A Simple, Inexpensive Metabolism Cage for Small Mammals

By utilizing ordinary laboratory equipment and a spherical feces-urine separator, a simple, inexpensive metabolism cage for small mammals can be constructed. A hardware cloth animal cage over a cylindrical battery jar containing a spherical feces-urine separator affords the following advantages not commonly found in commercial metabolism cages: 1) complete separation of feces and urine through minimal contact, 2) minimal evaporation of urine due to proximity of storage vessel and lack of exogenous air currents, and 3) extremely low cost of less than five dollars. The metabolism cage is designed to allow measurement of fluid intake, and to separate and collect feces and urine for numerous qualitative and quantitative determinations. In addition, the metabolism cage permits observation of the animal, feces, and urine at all times, is readily cleaned or sterilized, and is easily fashioned from common laboratory equipment.     

Mouse urine collection using clear plastic wrap

Qualitative urinalysis using Multistix reagent strips for the detection of urinary pH, protein, glucose, bilirubin, blood, ketone, urobilinogen and creatinine can be carried out with a few drops of mouse urine. The use of metabolic cages is not practical for such qualitative studies particularly when several animals are involved. Here we describe two different methods for collecting pure mouse urine. The single animal method (SAM) involves allowing a single mouse to urinate on Glad cling wrap outside of the animal cage. The multiple animal method (MAM) involves partitioning seven mice into seven different make-shift compartments laid out on top of the cling wrap and allowing them to urinate. The voided urine, in each case, is then aspirated into micro-centrifuge tubes using a Pipetman. Without coercion pure urine was obtained as early as 12 s. Volumes in the range of 10-250 microl were obtained. Modifications of the SAM could prove useful for rat or mouse urine collection under conditions of microgravity.     

Capillary tube urinalysis for small animals.

A simple method was devised using a capillary tube to collect urine for urinalysis. This method offers the following advantages: the animal is not adversely influenced; the collection and analysis of the urine is repeatable; the amount of urine required is small; because of the simplicity of the method, the number of samples tested in a given time can be increased; and contamination with foreign material is reduced, permitting more accurate evaluation of the urinary sediments.     

Sex difference in attraction thresholds for volatile odors from male and estrous female mouse urine

Volatile urinary odors from opposite sex conspecifics contribute to mate recognition in numerous mammalian species, including mice. We used a simple habituation/dishabituation testing procedure to ask whether the capacity to detect and investigate decreasing concentrations of volatile urinary odors is sexually differentiated in mice. Beginning 2 months after gonadectomy and in the absence of any sex steroid treatment, adult, sexually naive male and female CBA x C57Bl/6 F1 hybrid mice received two series of daily tests that involved the presentation of different dilutions of urine from C57Bl/6 males followed by urine from estrous females. Each test session began with three consecutive presentations of deionized water (10 microl on filter paper for 2 min, behind a mesh barrier which prevented direct physical access, in the home cage at 1-min intervals) followed by three presentations of one of five different dilutions of urine (a different dilution on each test day). Males and females showed equivalent, significant habituation/dishabituation responses (low investigation times for successive water presentations; increased investigation of the first urine stimulus, followed by a decline in successive urine investigation times) to both male and female urine/water dilutions of 1:1, 1:10, and 1:20. However, only female mice responded reliably to 1:40 and 1:80 dilutions of both types of urine, pointing to a sex dimorphism in the detection and/or processing of biologically relevant, volatile urinary odors by the main olfactory system.     

MPTP in mice: treatment, distribution and possible source of contamination

Mice were treated daily with [3H]MPTP (30 mg/kg, 1 uCi, s.c.) for 1, 3, and 10 days to determine the fate and localization of tritiated compounds. An untreated mouse was housed either in the same cage ("cage-mate control") or in an adjacent cage separated by mesh-wire ("near-neighbor control"). The radioactivity measured in blood, brain, liver, and remaining body of [3H]MPTP-treated mice was dependent on the total dose of the drug the animals received and did not vary with the type of tissue analyzed. Significant amounts of radioactivity were found in the tissues of the "cage-mate control" mice, but not of the "near-neighbor control" mice. The route of transmission appears to be through the urine, as the urine of [3H]MPTP-treated mice was highly radioactive after the drug injection. Only traces of radioactivity were found in their feces and there was no increase in the background radiation in the environment of the cages, indicating that the tritiated compounds were not exhaled. Proper disposal of urinary products of MPTP-treated animals is therefore necessary to reduce the risk of possible drug contamination in humans.     

Synergistic effects of nitric oxide and prostaglandins on renal escape from vasopressin-induced antidiuresis

Recent results from our laboratories indicate that renal escape from AVP-induced antidiuresis is accompanied by marked downregulation of kidney aquaporin-2 (AQP2) and AVP V2 receptors. The present studies evaluated the effect of nitric oxide (NO) and PG synthesis blockade on escape from antidiuresis. dDAVP-infused rats were water loaded (WL) for 5 days. l-NAME, an NO synthesis inhibitor, or diclofenac, a cyclooxygenase inhibitor, was infused subcutaneously beginning 1 day before WL. As early as 2 days after WL, urine volume increased and urine osmolality decreased, indicating the onset of escape. Endogenous NO synthesis, measured as urinary NO2 + NO3 excretion, was significantly increased in the WL group compared with the non-WL controls during all 5 days of WL. l-NAME (20 mg. kg(-1). day(-1)) markedly decreased urine volume on days 4 and 5 of WL, indicating inhibition of the escape phenomenon. Kidney AQP2 protein was significantly increased by this dose of l-NAME as well. A lower dose of l-NAME (10 mg. kg(-1). day(-1)) or diclofenac (2.5 mg. kg(-1). day(-1)) did not significantly affect the escape phenomenon by itself, but the combination of l-NAME and diclofenac showed a marked inhibitory effect on the escape phenomenon, which was also accompanied by a significant increase in kidney AQP2 expression. These results therefore suggest that renal NO and PG both play important roles in escape from AVP-induced antidiuresis by acting synergistically to downregulate kidney AQP2 expression.     

Single-mouse urine collection and pH monitoring system.

A glass cage with minimal surface area was designed and used to house mice for 24-hour urine collections. An experiment was performed with a radio-labeled compound excreted in the urine to assess the collection efficiency of the cage. In this experiment 74.2 +/- 6.5% of the excreted radioactivity was recovered in the urine, with 25.8 +/- 6.5% found adhering to the cage surfaces. When a flow-through pH electrode, meter, and recorder were attached, the system provided a continuous pH versus time urination record.     

Experimental animal urine collection: a review

Animal urine collection is a vital part of veterinary practice for ascertaining animal health and in scientific investigations for assessing the results of experimental manipulations. Untainted animal urine collection is very challenging, especially with small rodents, and is an almost impossible task under conditions of microgravity. The fundamental aspects of urine collection are: (1) ease of collection, (2) quality of sample, (3) prevention of contamination, (4) severity of procedures used, (5) levels of pain caused to the animal and (6) refinement of methods to reduce stress, pain or distress. This review addresses the collection of urine for qualitative and quantitative purposes from rodents, rabbits, felines, canines, avian species, equines, porcines, ungulates and certain non-human primates, with animal welfare in mind. Special emphasis has been given to rodents, canines and non-human primates, since they are the animals of choice for research purposes. Free catch (voluntary voiding), methods with mild intervention, surgical methods, modified restraint, cage and special requirement methods have been reviewed here. Efforts need to be taken to provide appropriate animal husbandry and to nurture the animals in as natural an environment as possible since experimental results obtained from these research subjects are, to a great extent, dependent upon their well-being. A continuous refinement in the procedures for collecting urine from experimental animals will be the most efficient way of proceeding in obtaining pure urine specimens for obtaining reliable research data.     

Hydration status affects urea transport across rat urothelia

Although mammalian urinary tract epithelium (urothelium) is generally considered impermeable to water and solutes, recent data suggest that urine constituents may be reabsorbed during urinary tract transit and storage. To study water and solute transport across the urothelium in an in vivo rat model, we instilled urine (obtained during various rat hydration conditions) into isolated in situ rat bladders and, after a 1-h dwell, retrieved the urine and measured the differences in urine volume and concentration and total quantity of urine urea nitrogen and creatinine between instilled and retrieved urine in rat groups differing by hydration status. Although urine volume did not change >1.9% in any group, concentration (and quantity) of urine urea nitrogen in retrieved urine fell significantly (indicating reabsorption of urea across bladder urothelia), by a mean of 18% (489 mg/dl, from an instilled 2,658 mg/dl) in rats receiving ad libitum water and by a mean of 39% (2,544 mg/dl, from an instilled 6,204 mg/dl) in water-deprived rats, but did not change (an increase of 15 mg/dl, P = not significant, from an instilled 300 mg/dl) in a water-loaded rat group. Two separate factors affected urea nitrogen reabsorption rates, a urinary factor related to hydration status, likely the concentration of urea nitrogen in the instilled urine, and a bladder factor(s), also dependent on the animal's state of hydration. Urine creatinine was also absorbed during the bladder dwell, and hydration group effects on the concentration and quantity of creatinine reabsorbed were qualitatively similar to the hydration group effect on urea transport. These findings support the notion(s) that urinary constituents may undergo transport across urinary tract epithelia, that such transport may be physiologically regulated, and that urine is modified during transit and storage through the urinary tract.     

Changes in androgenic steroid profile due to urine contamination by microorganisms: a prospective study in the context of doping control

Urine contamination by microorganisms may affect the interpretation of urinalysis in different areas of clinical diagnosis. This is particularly relevant in doping control. A prospective study was designed to assess the effects of urine contamination by selected pathogens on the endogenous androgenic steroid profile. Pooled urine from a healthy male volunteer with standard steroid profile compared with reference values for the Caucasian population was sterilized by filtration and stored in sterile glass tubes. Aliquots were inoculated with known amounts of 15 different organisms (bacteria, fungi, and moulds) and incubated at 37 degrees C for 2 weeks. Different markers of urine contamination, such as pH, deconjugation of steroids, and metabolic by-products, were determined. Alkalization of urinary pH was not a reliable indicator of urine contamination as several organisms grew in this medium and no alteration of this parameter was found. In uncontaminated urine, less than 10% of steroid glucuronide conjugates were spontaneously hydrolyzed. Higher rates of hydrolysis for sulfate conjugates were found. An unconjugated fraction higher than 10% of the total amount of testosterone was a reliable indicator of urine contamination. However, microbial production of testosterone or epitestosterone was not detected. In contrast, a few organisms were able to synthesize 5alpha-androstanedione, 5beta-androstanedione, and androstenedione using endogenous steroids as substrates.     

Evaluation of individually ventilated cage systems for laboratory rodents: occupational health aspects

New ventilated caging systems for laboratory animals were compared with conventional caging regarding allergen distribution, ergonomic suitability, cage environment and animal welfare. This paper presents occupational health evaluations. Mice were placed in individually ventilated cage (IVC) systems, a ventilated cabinet, and in cages on open shelves (conventional husbandry). The IVC systems were studied at negative and positive airflow. Aeroallergens were sampled on filters (n = 204, including controls) in undisturbed rooms and during cage changing. Concentrations of mouse urinary allergen (Mus m 1) in filter eluates were measured using sandwich ELISA. An ergonomic evaluation was performed with measurement of traction forces. Staff exposure during cage changing was high in all systems, range 116-4430 ng Mus m 1/m3. In undisturbed animal rooms, allergen levels were orders of magnitude higher when using conventional caging compared with ventilated systems; P < 0.001. At positive pressure both IVCs leaked allergen (median Mus m 1 concentration was < 0.08 ng/m3 at negative, but 6.5 ng/m3 (IVC1) and 0.8 ng/m3 (IVC2S) at positive pressure). The IVC systems had ergonomic disadvantages compared with the conventional husbandry and the ventilated cabinet, for instance with cages in unsuitable working heights. Ventilated husbandry solutions reduce levels of airborne allergen substantially at negative pressure, but are ergonomically less suitable. To prevent allergen exposure during cage changing, we propose that this procedure should be performed under ventilated conditions. Producers and users must cooperate in optimizing animal caging systems for both animals and staff.     

Compensation of escape direction in unilaterally cercus-ablated crickets, Gryllus bimaculatus, is associated with the distance walked during recovery period

In response to an air puff stimulus, intact crickets, Gryllus bimaculatus, make an escape almost 180 degrees opposite to the stimulus source. In order to verify our previous hypothesis that a self-stimulation of the wind-sensory system is necessary for a compensational recovery of the escape direction (behavioral compensation) in unilaterally cercus-ablated crickets, we investigated the relationship between the conditions of rearing after a unilateral cercal ablation and the degree of behavioral compensation. A unilaterally cercus-ablated cricket reared in a large cage to permit free locomotion showed a significantly higher degree of recovery of escape direction compared with those reared under restrained conditions in a small glass vial. However, the degree of behavioral compensation in a cricket reared alone in a large cage was smaller than that of crickets reared in a cage of the same size with 5-6 other cercus-ablated crickets. Mutual stimulation possibly increased the extent of locomotion of crickets reared in a group and improved the degree of compensational recovery of the escape direction. To ascertain this, the distance a cricket moved during the recovery period was associated with the degree of compensational change of the escape direction. The result suggests that the degree of compensation of the escape direction clearly depended on the distance walked by the crickets. The compensation seemed not to be caused by other factors such as chemical ones in the case of group rearing because forced locomotion induced by touch stimulation on the body surface was solely effective in improving the escape direction.     

Bacterial effect of hydrogen peroxide on urinary tract pathogens.

Bacterial contamination of urinary drainage bags is a frequent source of bladder bacteriuria in patients with indwelling catheters. Previous work demonstrated that the addition of 30 ml of 3% H2O2 prevented bacterial contamination of urinary drainage bags for up to 8 h in patients with urinary infections (greater than 10(5) colony-forming units per ml). Survival curves of a variety of organisms in filter-sterilized urine with various concentrations of H2O2 (0.6 to 0.01%) were constructed. Organisms with high cellular catalase activity (Staphylococcus aureus, Serratia marcescens, and Proteus mirabilis) required 30 to 60 min of exposure to 0.6% H2O2 for a reduction of 10(8) to less than 1 colony-forming unit per ml, whereas Escherichia coli, Streptococcus sp., and Pseudomonas sp. required only 15 min of exposure. The efficacy of H2O2 in urine was maintained despite exposure to room temperature for 5 days and reinoculation with bacterial suspensions. H2O2 is inexpensive and relatively nontoxic, and these data suggest that periodic instillation of H2O2 into urinary drainage bags may eliminate a source of bladder bacteriuria and environmental contamination.     

Effects of water dilution, housing, and food on rat urine collected from the metabolism cage

The objective of the study reported here was to investigate three factors that may affect the amounts of water consumed and urine excreted by a rat in the metabolism cage: water dilution, housing, and food. Young F344/N rats (eight per group) were used for all experiments. Food was withheld from rats before each 16-h urine collection, then rats were transferred into a metabolism cage. For trial A (water dilution), urine was collected from rats supplied with dyed water (0.05%, vol/vol). This was repeated three times over a 2-week period. Dye in water or urine was quantified, using a spectrophotometer. For trial B (housing), rats were individually housed in wire cages for 3 weeks before the first urine collection. Then they were group housed in the solid-bottom cage (four per cage). After 2 weeks of acclimation, urine collection was repeated. For trial C (food), one group of rats was provided with food, the other was not, during urine collection. About 8% of urine samples of small volume (< or = 3 ml) from trial A were contaminated with drinking water up to 13% of volume. The average urine volume associated with individual housing was approximately twice as large as that associated with group housing. When food was provided during urine collection, rats consumed similar amounts of water but excreted significantly smaller amounts of urine than did rats without food. It was concluded that water dilution of a urine sample from a sipper bottle is relatively small; rats individually housed in wire caging before urine collection can consume and excrete a larger quantity of water, compared with rats group housed in solid-bottom cages; and highly variable urine volumes are, in part, associated with lack of access to food during urine collection.     

Radiation metabolomics. 4. UPLC-ESI-QTOFMS-Based metabolomics for urinary biomarker discovery in gamma-irradiated rats

Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.     

The urinary excretion of prostaglandins and thromboxane B2 in healthy volunteers: a study in males and females, and on the influence of seminal fluid contamination

Radioimmunoassay measurements of prostaglandins (PGs) E2, F2 alpha, 6-keto-PGF1 alpha and thromboxane (Tx) B2 in 24 h urine specimens from a male and a female healthy volunteer on several consecutive days revealed a dramatic increase of PGE2, PGF2 alpha, 6-keto-PGF1 alpha on days, upon which they had sexual intercourse; only TxB2 remained stable. Furthermore, the PGE2/PGF2 alpha ratio rose to values greater than 0.5 on days with sexual intercourse. This was found to be due to contamination of the urine samples by seminal fluid. Two 24 h urine samples from each of 26 healthy male and female volunteers (HV) revealed higher (p less than 0.01) mean PGE2 and PGF2 alpha values in males than in females. The results show that the interpretation of the urinary PG excretion as a measure of renal PG synthesis should be considered carefully, and that a PGE2/PGF2 alpha ratio greater than 0.5 indicates probable seminal contamination of urine.     

New Ventilated Isolation Cage

A multifunction lid has been developed for a commercially available transparent animal cage which permits feeding, watering, viewing, long-term holding, and local transport of laboratory rodents on experiment while isolating the surrounding environment. The cage is airtight except for its inlet and exhaust high-efficiency particulate air filters, and it is completely steam-sterilizable. Opening of the cage's feed and water ports causes an inrush of high velocity air which prevents back-migration of aerosols and permits feeding and watering while eliminating need for chemical vapor decontamination. Ventilation system design permits the holding in adjacent cages of animals infected with different organisms without danger of cross-contamination; leaves the animal room odor-free; reduces required bedding changes to twice a month or less, and provides investigators with capability to control precisely individual cage ventilation rates. Forty-eight cages can be conveniently placed on a standard NIH "shoebox" cage rack (60 inches wide x 28 inches deep x 74 inches high) fitted with a simple manifold exhaust system. The entire system is mobile, requiring only an electrical power outlet. Principal application of the caging system is in the area of preventing exposure of animal caretakers to pathogenic substances associated with the animal host, and in reducing handling of animals and their exposure to extraneous contamination.     

Effects of urine from pregnant and lactating female house mice on oestrous cycles of adult females.

It was demonstrated that mice treated with urine from pregnant or lactating females experienced longer periods of oestrus than did mice treated with water or urine from singly caged females. Application of urine by means of perforated capsules placed in the cage of the test mouse showed that the factor(s) responsible for the longer periods of oestrus was an airborne pheromone. The females experiencing longer oestrous periods ovulated (ova in oviducts), became pregnant and gave birth.     

Altered regulation of renal sodium transporters and natriuretic peptide system in DOCA–salt hypertensive rats

Although deoxycorticosterone acetate (DOCA)–salt hypertension is a volume dependent model of hypertension, it shows polyuria and natriuresis. It is expected that dysregulation of aquaporin water channels (AQPs) and sodium transporters associated with natriuretic peptide (NP) system may play an escape role in sodium retaining state. One week after left unilateral nephrectomy, rats were subcutaneously implanted with silastic DOCA (200 mg/kg) strips. Physiologic saline was supplied as a drinking water to all animals. 4 weeks after operation, the protein expression of AQPs, sodium transporters, and endopeptidase (NEP) was determined in the kidneys by semiquantitative immunoblotting and immunohistochemistry. The mRNA expression of NP system was determined by real-time polymerase chain reaction. The amount of urinary ANP excretion was measured by radioimmunoassay. In DOCA–salt rats, urine osmolality was decreased while urinary excretion of sodium was increased. The expression of AQP1-3 as well as that of α-1 subunit of Na,K–ATPase, NHE3, NKCC2 and NCC was decreased in the kidney. The mRNA expression of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) was increased in the kidney. The expression of NEP was decreased, and urinary ANP excretion was increased. Downregulation of AQPs and sodium transporters may contribute to mineralocorticoid escape in DOCA–salt hypertension. Increased expression of natriuretic peptides associated with downregulation of NEP may play a role in natriuresis.