Qualitative Urinalyses in Puppies 0 to 24 Weeks of Age
Suggestions for interpreting qualitative urinalyses from puppies have been based on limited results obtained in the laboratory setting. Proteinuria, glucosuria, and decreased concentration of urine have been considered normal in puppies <8 weeks of age due to immature renal function. In this study, the authors reviewed 149 voided urine samples from 118 different, apparently healthy, random-source puppies. The primary finding was that mean urine specific gravity (USG) was significantly lower in 0- to 3-week-old puppies when compared to puppies 4 to 24 weeks old. Mean USG in all other age groups was >1.030. There was no difference in the frequency of positive protein or occult blood dipstick results among age groups, and there were no positive glucose, ketone, bilirubin, or urobilinogen reactions in any samples analyzed. Urine sediment results are reported for 41 samples. Epithelial cells and white blood cells were the most common sediment findings in these 41 voided samples, observed in 34 (83%) and 18 (44%) samples, respectively. Crystals were observed in 15 (37%) samples, whereas casts, bacteria, and red blood cells were observed less commonly.
Introduction
Practicing veterinarians occasionally encounter puppies with urinary tract disorders. A urinalysis, including urine specific gravity (USG), urine dipstick analysis, and sediment examination, is a mainstay of the diagnostic evaluation.1–3 Previous studies have demonstrated a relationship between age and renal function and have characterized the maturation of canine kidneys.4–7 In addition, quantitative urinalyses based on 48-hour urine collection have been reported in a small sample of healthy, growing beagle puppies from 9 to 27 weeks of age.8 Most reported studies have utilized sophisticated measurements of glomerular or tubular function or timed urine collection, employed in small numbers of puppies. However, recommendations for interpreting urinalysis results from immature puppies have been extrapolated from these limited results. Specifically, proteinuria, glucosuria, and decreased urine concentration have been considered to reflect immature renal function and have been interpreted as clinically insignificant in urinalyses from neonatal or juvenile puppies.129
However, to the authors’ knowledge, there are no available reports of random qualitative urinalyses from large numbers of puppies. Collection of urinalysis results from apparently healthy puppies will aid in the interpretation of urinalyses from ill puppies or puppies with urinary tract disease. The purpose of this study was to determine the results of qualitative urinalyses, including USG, biochemical dipstick findings, and urine sediment findings as performed in the veterinary practice setting, in healthy, random-source puppies from 0 to 24 weeks of age.
Materials and Methods
Selection of Animals
Apparently healthy puppies 0 to 24 weeks of age were randomly identified at various facilities during a 6-month period. Puppies included those held for adoption at animal shelter facilities and local pet stores and those owned by clients, students, or staff at the University of Tennessee’s College of Veterinary Medicine Veterinary Teaching Hospital and several local veterinary clinics. Permission for urine collection was granted by the facility owner or puppy owner in all cases.
Puppies were regarded as healthy based on physical examination and history, if available. Age (known or estimated), gender, and breed (if purebred) were recorded. If exact age was not known, age was estimated using owner/manager information, eyes, body size, and dentition. Dogs of unknown breed origin were considered to be of mixed breed. In order to increase sample size in all age groups, repeated samples were collected from some client-owned puppies. Multiple samples were collected only if the exact age was known and at least 7 days had passed between collections.
Urine Collection Methods
Puppies from the animal shelter were walked outside either on a leash or in a fenced area for a period of 10 to 15 minutes. Voided urine sample was obtained using a long-handled spoon that was placed in the path of the urine stream. The collection spoon was cleaned with disinfectant and dried between samples. Urine samples from puppies at commercial pet stores were obtained in a variety of ways: 1) Puppies remained in their crates after the trays were cleaned and disinfected. The trays were replaced without newspaper, and when the puppies urinated, the sample was immediately collected off the bottom of the tray using a sterile syringe. 2) Puppies were placed in an open plastic storage bin. Immediately after the puppy voided, the sample was collected with a sterile syringe. 3) Puppies were placed on a cleaned floor in a “play room” and allowed to play for a period of 10 to 15 minutes. If urination occurred, the sample was immediately collected from the floor using a sterile syringe.
All collected samples were transferred to a sterile, 5-mL, polypropylene vial.a Samples were placed on ice for no longer than 4 hours until they were ready for processing. Time of collection, method of collection, puppy identification, and source were recorded for each sample.
Urinalysis and Urine Sediment Examination
Urine samples were removed from ice for approximately 20 minutes and allowed to reach room temperature (25°C) before analyzing. Protein, pH, glucose, blood, ketone, bilirubin, and urobilinogen were measured by dipstickb biochemical analysis. If only 1 to 3 mL of urine was available for a given sample, USG, protein, glucose, pH, and occult blood assessments were sequentially prioritized. Any colorimetric change detected was defined as “positive” for the purposes of this study. For protein, “significant” proteinuria was further defined as any protein detected in urine with USG <1.035 and ≥2+ protein in urine with USG ≥1.035. Measurement of USG was performed using a hand refractometer.c
If at least 3 mL of urine was collected, a 3- to 4-mL aliquot of urine was placed in a polystyrene conical tube,d and the sample was centrifugede for 5 minutes at 1000 rpm. The supernatant was poured off, and the pellet was resuspended in the urine that remained in the bottom of the tube. Using a polyethylene transfer pipet,f a 100-μL drop was transferred to a microscope slide and covered with a coverslip; the samples were observed by a single individual (Faulks), first on low power (10×) and then on high power (40×). The presence of white blood cells (WBCs), red blood cells (RBCs), epithelial cells, crystals, and bacteria was reported either numerically or quantitatively per high-power field (HPF); the presence of casts was reported and quantitated under the low-power objective.
Statistical Analysis
Results were analyzed in the following age groups: 0 to 3 weeks, 4 to 6 weeks, 7 to 9 weeks, 10 to 12 weeks, 13 to 15 weeks, 16 to 20 weeks, and 21 to 24 weeks. Age groupings were selected to assess multiple, small (3-week) age ranges in puppies <16 weeks of age. The range of ages in each of the older groups is expanded slightly because of increased difficulty in estimating specific ages in those puppies. Descriptive statistics only are given for urine sediment findings and urine biochemical parameters including glucose, ketones, bilirubin, and urobilinogen. Differences between age groups for mean USG were detected by one-way analysis of variance (ANOVA) and the Tukey test for multiple comparisons using an analytic software program.g Differences between age groups for median urine pH, protein dip-stick, and blood dipstick results were detected by Kruskal-Wallis one-way ANOVA on ranks and Dunn’s method for multiple comparisons.g
Results
Puppy age characteristics, gender, and source are summarized by age group in Table 1.
Number of samples, number of repeat samples, gender of samples, and sample collection method are summarized by age group in Table 2.
Animals
Overall, 149 urine samples were collected from 118 different puppies. Multiple (two to three) samples were collected from 17 puppies at different ages. Four and six samples were collected at different ages from one puppy each. Gender was recorded for 115 puppies; 69 were female (seven spayed), and 46 were male (seven neutered). Of the 118 puppies, exact ages were known for 31 (27%).
In the 0 to 3 week age group, there were 33 samples collected from 26 different puppies. The sample population in this age group included Welsh corgis (n=13), English bulldogs (n=4), beagles (n=3), and mixed-breed dogs (n=6). Exact ages were known for 20 puppies, whereas an age <2 weeks was estimated for six puppies whose eyes were unopened at the time of sample collection. All samples in the 0 to 3 week age group were obtained from puppies nursing or fed liquid milk replacer, except for four samples that were obtained from neonates that had not yet nursed.
There were 29 samples from 22 different puppies in the 4 to 6 week age group. Puppies in this age group included Welsh corgis (n=7), beagles (n=5), and mixed-breed dogs (n=10). Four puppies in this group were still nursing at the time of sampling.
There were 17 samples from 17 different puppies in the 7 to 9 week age group; puppies from which urine samples were studied in this age group included Jack Russell terriers (n=2), a Pekeapoo (n=1), and mixed-breed dogs (n=14).
There were 41 urine samples from 41 different puppies in the 10 to 12 week age group; puppies from which urine samples were studied in this group included 32 mixed-breed dogs and nine purebred puppies, including the Australian shepherd (n=3), Jack Russell terrier (n=2), and Pomeranian, Shetland sheepdog, Finnish spitz, and Yorkshire terrier (n=1 each).
There were nine samples from eight different puppies in the 13 to 15 week age group; puppies from which urine samples were studied in this group included six mixed-breed dogs, a Jack Russell terrier, and a West Highland white terrier.
In the 16 to 20 week age group, there were 11 urine samples from 10 different puppies; puppies from which urine samples were studied in this group included eight mixed-breed dogs and two Jack Russell terriers.
There were nine urine samples from nine different puppies in the 21 to 24 week age group; puppies from which urine samples were studied in this group included six mixed-breed dogs and a Jack Russell terrier, Dalmatian, and Australian cattle dog. Diet was not recorded for all dogs ≥7 weeks of age and is not reported here.
Of all the different dogs ≥7 weeks of age from which samples were collected, only 15 were purebred dogs; breeds represented more than once included the Jack Russell terrier (n=4) and the Australian shepherd (n=3).
Urine Specific Gravity
Urine specific gravity measurements were determined in all samples and ranged from 1.003 to 1.055. There was a significant difference in mean USG between puppies in the 0 to 3 week age group and those puppies ≥4 weeks of age [P<0.001; Figures 1 and 2; Table 3]. Four samples were from newborn puppies <2 hours old that had not yet nursed; the USG measurements (1.027 to 1.035) from these samples were higher than those of 25 of the remaining 29 samples from other puppies in the 0 to 3 week age range.
Biochemical Dipstick Analyses
Complete biochemical dipstick analyses were performed on 136 urine samples. Incomplete analyses were performed on 13 urine samples due to the small volume of urine collected. Nine of the incomplete samples were from puppies 0 to 3 weeks old [Table 2]. There were no significant differences in median pH values among age groups [Table 3]. Protein was found in varying amounts (trace to 3+) in 145 of 148 (98%) samples. There were no differences in the frequency of positive qualitative protein analysis among age groups. Occult blood was positive on dipstick analysis in 23 of 138 (17%) samples. All but one of the occult blood positive results were classified as “nonhemolyzed or hemolyzed trace.” There was also no difference in the frequency of positive occult blood analyses among age groups. Of the remaining indices analyzed by dipstick method (glucose [n=147], ketones [n=136], bilirubin [n=136], urobilinogen [n=134]), no positive reactions occurred in any samples analyzed.
Urine Sediment Examination
Urine sediments were evaluated in 41 samples that were collected by free catch methods. Sediment results are not reported for any samples collected from floor or cage surfaces. Sediment findings in each age group are summarized in Table 4. Small numbers (0 to 5 cells per HPF) of RBCs were observed in 38 of the samples. Two samples had 10 to 20 RBCs per HPF. White blood cell estimates were <5 per HPF in 18 samples; 5 to 10 per HPF in five samples; 10 to 20 per HPF in two samples; 20 to 40 per HPF in seven samples; 40 to 70 per HPF in five samples; and were too numerous to count in four samples. In the 18 samples with >10 WBCs per HPF, 16 were from female puppies. Epithelial cells were observed in 34 total samples and included squamous epithelial cells (in 30 samples) and transitional cells (in 21 samples). Suspect bacteria were observed in the sediment of four samples; three of the four samples were from female puppies ≤12 weeks of age. Crystals were observed in 15 samples. Thirteen of the samples contained struvite crystals. These samples had pH values ranging between 6.5 and 8.5. Two samples had calcium oxalate crystals present, and both had pH values of 6.0. Small numbers of granular casts were observed in seven samples.
Discussion
Puppies occasionally present to clinicians for urinary tract problems, including juvenile renal disease, polyuric disorders, urolithiasis, and urinary tract infection. The urinalysis is a key component in the diagnostic evaluation of such puppies. Recognition of abnormal urinalysis results depends on a definition of expected normal findings, which have not been studied in a large number of puppies. In contrast to previously published recommendations,129 the authors found that qualitative urinalysis indices can be interpreted, as for adult dogs, in puppies ≥4 weeks of age.
Assessment of results is somewhat limited by the use of random-source puppies in this study; the authors were not able to confirm the exact ages of all puppies and did not do extensive screening tests or biochemical analyses to confirm the health of the puppies. In addition, purebred dogs predominated in the 0 to 3 week age group, because access to very young puppies was provided by breeders. Voided urine samples were evaluated in this study because this collection method posed the least amount of risk to the youngest puppies and represented the most likely sample type in pediatric patients to be analyzed by practicing veterinarians.1 Voided samples are satisfactory for routine urinalysis screening, especially for assessing urine concentration and metabolic indicators such as pH, glucosuria, bilirubinuria, and ketonuria. Interpretation of avoided sample can be followed up with analysis of a cystocentesis sample when indicated. Biochemical (i.e., dip-stick) results from 15 samples collected from surfaces, such as a cage or room floor, were included in the study. The practical reality is that samples from small, untrained puppies often must be collected in this manner. The authors recognize the possibility that disinfectants or other floor cleaners could alter some dipstick results, but they feel this is unlikely.
With the exception of using a standardized urine volume, methods of analysis were those typically employed for routine qualitative urinalyses. The centrifugation of varying urine volumes for sediment examination was necessary in this study population because of the small amount of urine voided by many puppies, but this limits the comparison of sediment findings between individual or groups of puppies. Similar volumes, however, are likely to be assessed by practicing veterinarians or other investigators.
Urine specific gravity is a simple, inexpensive, and useful clinical indicator of hydration status, polyuric disorders, and renal concentrating or diluting function. In combination with other components of the urinalysis, USG also is useful in estimating the severity of loss of substances such as protein in urine.10 Although measured osmolality is more precise than refractive indices, hand-held refractometer measurement is the most common, clinically applied method for estimating urine concentration.11 Results of this study suggest a difference in the typical USG in puppies 0 to 3 weeks of age in comparison to those ≥4 weeks of age. Although several reports support an increase in concentrating ability with age, random USG from multiple age ranges has not been reported in puppies. In one study of 26 puppies at various ages, the urine concentrating system was assessed by measurements of urine osmolality, plasma osmolality, and renal medullary osmolality.5 In these anesthetized puppies instrumented for micropuncture studies, urine-to-plasma osmolality was approximately 2.0 in three puppies <10 days old, 2.0 to 4.0 in 16 puppies 10 to 50 days old, and approximately 3.0 to 7.0 in seven puppies >50 days old. The osmolality of the inner medullary gradient also increased with age, although in a less consistent fashion.5 While individual osmolalities were not given, plasma osmolality is typically around 300 mOsm/kg, so these urine osmolalities correlate with approximately 600 to 1200 mOsm/kg in younger puppies and 900 to 2100 mOsm/kg in older puppies.5 Similarly, mean urine osmolality in 4- to 6-week-old kittens was 1424 mOsm/kg, and it was 2432 mOsm/kg in 7- to 12-week-old kittens in another study.12 Osmolalities of approximately 1100 mOsm/kg and USG of ≥1.030 are clinically associated with adequate renal concentrating function in dogs.1013 In the authors’ study, mean USG in puppies >4 weeks of age was comparable to that of adult dogs and was greater than the expected 1.030. These results conflict with previous assertions that USG ranges from 1.006 to 1.017 in puppies <8 weeks of age and does not reach values >1.030 until after 8 weeks.2
In addition, most of the puppies in the 0 to 3 week age range were either nursing or receiving a milk replacer, which may influence urine volume. Four samples were from newborn English bulldog puppies <2 hours of age that had not yet nursed. Interestingly, the USG measurements in these samples (1.027 to 1.038) were higher than that of other puppies in the 0 to 3 week age range. Water intake and USG can be influenced by feeding, diet, activity, and circadian rhythms;14 thus, the high USG in these samples could be a consequence of the puppies not having nursed. Urine specific gravity as high as 1.025 has been reported in a near-term canine fetus; however, the mean USG in 11 fetuses was 1.014.7 A larger number of newborn puppies, including those that had nursed and those that had not, would be needed to further interpret this finding.
Significant variation in urine concentration depending on time of day and age was observed in one study of multiple, random urine samples from 89 dogs >6 months of age.14 The range of USG in puppies >3 weeks of age in the authors’ study (1.010 to 1.055) is comparable to the USG found in the older dogs (1.006 to >1.050),14 and variation between samples was wide in all age groups [Figure 2]. In that study, urine samples collected in the morning were more concentrated than those collected in the evening, and urine concentration at both time intervals declined with advancing age.14 Due to multiple sources of puppies in this study, the authors were unable to standardize the time of collection for all puppies. A large range of variability in USG is expected in healthy dogs with free access to water or nursing; it may still be optimal to check multiple samples in puppies to adequately interpret urine concentrating ability. The authors cannot extrapolate from random USG measurements whether puppies can reach maximum concentration or dilution needed in the face of dehydration or volume overload.
In addition, although puppies >3 weeks of age appear able to produce concentrated urine, individual renal functions mature at different times in the postnatal period,4–6 and puppies may not be able to respond to rapid changes in body water or sodium content.615 However, the historical perception that neonates have limited or impaired renal response to homeostatic needs is currently being revised based on observations in preterm and older human infants.1617
Glucose can appear in urine if the plasma glucose exceeds the renal threshold or if renal tubular resorption is decreased. Because of immature tubular function, normoglycemic glucosuria has been expected in normal young puppies19 and has been described as a “frequent” finding in puppies ≤8 weeks of age.2 In one study of tubular function in young puppies, glucosuria was detected by chromatography in urine from five of 10 5-day-old puppies but was not detected in urine from three puppies ≥21 days of age.4 Transient glucosuria was attributed to a greater proportion of immature nephrons in puppies <14 days of age in another study.18 However, glucosuria was not detected by routine glucose reagent (i.e., dipstick) methods in any samples in the authors’ study, including those from puppies ≤3 weeks of age.
Proteinuria was detected by the dipstick method in almost all (145/148; 98%) samples; protein was recorded as trace in 69 (47%) of those. Using the dipstick method, the primary protein detected is albumin. It is important to note that proteinuria should be interpreted in conjunction with urine concentration, urine sediment findings, and pH. Small amounts of protein may be normal in concentrated urine; false-positive reactions may be observed in alkaline urine.10 In this study, significant dipstick protein was detected most commonly in the 0- to 3-week-old puppies. Although protein excretion may reflect abnormalities of glomerular permeability or tubular resorptive function in puppies, proteinuria as defined in this study was likely due to the urine concentration or pH in this group, but it also may have been derived from cellular components found in voided urine in some samples. Using quantitative protein determination on pooled voided urine, 24-hour protein excretion and urine protein:creatinine ratios declined steadily from 9 to 27 weeks of age in beagle puppies; proteinuria in excess of normal adult measurements was not found at any age.8 However, physiological proteinuria is reportedly expected in animals during the first few days of life as colostral proteins are absorbed in the intestine and excreted in the urine.9
While there was no macroscopic hematuria in any urine samples, occult blood was trace to 1+ positive on dipstick analysis on 23 of 138 (17%) samples run. A positive reaction indicates intact RBCs, hemoglobin, or myoglobin; the reagent strips are most sensitive to hemoglobin. Unfortunately, urine sediment examinations were not performed on many of these samples to confirm hematuria, because the sample volume was small. When sediment examination was done, excess RBCs were observed in only two samples, so it is unclear why occult blood was positive in so many samples.
Urine sediment is reported for only 41 of the 149 samples, so these results may not accurately reflect a larger population of puppies. It can be difficult to collect adequate volumes of urine for centrifugation in this population, since untrained puppies void small amounts of urine. Variation in the amount of sediment may have occurred in this study; however, excess cells were conservatively defined to minimize fine variations between samples. White blood cells in excess of 10 per HPF were found on 18 of the 41 (44%) samples. Most (16/18; 89%) samples with this degree of pyuria were from female puppies, and the majority (13/18; 72%) were from puppies ≤12 weeks of age. The authors did not observe external evidence of vaginitis on routine physical examination in the puppies studied. While urinary tract or vaginal inflammation cannot be completely ruled out without further evaluation, a more likely cause for the increased number of WBCs would be urethral or genital contamination in voided samples.19 Contamination of urine with WBCs may be more likely in young puppies than in other dog populations, particularly because of their litter environment and lack of grooming. Pyuria of a similar magnitude (WBCs >10 per HPF) was observed in voided midstream specimens from only two of 20 dogs in another study; however, the ages of the dogs were not reported.20 Suspected bacteria were observed in five of the 41 samples in the authors’ study; four samples were from female puppies ≤12 weeks of age. This is not a surprising finding as voided urine samples may be contaminated with bacteria from the distal urethra, genital tract, or skin.20 Culture of urine specimens collected by cystocentesis and further evaluation of vaginal cytopathology would be necessary in order to determine if urinary tract or genital infection was present in any of these puppies.
An occasional granular cast was found in seven samples. Casts may be observed in healthy animals, and this was not considered a significant finding. Epithelial cells were observed in many (34/41; 83%) of the samples examined, but they were not present in increased numbers and were considered insignificant, again related to the method of collection. Sample sizes were considered too small within age groups to determine significant differences between age groups for these findings.
Crystals are reportedly a common finding in puppies.1 Crystals were observed in 15/41 (37%) samples in this study. Formation of crystals is influenced by a variety of factors, including pH and temperature. Sixteen of the samples contained struvite crystals and had pH values ranging between 6.5 and 8.5, a range at which struvite crystals are likely to precipitate. Two samples had calcium oxalate crystals present, and both had pH values of 6.0. Refrigeration and transport time may have contributed to in vitro crystalluria.10 Urine samples were placed on ice after collection, which may have enhanced crystal formation, but they were brought to room temperature prior to analysis.
Conclusion
With the exception of USG measurements in puppies <4 weeks of age, results of random qualitative urinalyses in puppies appear to be similar to those of healthy adult dogs. The study was designed to reflect the most common scenario presented to veterinary practitioners and the usefulness of a random voided urine sample as part of the initial evaluation of such a patient. However, this design limits the interpretation of dipstick protein, occult blood, and sediment findings; proteinuria and positive occult blood reactions should not be overinterpreted in voided urine samples. In addition, while the overall number of animals studied is greater than in other single studies, the number of animals within each age group and the number of urine sediments examined are small. Thus, results from a larger number of puppies under known dietary and other conditions would be necessary to further confirm the authors’ findings.
Cryogenic 5-mL vial; Corning Inc., Acton, MA
Multistix; Bayer Corporation, Pittsburgh, PA
A-300-CL; Atago Company, Ltd., Tokyo, Japan
Urinalysis Tube 15 mL; Fisher Scientific, Pittsburgh, PA
Damon IEC HN-SII; GMI Inc., Clearwater, MN
Transfer Pipet 13-711-5A; Fischer Scientific, Pittsburgh, PA
Sigmastat version 2.03; SPSS Science, Chicago, IL
Acknowledgments
The authors thank Doris Millsap for her technical assistance, as well as the many owners or agents of puppies provided for study.
Citation: Journal of the American Animal Hospital Association 39, 4; 10.5326/0390369
Citation: Journal of the American Animal Hospital Association 39, 4; 10.5326/0390369
Columns represent mean urine specific gravity (USG) measurements in random urine samples from healthy puppies in each age group (n=number of samples).
Scatterplot of urine specific gravity (USG) measurements in random urine samples from healthy puppies in age groups from 0 to 24 weeks. The horizontal bar represents the median value.
