhibitory effects of urine concentration and pH on E. coli growth., Hypothesis/objectives: To determine the effect of different pH and urine concentrations on E. coli growth in vitro., Animals: Voided urine samples from 10 apparently healthy spayed female dogs were used., Methods: A matrix of 9 urine specific gravity (USG; 1.010, 1.020, and 1.030) and pH (5.5, 7.0, and 8.5) combinations was prepared by diluting and titrating filtered voided urine samples. Three E. coli isolates were obtained from urine of female dogs with signs of lower urinary tract infection and cultured at different urine pH and USG combinations in wells of a microtiter plate. The number of E. coli colony-forming units (CFU) per mL of urine was calculated after aerobic incubation of the urine at 37°C for 18 hours, and statistically compared., Results: Significant differences were identified in the mean log CFU/mL among different combinations of pH and USG. The lowest log CFU/mL were observed in alkaline concentrated urine (pH 8.5 and USG 1.030)., Conclusions and Clinical Importance: Escherichia coli in vitro growth was higher in neutral to acidic and diluted urine compared to alkaline and concentrated urine. The impact of non-alkalizing diluting diets on the incidence of E. coli lower urinary tract infections should be further explored., (Copyright © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

This combined retrospective-prospective study evaluated the correlation between canine urine color (UC) and urine specific gravity (USG). Urine color was positively correlated with USG, but the relationship was not significantly improved by use of a UC chart. Urine color as an indicator of USG is limited as 20% of dark-yellow samples had a USG < 1.030.

Urine osmolality (UOsm) is considered the most accurate measure of urine concentration and is used to assess body fluid homeostasis and renal function. We performed analytical validation of freezing point depression measurement of canine UOsm, to establish reference intervals (RIs) and to determine the effect of age, sex, and reproductive status on UOsm in dogs. Clinically healthy dogs ( n = 1,991) were retrospectively selected and stratified in groups by age (young [0-12 mo], adults [13-84 mo], and seniors [>84 mo]), sex (females and males), and reproductive status (intact and neutered). RIs were calculated for each age group. Intra- and inter-assay coefficients of variation were <1% in all cases. Good linearity ( r 2 = 1, p < 0.001) and recovery (89-98%) were observed. The limit of detection and limit of quantification were zero. Urine specific gravity and UOsm had a highly significant positive correlation ( r = 0.96, p < 0.001) but had inconsistent agreement. The 95% RI for canine UOsm was 369-2,416 mOsm/kg in young and adult dogs, and 366-2,178 mOsm/kg in seniors. Senior dogs had a significantly lower UOsm than young and adult dogs ( p < 0.000). Neutered females had a significantly lower UOsm than intact female dogs ( p < 0.002). These results indicate that the method evaluated is adequate for UOsm measurement and that RIs based on age and reproductive status should be used in dogs.

Gonadotropin-releasing hormone (GnRH) is a 10-residue peptide hormone that induces secretion of luteinizing hormone (LH) and follicle-stimulating hormone into the blood from the pituitary gland. In males, LH acts on the testes to produce testosterone. The performance-enhancing potential of testosterone makes administration of exogenous GnRH a concern in sports doping control. Detection of GnRH abuse is challenging owing to its rapid clearance from the body and its degradation in urine. Following recent investigations of GnRH abuse in racing greyhounds in New Zealand, we carried out a GnRH administration study in greyhounds in an attempt to identify GnRH metabolites that might provide more facile detection of GnRH abuse; little information is available on in vivo metabolites of exogenous GnRH in any species and none in dogs. We identified three C-terminal GnRH metabolites in urine: GnRH 5-10, GnRH 6-10, and GnRH 7-10. These metabolites and intact GnRH, which was also detected in urine, were all excreted over a 1-3 h period after GnRH administration. Two of the GnRH metabolites - GnRH 5-10 and GnRH 6-10 - were more stable in urine than intact GnRH offering improved potential to detect GnRH administration. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Computed tomography (CT) is able to measure the attenuation of urine in Hounsfield units (HU) on abdominal imaging studies. This study was designed to measure the correlation of urine attenuation with urine specific gravity in urine samples of 40 dogs, providing a noninvasive measure of urine concentration. The HU of urine explained 72% of the variance in measured urine specific gravity [ R 2 = 0.72, F(1,38) = 95.55, P < 0.001]. This noninvasive measurement can be used to estimate urine concentration in dogs undergoing abdominal CT imaging.

The objective of this prospective crossover study was to investigate the effects of a high-protein diet on canine urinary corticoid-to-creatinine ratio (UCCR). The hypothesis was that meal-induced hypercortisolism is, as has been shown in humans, a predictable and consistent finding in healthy dogs. Eight clinic-owned beagles were randomly assigned to one of two groups. The allocation to the groups defined the sequence of a protein-enriched meal (meal A) or no meal on the first and second days, whereas on the third day all dogs again received an identical meal (meal B) to test reproducibility. Urinary corticoids were measured using a solid-phase, competitive CLIA on unextracted urine. Contrary to our expectations, consistent incremental responses of the UCCR were not observed (meal A vs. no meal [anova]: absolute increase, F = 2.546, p = 0.162; relative increase, F = 4.084, p = 0.09; AUC (UCCR) , F = 0.279, p = 0.616). Nevertheless, the robust increases in two dogs above 60% of baseline suggest that the collection of urine prior to feeding likely increases the specificity of the UCCR to discriminate between dogs with and without hypercortisolism., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)

In dogs, there is no diagnostic test to identify and differentiate fetal fluids from maternal urine in the event that a clear-yellowish vulvar discharge is observed pre-whelping. The objective of this study was to find a test that could easily and accurately identify rupture of the fetal membranes preceding parturition. Maternal urine, and amniotic fluid (AMF) and allantoic fluid (ALF) from only one fetus per bitch, were collected intraoperatively during Cesarean section. Specific gravity (SG) was analyzed with a refractometer, whereas the presence of leukocytes, protein, glucose, ketones, bilirubin, urobilinogen, nitrite, erythrocyte/hemoglobin (Hb), and the pH were assessed using a urine dipstick (Combur-Test ® ). Combined calcium and magnesium (Ca/Mg) content were evaluated with the Total Hardness Test. The AmniSure test, which detects rupture of fetal membranes in women on the basis of the presence of human placental alpha microglobulin-1, was also performed on canine AMF, ALF, and urine. Data were analyzed using the Fisher's exact test, Wilcoxon signed-rank test, and Pearson's correlation. Sensitivity, specificity, and positive and negative likelihood ratios (LR) were calculated for parameters with significant difference between urine and both fetal fluids. Maternal urine had higher SG and lower leukocyte, protein, Hb, and Ca/Mg content than AMF and ALF. Glucose was more often present in AMF (n = 17) and ALF (n = 12) than in urine (n = 1), whereas ketone bodies were rarely detected in ALF compared with urine. Bilirubin content was higher in urine and ALF than in AMF. AMF pH was less variable and higher than the pH of ALF or urine. The AmniSure was negative in all samples tested. Sensitivity and specificity for SG and for the detection of leukocytes, protein, glucose, Hb, Ca/Mg, and glucose without ketones in urine and fetal fluids were between 42% to 100% and 65% to 100%, respectively. Best positive LR was achieved for the detection of glucose without ketones and best negative LR for SG of 1.022 or less. In conclusion, the AmniSure test, which is used in humans with high diagnostic accuracy, cannot identify AMF and ALF in dogs. On the basis of our results in 26 dogs undergoing Cesarean section, the presence or absence of fetal fluids could be best determined by a positive glucose test without ketone bodies or by SG higher than 1.022, respectively. These tests may serve as additional tools to recognize parturition if clear-yellowish vulvar discharge is present in a term pregnant bitch, but their accuracy and practicability in the clinical setting need to be confirmed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A pharmacokinetic/pharmacodynamic modelling approach was used to determine a dosage regimen which maximizes diuretic efficiency of torasemide in dogs. Kinetic profiles of plasma concentration, torasemide excretion rate in urine (TERU) and diuresis were investigated in 10 dogs after single oral administrations at 3 dose levels, 0.2, 0.8 and 1.6 mg/kg, and an intravenous injection of 0.2 mg/kg. Endogenous regulation was evidenced by a proteresis loop between TERU and diuresis. To describe the diuresis-time profile, TERU served as input into a turnover model with inhibition of loss of response, extended by a moderator acting on both loss and production of response. Estimated maximum inhibition of loss of response, I max , was 0.984 showing that torasemide is an efficacious diuretic able to suppress almost total water reabsorption. A TERU 50, value producing half of I max , of 1.45 μg/kg/h was estimated from the model. Pharmacokinetic and pharmacodynamic parameters were used to simulate the torasemide dose-effect relationship after oral administration. Model predictions were in good agreement with diuresis measured in a validation study conducted in 10 dogs, which were administered oral doses of 0.15, 0.4, 0.75, 1.5 and 4.5 mg/kg for 5 days. Finally, oral dose associated with the highest daily diuretic efficiency was predicted to be 0.1 mg/kg., (© 2016 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.)

Background: Acute kidney injury (AKI) is a common problem in small-animal patients and carries a guarded prognosis with substantial morbidity and mortality, particularly in oligoanuric dogs. Fenoldopam, a selective dopamine agonist, has been shown to increase urine output in healthy dogs and cats; however, the mechanism of action is unknown., Hypothesis/objectives: To evaluate the effect of fenoldopam infusion on glomerular filtration rate (GFR) and fractional excretion of sodium (FeNa) in healthy dogs., Animals: Ten healthy, privately owned dogs., Methods: Randomized, crossover design with negative control. Ten healthy dogs were given fenoldopam diluted in 5% dextrose (D5W) as a continuous IV infusion of 0.8 μg/kg/min for 5 hours and a control infusion of D5W alone, 7 days apart. Glomerular filtration rate was measured by exogenous iohexol clearance, beginning 1 hour after the start of the fenoldopam infusion. Fractional excretion of sodium (FeNa) was measured before and after the infusion. Glomerular filtration rate and change in FeNa were compared between treatment days., Results: Fenoldopam infusion resulted in a significantly increased (P = .0166) GFR (median GFR, 3.33 mL/min/kg) in healthy dogs compared with D5W infusion (median GFR, 2.71 mL/kg/min). Fenoldopam also resulted in a significantly increased (P = .0148) FeNa (mean change, 0.106), whereas infusion of D5W alone did not (mean change, 0.016)., Conclusions and Clinical Importance: In healthy dogs, fenoldopam significantly increased GFR and FeNa compared with infusion of D5W alone. No adverse effects were seen., (Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Many studies have reported the aversive reactions of prey towards a predator's odour signals (e.g. urine marks), a behaviour widely thought to reduce the risk of predation by the predator. However, because odour signals persist in the environment, they are vulnerable to exploitation and eavesdropping by predators, prey and conspecifics. As such, scent patches created by one species might attract other species interested in information about their enemies. We studied this phenomenon by examining red fox investigation of odours from conspecifics and competing species in order to understand what prey are responding to when avoiding the odours of a predator. Surprisingly, foxes showed limited interest in conspecific odours but were highly interested in the odours of their competitors (wild dogs and feral cats), suggesting that odours are likely to play an important role in mediating competitive interactions. Importantly, our results identify that simple, dyadic interpretations of prey responses to a predator odour (i.e. cat odour = risk of cat encounter = fear of cats) can no longer be assumed in ecological or psychology research. Instead, interactions mediated by olfactory cues are more complex than previously thought and are likely to form a complicated olfactory web of interactions., (© 2016 The Author(s).)

Background: Inadequate dietary selenium (Se) intake in humans and animals can lead to long term health problems, such as cancer. In view of the owner's desire for healthy longevity of companion animals, the impact of dietary Se provision on long term health effects warrants investigation. Little is currently known regards biomarkers, and rate of change of such biomarkers in relation to dietary selenium intake in dogs. In this study, selected biomarkers were assessed for their suitability to detect changes in dietary Se in adult dogs within eight weeks., Results: Twenty-four dogs were fed a semi-purified diet with an adequate amount of Se (46.1 μg/MJ) over an 8 week period. They were then divided into two groups. The first group remained on the adequate Se diet, the second were offered a semi-purified diet with a low Se concentration (6.5 μg/MJ; 31% of the FEDIAF minimum) for 8 weeks. Weekly urine and blood was collected and hair growth measurements were performed. The urinary Se to creatinine ratio and serum Se concentration were significantly lower in dogs consuming the low Se diet from week 1 onwards, by 84% (adequate 25.3, low 4.1) and 7% (adequate 257 μg/L, low 238 μg/L) respectively. Serum and whole blood glutathione peroxidase were also significantly lower in dogs consuming the low Se diet from weeks 6 and 8 respectively. None of the other biomarkers (mRNA expression and serum copper, creatine kinase, triiodothyronine:thyroxine ratio and hair growth) responded significantly to the low Se diet over the 8 week period., Conclusions: This study demonstrated that urinary Se to creatinine ratio, serum Se and serum and whole blood glutathione peroxidase can be used as biomarkers of selenium status in dogs. Urinary Se to creatinine ratio and serum Se concentrations responded faster to decreased dietary Se than the other parameters. This makes these biomarkers candidates for early screening of long term effects of dietary Se provision on canine health.

Aims: To compare the performance of five refractometers for determination of urine specific gravity in cats and dogs, with reference to weight of total solids and pycnometer analysis., Methods: Urine samples from 27 cats and 31 dogs submitted for routine urinalysis were included. Urine specific gravity was determined with five refractometers. Four were optical, hand-held refractometers with a temperature compensation method and one was a digital model. Urine was dried to determine the precise weight of total solids. The total solids (g/L) were converted to an estimated specific gravity by division with 2.33. Urine specific gravity of four feline and seven canine samples were analysed with a pycnometer. Limits of agreement analysis was used to evaluate the agreement between specific gravity (analysed as specific gravity minus 1) measured by the refractometers and estimated from dried total solids, or pycnometer results., Results: The five refractometers reported clearly different results from each other. Proportional negative bias was noted for refractometer results compared to estimated specific gravity from total solids and a constant negative bias compared to pycnometer results. The two refractometers designed for cat urine reported similar and lowest specific gravity results with a mean negative bias of 0.007 and 0.008 units compared to estimated specific gravity from total solids, and a mean negative bias of 0.006 units compared to pycnometer results., Conclusions: Refractometer results did not increase consistently with increasing urine specific gravity compared to reference methods or to other refractometers. Two feline refractometers reported consistently lower specific gravity results than reference methods and other refractometers., Clinical Relevance: Because of this imprecision, veterinarians should not use precise cut off values such as 1.030 or 1.035 for evaluation of renal concentrating ability in dogs and cats. Veterinarians should consider the variability of refractometric specific gravity results in their clinical assessment. Two feline refractometers appeared to report falsely low specific gravity results.

Background: The urine protein:creatinine ratio (UPC) is used to quantify urine protein excretion and guide recommendations for monitoring and treatment of proteinuria., Hypothesis/objectives: Home urine samples will have lower UPCs than hospital samples. The objectives were to compare UPCs of samples collected in each setting and to determine whether environment of sample collection might affect staging, monitoring or treatment recommendations., Animals: Twenty-four client-owned dogs., Methods: Prospective, nonmasked study. Clients collected a urine sample from their dog at home and a second sample was collected at the hospital. Dogs receiving corticosteroids or angiotensin-converting enzyme inhibitors were excluded, as were those with urine samples of inadequate volume, no protein on dipstick analysis, or active urine sediment. Samples were refrigerated after collection, dipstick and sediment evaluations were completed and each sample was frozen at -80°C within 12 hours. UPCs were performed on frozen samples within 2 months., Results: From 81 paired samples, 57 were excluded. Of the remaining 24, 12/24 (50%) had higher hospital sample UPCs, 9/24 (38%) had identical UPCs, and 3/24 (12%) had lower hospital UPCs. The UPCs of hospital samples were higher than home samples for the total population (P = .005) and the subset with UPC > 0.5 (P = .001)., Conclusions: Setting and related circumstances of urine collection in dogs is associated with UPC differences; results are usually higher in hospital than in home samples. This difference has the potential to affect clinical interpretation., (© 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Urinalysis data in preclinical toxicology studies can be influenced by preanalytic and analytic factors which have the potential to confound interpretation. There is a paucity of information regarding positive reagent strip urinary blood reactions in healthy nonhuman primates (NHP) and Beagle dogs used in preclinical toxicology studies., Objectives: The objectives were (1) to establish historical control data for reagent strip urinary blood reactions in healthy NHP and Beagle dogs, (2) to determine the incidence of positive urinary blood reactions during predose and dosing phases, and (3) to determine if collection practice was a relevant parameter., Methods: Historical control data from 2 institutions in the biopharmaceutical industry were retrospectively analyzed for reagent strip urinary blood reactions in healthy NHP and Beagles. The incidence of positive results between the 2 institutions with different urine collection practices and between males and females was compared., Results: The incidence of positive urinary blood reactions in NHP was comparable between institutions (≤ 14% in males; ≤ 33% in females), while the incidence of positive urinary blood reactions in Beagles was more variable (≤ 77% in males; ≤ 69% in females), and higher in females during the dosing phase., Conclusions: Positive urinary blood results that could potentially be misinterpreted as toxicologically relevant were identified in healthy NHP and Beagles during predose and dosing phases. Different incidences of positive results between the 2 institutions were likely related to collection practices. Strategies to reduce feces and food contamination of collected urine samples should help minimize false-positive urinary blood reactions., (© 2015 American Society for Veterinary Clinical Pathology.)

Objective: To compare the diuretic effects of subcutaneous (s.c.) administration of furosemide to conventional methods of administration including intravenous (i.v.), per os (p.o.), and constant rate infusion (CRI) in healthy dogs., Design: Prospective, randomized, cross-over study., Setting: Veterinary university research facility., Animals: Seven healthy, adult mongrel dogs (3 males, 4 females)., Intervention: Each dog in the study was randomly assigned to receive a 2 mg/kg dose of furosemide via a single s.c., i.v., or p.o. dose at the beginning (time 0) of an 8-hour study, or via CRI during an 8-hour study period. Urine was collected by emptying the bladder using an indwelling catheter and blood samples were obtained via venipuncture at time 0 for baseline measurements and at 1, 2, 4, 6, and 8 hours into the study. Hourly urine output was calculated in all dogs for each study. Complete blood count, plasma total protein, blood urea nitrogen, creatinine, and renin concentration were measured for each sample., Measurements and Main Results: The s.c. administration of furosemide resulted in a urine output per hour (UOP/h) that peaked at 1 hour with UOP/h returning to baseline at 4 hours after injection. Following i.v. administration, UOP/h also peaked at 1 hour but returned more rapidly to baseline levels at 2 hours after injection. With p.o. administration, UOP/h reached a maximum UOP/h at 2 hours but time to return to baseline levels was prolonged to 6 hours after administration. With CRI administration, the time to the maximum UOP/h was delayed to 4 hours after injection but UOP/h was then maintained throughout the study period., Conclusions: Total urine output following s.c. administration of furosemide in healthy dogs was similar when compared to the i.v. and p.o. route. Subcutaneous route may be an effective means for administration of furosemide in dogs, particularly when i.v. access is difficult., (© Veterinary Emergency and Critical Care Society 2015.)