How to obtain individual urine samples from undisturbed marmoset families

American Journal of Primatology Blz223-;2B0 (f998)

TECHNICAL NOTE

How to Obtain lndividual Urine Samples FromUndisturbed Marmoset Families

G. ANZENBERGER AND H. GOSSWEILERAnthropologicgl Institute ond. Museum and, Institute of Psychology,Uniuersity of Zarich, Switzerland

An apparatus and a method are described, which allow simultaneous urinecollection from all individual members of undisturbed marmoset families.The monkeys have been trained, after leaving their sleeping box, to entersingle adjacent compartments where they are rewarded. There the mon-keys micturate within minutes and the clean urine runs directly intocryotubes. These simultaneously collected urine samples open up the op-portunity for investigation ofthe relationship between endocrine functionand behavioral dynamics within entire marmoset groups.@ 1993 Wiley-Liss, Inc.

Key wordsz Callithrir jaachus, Callitrichidae, urine collection, non-invasive method

INTRODUCTIONIt has always been a highly desirable approach in studying primate behavior

to combine ethological and physiological aspects within the same study. Unforüu-nately, the physiological aspect, almost inevitably requiring restraint or invasivetechniques, often impedes the addressing of more complex ethological aspects. onepossibility for dealing with such problems is to reduce the discomfort and stress fortest individuals (e.g., during blood sampling) by positive reinforcement [Hearn,1983; vertein & Reinhardt, 19891. However, the dilemma mentioned above wascertainly also the reason for developing non-invasive techniques such as determin-ing steroid hormones by means of urine samples (for an elegant method of urinesampling by instrumental conditioning in captive vervet monkeys see Kelley andBramblett [1981]), and research on captive callitrichids.has employed a wide arrayof different collection methods. In order to obtain urine samples, monkeys wereeither taken from their social units and placed for some time in metabolism cageslEpple & Katz,1984; French et al., 19841, or individuals were lured into smällcages [Evans.,& Hodges, Lg84; Heistermann et al., 1989], or the cage floor wascovered with pans [French et al., 1989] or plastic mats, respectively [Brand, 1981;Epple et al:, 19911, and the voided urine was then collected with pipettes, or aperson stepped into cages and followed single individuals with a sampling con-tainer [Savage et al., 1988; Ziegler et al., 1990]. In all of these studies, urine

Received for publication October 8, 1992; revision accepted March 30, 1gg8.

Address. reprint re-quests_ !o -Qu1tl Arzenberger, Anthropological Institute, University Zürich-Irchel,

Winterthurerstr. 190, CH 8057 Zürich, Switzerland.

O 1993 lYiley-Liss, Inc.

224 I Anzenberger and Gossweiler

samples of only one or two individuals per social unit could be collected at the sametime. On the other hand, captive callitrichids with their unique organization infamily groups show a variety of specific traits and complex social dynamics whichwould be worth investigating (e.9., the presence of twins, twin fights, competitiveconflicts between elder siblings, reproductive suppression ofprogeny, inbreedingavoidance among family members, and expulsions of offspring around the post-partum ovulation ofthe alpha-female), and an etho-endocrinological approach tothese topics would lead to a more integrated understanding of the underlyingcausal mechanisms. In order to obtain the essential endocrinological data sets werely on stress-free and simultaneously collected individual urine samples fromentire family groups of callitrichids. In common marmosets (Callithrix jacchus),this has been achieved with the method and apparatus to be described.

MATERIALS AND METHODSSubjects and Housing Conditions

Common marmosets are kept at the Department of Psychology as "naturally"as the restricted conditions of captivity allow. Each family group is housed invisual isolation from the others, in a separate room (between 35 ms and 45 m3)which is equipped with a suspended runway system made of natural branches.This runway system is densely furnished with freshly foliated twigs on a weeklybasis, except during the winter months. The floor is covered with a 5-10 cm thicklayer of mulch, containing enough microorganisms that faeces and food remnantsneed not to be removed but are processed overnight. The diet consists of a broadvariety of fresh fruit and vegetables and is supplemented with insects, quail eggs,and fresh, dead, small cichlids. The group size of our marmoset families rangesfrom 4 to 16 individuals. To date the apparatus described below has been used tocollect urine samples from 45 individuals from 6 different families.

Urine Collecting ApparatusThe urine collecting apparatus (UCA) is shown in Figure 1. The main part of

the UCA is constructed of clear PVC (2 mm thick) and consists of tunnel-shapedcompartments adjacent to each other. Each compartment (Fig. la, A; 10 cm W x20 cm H x 25 cm D) has sliding doors at each end (Fig. 1a, B). The sliding doorscan be operated by a pull and string system and can be frxed at the closed or openposition by a locking lever. An entrance gallery running throughout the entirewidth of the apparatus (Fig. 1a, C; 13 cm depth) can be joined with a catwalk whichconnects the hutch box and the monkeys'room (see below and Fig. 2). The UCAdepicted consists of 6 compartments; for families with more than 6 individuals twoor more units can be combined.

The bottom grid of the UCA is made of stainless steel wire mesh (2 mm thick; .

meshes 2 cm x 2 cm) which can be totally removed (Fig. 1a, D). Underneath thatbottom grid.there are two different installations. First, at the front edge there is aremovable metal container inserted, which is used to hold some tidbits for themonkeys (Fig. la, E). Second, there is a flat multi-V-shaped duct made of opaquePVC, whereby the width of the Vs corresponds with the width of the compartmentsabove (Fig. la, F). This duct collects the voided urine and leads it immediatelythrough a funnel into a cryotube (1.8 ml; manufacturer: NUNC, Denmark). Thecryotube is held by a metal clip mounted on a magnetic coin-like plate; the lattercan be attached to small magnets inserted into a metal strip (Fig. la, G).

The UCA is a frxed installation in each marmoset family's room. In all cases,to prevent the monkeys from damaging the UCA, the apparatus is installed in aseparate part of the room which is protected by wire mesh and accessible by per-

Urine Collection from Marmoset Families I 225

b)

Fig. 1. Urine collecting apparatus. a: Photographic side view, showing (A) adjacent compartments; (B) slidingdoors with pull and string system (two front and two back doors in open position); (C) entrance gallery; (D)

bottom grid; (E) food container; (F) urine collecting duct; (G) cryotubes with funnels. b: Technical sketch showingdimensions. See text for further explanations.

226 I Anzenberger and Gossweiler

DURING DAY & NIGHT IN THE MORNING

Fig. 2. Diagram showing layout of the urine collecting apparatus and two different operational situations(schema not dram to scale). a: Pivoting door blocks the way to the apparatus. The monkeys can freely movebetween hutch box and room. b: Pivoting door blocks the way to the monkeys' room. The monkeys haveio passthrough the apparatus on their way out. See text for further explanations.

sons. A catwalk tunnel connects the monkeys'room with the hutch box as well aswith the UCA (see Fig. 2). A pivoting door inserted in the catwalk tunnel can befixed in three positions. In the first position (not shown in the figure) the monkeyshave free access to the room, to the hutch box, and to the UCA. In the secondposition the entrance to the UCA is blocked, allowing the monkeys to move freelybetween the room and the hutch box (Fig. 2a); this was the usual situation duringday and night. In the third position the door blocks the catwalk tunnel between theroom and the hutch box. Now, in order to move into the room the monkeys have topass through the apparatus (Fig. 2b); this corresponds to the situation in themorning before urine collecting.

Training MethodDuring the first week of the training, the pivoting door and the front and back

sliding doors were kept open all of the time. At the same time, mealworms, theearly morning tidbits for the monkeys, were now offered in the food containerinserted in the UCA (cf. Fig. 1a, E), rather than in the normal food dishes. Themain goal of the first week of training was thus to familiarize the monkeys withthe apparatus as an extension of their home cage and as a source of mealworms.During the second week the pivoting door and the front sliding doors were keptclosed in the morning for about 15 min. This arrangement allowed the monkeys tomove only from the hutch box to the apparatus, where they could feed on meal-worms. By this time the monkeys seemed already to be quite familiar with theapparatus. At the beginning of the third week we started to close the back slidingdoor as soon as a monkey entered a compartment and this caused no obvious signsof stress or panicking. By the end of the third week of training, it was not unusualto collect individual urine samples from an entire family.

Urine Collecting MethodIn the morning a person enters the monkeys' room in darkness and switches

the pivoting door (cf. Fig. 2b). After the lights go on the monkeys separate spon-taneously and individually into the compartments (Fig. 3) and the person closes

Urine Collection from Marmoset Families I 227

the sliding doors behind them. There is a strong tendency in the monkeys to leavethe hutch box in a certain order and to occupy their "own" compartment. Then, themonkeys start feeding on the mealworms and soon afterwards they void theirmorning urine. Normally, all group members do so within about 10 min.

After micturation, the front sliding door is opened and individuals are releasedinto the monkeys'room. The filled cryo tube is taken, capped, labeled with theindividual's code and the date, and deep frozen immediately. Later the apparatusis cleaned. The removable bottom grid, the PVC urine duct, and the funnels aretaken out, kept for several minutes submerged under very hot water, rinsed well,and dried. Then the apparatus is prepared for the next morning.

Cross contamination of samples is practically excluded by the construction ofthe apparatus, but is carefully checked for by the person operating the apparatus.Due to the fact that urination almost always precedes defecation, no fecal materialenters the cryotube. Cross contamination by urine of small infants is also unlikely,because marmoset infants younger than 4-5 weeks of age are normally carried bya caregiver and do not urinate whilst being carried. At the same time infants usea separate compartment as early as 6 weeks of age-that is, there is only a shorttime span during which a cross contamination of urine samples can result. In theevent of cross contamination, samples are discarded.

DISCUSSIONThe apparatus and method described offer some advantages for monitoring

endocrine profiles by means of non-invasive urine sampling. Before addressingthese advantages we would like to stress some points regarding the training of themonkeys. First of all, it is important to let the monkeys explore the new installa-tion at a self-determined pace (i.e., simply to leave the apparatus open for severaldays without a person present). Later, when starting to lock the monkeys in, it iscrucial that a person watches them very closely and lets them go as soon as theyshow any signs of discomfort, such as frequently turning around or manipulatingdoors. After a few days the monkeys stopped behaving in this way. Moreover, afterhaving been released into the monkey room, they readily returned to the appara-tus as long as the front sliding door was open. In other words, the monkeys seemedno longer to be bothered at all by being closed in. Therefore we are almost certainthat this kind of urine collection is stress-free for the monkeys. Another observa-tion may illustrate the monkeys'familiarity with the apparatus. When the groupleaves the hutch box, preambulatory infants are normally carried by the mother.If she tries to get rid of them while still in the apparatus we simply open the backsliding doors of both parents' compartments, thus allowing them to transfer theinfants, They then go back into their compartments, now with the father carryingthe infants.

Although the urine collection technique presented has been developed to beused with ofle of the smallest primate species, there is no doubt that it could beadapted for use with larger primate species. In one of our marmoset groups theapparatus had to be attached to the front wall of the monkeys' room, without animmediate connection to the hutch box. Nevertheless, all group members readilyentered the apparatus after leaving the hutch box and started feeding on meal-worms. Thus, the procedure should also work with species where group membersdo not have the natural "advantage" of sleeping together in precisely the samespot. In our opinion one essential prerequisite for the training ofthe monkeys andfor the subsequent operation of the apparatus is that all group members remainclose together and none have to be singled out (i.e., that social facilitation is at

228 I Anzenberger and Gossweiler

Fig. 3. A family of mamosets (a) leaving the hutch box (on the right edge of the photograph) and (b) separatingthemselves into the single compartments ofthe urine collecting apparatus.

work). For individuals born after the apparatus has been installed, urine samplingbecomes part of their normal daily routine.

We would now like to address and discuss three specifrc advantages of the

Urine Collection from Marmoset Families / 229

collection technique described. First, the method is entirely non-invasive, and it isnot obviously stressful to the monkeys. With the apparatus presented, one does notneed either to single out and put individuals in metabolism cages or step into cagesand follow single individuals with a container in order to sample urine. There is nolimit to the number of individuals monitored per social unit. This means one canrelate social events and complex social dynamics to their endocrine correlateswithin entire families of callitrichids, without disturbing the normal daily life ofthe group in any respect. Second, urine samples can be collected seven days a week,and our experience shows that the method works reliably, with hardly any samplesmissed. Thus, the method provides ideal data sets for longitudinal studies. Inaddition, the method is also highly reliable with respect to possible contaminationsof the urine, because the urine is voided almost directly into the cryo tube. Themost important point, however, regarding intra- as well as inter-individual com-parisons of the endocrine data is that we get individual morning urine samplesvoided by all group members within a very narrow time span. Third, the presentmethod and apparatus allow one to collect fecal samples as well, and we do this forregular parasitological and bacteriological checkups. In this case, the monkeysremain confined after having urinated, and they typically defecate soon after-wards. Such fecal samples could also be used for determination of steroid hor-mones, as has been done for several primate species [e.g., Wasser et al., 1991;Ziegler et al., 19891.

In sum, the present urine collection technique provides an interface for inves-tigating complex behavioral mechanisms within groups and for simultaneouslymonitoring the endocrinological correlates within individuals. This approach sub-stantially broadens the causal analysis of behavioral mechanisms and leads to abetter and more integrated understanding of social dynamics within such highlycomplex associations as primate groups are.

CONCLUSIONS

1. The apparatus and method described allow a timed and individual urinesampling of all members of marmoset groups.

2. Matched urine samples from entire marmoset groups open up the possibil-ity of closely monitoring complex social dynamics in a non-invasive way.

3. The system presented seems to be adaptable for the use in other facilities aswell as with other primate species.

ACKNOWLEDGMENTSDue to her outstanding engagement and sensitivity, our technician Kathrin

Weiss has been of invaluable help during the training of the monkeys as well asduring routine sampling procedures afterwards. Ms. Deborah Curtis and Dr. C.R.Menzel kindly corrected the English.

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