Micturition in Göttingen minipigs: first reference in vivo data for urological research and review of literature

Abstract

One possible symptom of overactive bladder (OAB) is urinary incontinence, which is generally considered to be an age-associated disease and which is rapidly increasing with demographic changes. Rodent models are commonly used for the investigation of lower urinary tract functions, although the use of these species has limitations in several translational aspects. In biomedical research and preclinical toxicology, Göttingen minipigs are used increasingly. But in urological research, only few data are available for Göttingen minipigs. To the best of our knowledge, this study is one of the first to provide reference data of micturition in female Göttingen minipigs. Micturition frequency and volumes were monitored and analyzed in five female Göttingen minipigs. Voided volume was 520 ± 383 mL (mean ± standard deviation of mean [SD]) and micturition frequency 6.17 ± 3.68 (mean ± SD). We also performed a review of the literature to compare our data with data from different species (humans, pigs, rats and mice). Our findings revealed that micturition volume and frequency of Göttingen minipigs are more comparable with that of humans, leading to the conclusion that Göttingen minipigs may be the better choice for translational research in different research fields, such as urology, neurology and nephrology, etc. The provision of in vivo reference values meets with the 3R concept of ‘reduction, refinement and replacement’ of laboratory animals, because they allow comprehensive statistical power calculations (reduction), planning of telemetric approaches (refinement), and generation of computer-based modulation for the development of intravesical drug delivery systems (replacement).

Keywords

pigs species comparison experimental design in vivo 3Rs

With demographic changes, age-associated diseases will become challenging, and urinary incontinence is one of the ‘four giants of geriatrics’.¹ With a high prevalence of 12% in Europe and 17% in the US, overactive bladder (OAB) is a common condition with increased age.^2,3 Patients who suffer from OAB complain about urinary urgency often accompanied by increased frequency and nocturia, and sometimes with incontinence episodes.⁴ The gold-standard for treatment of OAB is still the use of antimuscarinics which have good initial response rates.⁵ However, due to their adverse side-effects and decreasing efficacy in the long-term, overall effectiveness is limited and alternative therapies are desirable.⁵ In addition to the treatment limits, the cause of OAB is still unknown. As in other medical disciplines, the use of animal models can help answer questions about the disease pathogenesis. But the definition of OAB by itself highlights the difficulties for the development of animal models.⁶ In 2006, McMurray et al. pointed out that the understanding of normal physiological control of continence and pathophysiological dysfunction is mandatory for the investigation of urinary incontinence and that, therefore, animal models should be used which closely resemble the human lower urinary tract in its anatomical and physiological functions.^7,8 For the investigation of lower urinary tract function, different animal species have been used, particularly non-human primates, dogs, pigs, cats, rabbits, rats, guinea-pigs, mice and hamsters.⁷ Although rodent models are commonly used for the investigation of lower urinary function, their use has limitations in several translational aspects, for instance because of anatomical or diurnal differences.^9,10 Another aspect of the development of animal models is the difficulty of developing chronic models, especially in functional urology. Because of this, chronic models of OAB should have several features: stability for several weeks, robustness and reproducibility, permission to obtain unanesthesized in vivo measurements, and small tissue damage.⁷ Especially with regard to in vivo measurements, large animal models offer advantages for continuous monitoring by telemetry.⁷ In biomedical research and preclinical toxicological testing, pigs, especially Göttingen minipigs, are increasingly used in translational research as alternatives to dogs or monkeys.^11,12 In urological research only few data are available for Göttingen minipigs.^13–15

To the best of our knowledge, this study is one of the first to present reference data of micturition volume and frequency in Göttingen minipigs. We also performed a literature review to compare our data with data from different species (human, pigs, rats and mice).

Material and methods

Animals

Five female Göttingen minipigs aged 32 ± 3.2 months (mean + standard deviation of mean [SD]) from a disease-free breeding facility (University Göttingen, Göttingen, Germany) with an average weight of 54.3 ± 3 kg (mean + SD) were used in this study. The clinical health status of the animals was checked on arrival by a veterinarian. Blood samples were taken under anesthesia with prior overnight fasting with water ad libitum. For pre-sedation, minipigs received an intramuscular injection of 4 mg/kg azaperone and 0.1 mg/kg atropine. After 10 to 15 min, a second intramuscular injection of 15 mg/kg azaperone was given. Blood samples were used for standard blood counts (leukocytes, erythrocytes, platelets, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration and mean corpuscular hemoglobin). Fecal samples were investigated via native and flotation microscopy for endoparasites (worms and worm eggs, such as Trichuris spp., Strongylus spp. and Ascaris spp.) and via smears for Salmonella spp. All animals were acclimatized at least seven days prior to experiments.

Animal care

All procedures were performed in accordance with German legislation governing animal studies and the Guide for the Care of Animal Use of Laboratory Animals (NIH Publication No. 85–23, revised 2011).¹⁶ Official permission was granted by the governmental Animal Care and Use Office (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany). The five Göttingen minipigs were all housed in a certified facility (DIN ISO:9001/2008) with humidity and temperature-controlled environment and a 12:12 h light:dark cycle. Göttingen minipigs were housed singly, allowed contact with other pigs, or pair-housed on hardwood bedding (Lignocell F14; JRS, Rosenberg, Germany) in a pen of at least 3 m² in size and fed with energy-reduced, standardized minipig diet (ssniff Special diets, Soest, Germany). For enrichment nesting material (Voll Logging GmbH, Bad Camberg, Germany), a plastic ball and a metal chain (Bioscape GmbH, Castrop-Rauxel, Germany) were provided.

Experimental design

With metabolic cage studies, micturition frequency and volume of Göttingen minipigs were determined. We performed a literature review to establish reference data of micturition frequency and volume in humans, pigs, rats and mice.

Metabolic cage studies

Experimental set-up

Metabolic cages (Bioscape GmbH, Castrop-Rauxel, Germany) of 1 m × 1.15 m × 1.15 m (width × height × depth) in size were used. At the outlet, a collection reservoir was placed which was connected to a scale and corresponding software (Flow; Laborie, Montreal, Quebec, Canada) (Figure 1). Additionally, a video camera (Mobotix, Langmeil, Germany) with two motion sensor fields on the metabolic cage outlet was used to monitor micturition frequency and volume. Video analysis was performed with the corresponding software (sm-player portable version 0.6.9, sourceforge.net; Dice Holdings Inc, New York, NY, USA).

Figure 1.

Experimental set-up with metabolic cage (a), video camera (b), collection reservoir and flowmeter (c), personal computer (d).

Animal care during measurements

In line with animal welfare guidelines, the experiments were performed for no longer than 24 h in the metabolic cage, with regular diet and controlled water ad libitum. Thereafter, the minipigs were again transferred to a regular pen.

Literature review: reference data for micturition frequency and volume in humans, rodents and pigs

Literature research was performed in March 2014 using the Medical Literature Analysis and Retrieval System Online (US National Library of Medicine’s life science database; MEDLINE; PubMed), Thomson-Reuters’ Web of Science, and Google Scholar. MEDLINE and Web of Science searches were performed using the following combined terms: ‘frequency AND volume AND chart AND urology’ and ‘urology AND healthy AND micturition’ for literature concerning humans. MEDLINE search for literature concerning animals consisted of comparable filters, excluding references to human literature and following the combined term: ‘micturition AND healthy’. ‘Micturition AND animals not human AND healthy’ was the search term used with the Web of Science database. In Google Scholar, the search term used was ‘in title: micturition AND healthy’.

Statistical analysis

Results are shown as mean ± SD or median with range. Descriptive statistics and creation of figures were performed with GraphPad Prism5 software (GraphPad Software Inc, La Jolla, CA, USA).

Results

Metabolic cage studies

Blood samples showed no abnormalities and fecal samples were free of endoparasites. Feeding habits during metabolic cage studies were similar to those during free-moving maintenance, twice daily with a diet adjusted to 2% body weight. The total number of 24 h metabolic cage sessions was 35 with 221 single micturitions. Calculated micturition frequency was 6.17 ± 3.68 (mean ± SD) with a range from 0.0 to 14.0 micturitions in 24 h. The range of voided volumes was 100–2400 mL and the mean volume was 520 ± 383 mL (mean ± SD) (Figure 2).

Figure 2.

Micturition frequency and volume in Göttingen minipigs.

Intra-individual comparison

Pig 1 and Pig 2 showed high micturition frequencies (medians 7.5 and 10.5) with corresponding low micturition volumes (median values 400 and 500 mL) (Figures 3 and 4). The lowest micturition frequency investigated was for Pig 3, with a median value of two micturitions in 24 h. But voided volume in Pig 2 was higher than in Pig 1 and Pig 3 with a median of 1200 mL per void. Measurements of micturition volume revealed the highest median value for Pig 4, with 1500 mL per void, but showed an intermediate frequency of 5.5 voids in 24 h. Pig 5 showed a low frequency (4 micturitions) with an intermediate volume (450 mL per void) (Figures 3 and 4).

Figure 3.

Intra-individual comparison of micturition frequency in five female Göttingen minipgs (median with range).

Figure 4.

Intra-individual comparison of micturition volume in five female Göttingen minipigs (median with range).

Literature review

Study inclusion

First line database searches yielded 927 references in total, 324 from PubMed, 333 from Web of Knowledge, and 270 from Google Scholar. First, duplicates and triplicates were removed. In accordance with our inclusion and exclusion criteria (see Table 1) 826 references were excluded and 101 potentially relevant articles underwent full text screening (Figure 5). Secondary screening led to the exclusion of 90 articles due to subject disorders, measurement techniques, and other exclusion criteria (Table 1). Ten articles were included in the literature review (see flow chart, Figure 5). The following human studies were included (in chronological order): Kassis and Schick 1993, De Wachter and Wyndaele 2003, Ünsal and Cimentepe 2004, Van Haarst et al. 2004, and Eryildirim et al. 2006.^17–21 Four rat studies entered the final set (in chronological order): Malmgren 1888, Chai et al. 1999, Wang et al. 2000, and Huang et al. 2010.^22–25 Chen et al. 2005 was the only included study with mice²⁶ (Table 2).

Figure 5.

Review flow chart: micturition data from humans, pigs, rodents and mice.

Table 1.

Inclusion and exclusion criteria of literature review.

Humans		Animals
Inclusion	Exclusion	Inclusion	Exclusion
Ethical approval	No approval	Animal welfare approval	No approval
Healthy patients	Patients with LUTS, etc.	Healthy animals	Disorders, pathological animal models
Normal nutrition and drinking		Normal nutrition and drinking	Water with 3% glucose
Study groups mixed or 18 to 40 years	Patients younger than 18 and studies containing only older patient	Adult animals	Juvenile or senescent
Research paper	Review article, case reports	Research paper	Review article, case reports
No drug treatment	Drug treatment	No drug treatment	Drug treatment
1960–2013		1960–2013
Frequency–volume chart, urinary diaries, flowmetry	Urodynamics, cystometries, sensation studies	Metabolic cage measurements	Urodynamics, cystometries, cumulative studies or staining, sedation, fixed catheter, sham surgery

LUTS: lower urinary tract syndrome.

Table 2.

Included research articles – humans, rats and mice.

Reference	Species	n	Gender	Time interval	Measurement condition	Statistics	Age	Parameters
Humans
Kassis & Schick 1993¹⁷	Human	33	F	Day	Sitting	Mean + SD	18–56	Frequency + volume
De Wachter & Wyndaele 2003¹⁸	Human	15	F	24 h	Sitting	Mean + SD	18–24	Frequency + volume
Ünsal & Cimentepe 2004¹⁹	Human	72	M + F	Not assigned to hours	Sitting, standing, crouching	Mean + SD	18–44	Volume
Van Haarst et al. 2004²⁰	Human	675	M + F	24 h		Mean + SD	20–49	Frequency + volume
Eryildirim et al. 2006²¹	Human	30	M + F	Not assigned to hours	Sitting, standing, crouching	Mean + SD	28.6 ± 0.7 (mean + SD)	Volume
Rats
Malmgren 1988²²	Sprague-Dawley rats	9	F	24 h	Metabolic cage	Median + range	Adult (200–300 g)	Frequency
Chai et al. 1999²³	Wistar rats	57	M	4 h	Metabolic cage	Mean + SD	Adult (200–300 g)	Frequency
Wang et al. 2000²⁴	Sprague-Dawley rats	20	Not given	17 h	Metabolic cage	Mean + SEM	Adult (220–250 g)	Frequency + volume
Huang et al. 2010²⁵	Wistar rats	40	M	24 h	Metabolic cage	Mean + SD	3 months	Frequency + volume
Mice
Chen et al. 2005²⁶	129Sv-C57 Black/J6-background mice	10	F	24 h	Metabolic cage	Mean + SD	12 weeks	Frequency + volume

F: female, M: male, SD: standard deviation of the mean, SEM: standard error of the mean.

Study characteristics

Humans

In clinical research papers, two measurement methods were used to monitor micturition volumes and micturition frequency. Frequency/volume charts were used in Kassis and Schick 1993, De Wachter and Wyndaele 2003, and Van Haarst et al. 2004.^17,18,20 In the experimental set-up, subjects were handed micturition diaries to fill in all micturition times and volumes. Micturition frequency and volume were determined via computer calculation. Uroflowmetry was used in the studies of Ünsal et al. 2004 and Eryildirim et al. 2006.^19,21 Volunteers were asked to use a private room with a prepared measurement set-up for micturition allowing different voiding positions like sitting, standing or crouching (Figures 6 and 7).

Figure 6.

Review data of micturition volume in humans, Göttingen minipigs (recent study), rats and mice.

Figure 7.

Review data of micturition frequency in humans, Göttingen minipigs (recent study), rats and mice.

Rats

Malmgren 1988, Chai et al. 1999, Wang et al. 2000, and Huang et al. 2010^22–25 placed animals in metabolic cages and monitored fluid outcome with collecting and force transducing devices. In the Malmgren study only medians with range were given The mean of micturition volume from this study was estimated from median and range as postulated in Hozo et al. 2005 with the following equation:²⁷

x = \frac{a + 2 m + b}{4} + \frac{a - 2 m + b}{4 n}

Calculated micturition frequency in 24 h from Malmgren 1988 as postulated by Hozo et al. 2005, offers the following values of sample size (n) = 9, median (m) = 24, lowest value (a) = 17, and highest value (b) = 30. This gives:

x = \frac{17 + 2 * 24 + 30}{4} + \frac{17 - 2 * 24 + 30}{4 * 9} = 23.72

Micturition data from Malmgren 1988 was as follows: the given value for urine volume (mL × kg⁻¹× 24 h⁻¹) was 44 mL (29–64) (median and range) with a weight of between 200 and 300 g for each rat, which was a cumulative value and could not be included.

Mice

We could only find one study meeting all our inclusion criteria for micturition frequency and volume. Ten 129Sv-C57Black/J6-background mice, which were placed in metabolic cages connected to a digital scale for 48 h was used by Chen et al. 2005 in their study. The recording interval was 2 min. In 24 h, the micturition frequency was 15.9 ± 5.2 and the voided volume was 0.16 ± 0.03 mL (mean + SD) (Figures 6 and 7). Although mice were and are widely used, many studies had to be excluded because of cumulative calculations or the calculation of values from staining blots. The difficulty for the determination of micturition data in mice is the small amount of excreted urine, which is around 100 µL and which quickly evaporates.

Domestic pig/Göttingen minipig

We could not find any study presenting data from pigs (either domestic pig or Göttingen minipig). Neither the University of Göttingen nor Ellegard, Denmark (breeders of Göttingen minipigs) could provide reference data.

Discussion

For the investigation of lower urinary tract disorders like OAB, many animal models with different species have been used over time.⁷ The limits of rodent models have been discussed by different research groups, as well as the differences with humans, such as anatomy, volume or diurnal rhythm.^9,10,28 To the best of our knowledge, this study is one of the first to provide physiological reference data of micturition in the Göttingen minipig. Advantages of this species for scientific research are for example their physiological similarity to humans and the fact that they are of a smaller size than domestic pigs.²⁹ Therefore in biomedical research and preclinical toxicology testing, especially, Göttingen minipigs are increasingly being used in translational research as alternatives to dogs or monkeys.^11,12 Other reasons are the increasing resistance in society against the use of pet animals and non-human primates and the acceptance by society of the use of agricultural animals with less emotional attachment to humans.^29,30

Intra-individual data of the five pigs presented showed significant differences leading to the conclusion that in longitudinal or chronic studies each animal should function as its own control (Figures 3 and 4). In addition individual micturition patterns can be used for crossover study design.

The comparison of our own data with literature review data showed that the volumes and frequency of micturition in Göttingen minipigs are more similar to human micturition values than are those of rodents (Figures 6 and 7). These findings indicate that, for certain urological animal studies, Göttingen minipigs would be the better choice for translational research. In the future, the reference data provided may be used for planning new animal studies in different research fields such as urology, neurology or nephrology. With this study we have addressed the 3R concept of Russell and Burch (1959), stating a responsibility for ‘reduction, refinement and replacement’ in laboratory animal experiments. Because of the use of reliable reference values, a comprehensive statistical power calculation is possible, leading to fewer animal numbers (reduction). Göttingen minipigs in urological research can be used with telemetric approaches involving free-moving animals, which allows higher accuracy and less infection risk (refinement) in monitoring. In the future, the data provided can be used to generate computer-based modulations of drug release and enable the development of intravesical drug-delivery systems, and satisfy the replacement principle.^31,32

We are aware of the limitations of our study. Briefly, only a small number of female-only Göttingen minipigs were used, but the data presented consisted of 35 monitoring sessions with 221 single micturitions. We chose very restricted inclusion/exclusion criteria. The rationale of this paper was the discussion of translational comparability of different species with a focus on physiological function. Diagrams from 20 or more different studies would be really confusing, and yet the overall mean value would be nearly the same, so comparison of mean values among species would be the same even with less restrictive review criteria.

Footnotes

Declaration of conflicting interests

None declared.

Funding

Financial support was given by the Federal Ministry of Education and Research, Germany, grant no. 13N11308.

Acknowledgements

The authors thank the research group of the Department of Urology especially Katja Stollenwerk for technical support, and the Institute of Laboratory Animal Sciences especially Karl Heinz Riemek, Thaddäus Stopinski and Anna Woitok for excellent animal care. We would like to thank an anonymous reviewer for the constructive analysis of the manuscript and the helpful input.

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