The following are the abstracts of papers and posters presented at a joint EUPREN/EMRG meeting held at DPZ in Göttingen (6-8 December, 1995). The topic of the meeting was:
Recent advances in remote and non-invasive monitoring technologies offer
considerable potential for refining studies involving non-human primates, both
in terms of animal welfare and scientific quality. The range of speakers and
delegates from diverse scientific backgrounds facilitated invaluable
multidisciplinary information exchange.
Proceedings of the meeting will be
published and a follow-up is planned for November, 1996.
The organizers
gratefully acknowledge the financial support received from the EU (DGXI), the
pharmaceutical industry and from trade exhibitors.
Urinary endocrinology and social dynamics: a method for monitoring entire marmoset groups.
Gustl Anzenberger
Anthropologisches Institut und Museum der
Universitat Zurich
Winterthurerstrasse 190, 8057 Zuerich, Switzerland
Introduction: Determining steroid hormones by means of urine samples was the first non-invasive technique for socioendocrinological studies in primates. Accordingly, many different sampling methods have been developed by people studying callitrichids but urine samples of only one or two individuals per social unit could be collected at the same time. On the other hand, captive callitrichids with their unique organization in family groups show a variety of specific traits and complex social dynamics which would be worth investigating in the group context (e.g. the presence of twins, twin fights, competitive conflicts between elder siblings, reproductive suppression of progeny, inbreeding avoidance among family members, and expulsions of offspring around the post-partum ovulation of the alpha-female). In order to obtain the essential endocrinological data sets we rely on stress-free and simultaneously collected individual urine samples from entire family groups. In common marmosets (Callithrix jacchus), this has been achieved
Apparatus and methods: An apparatus and a method are presented, which allow simultaneous urine collection from all individual members of undisturbed marmoset families. The monkeys have been trained, after leaving their sleeping box, to enter single adjacent compartments where they are rewarded. There the monkeys micturate within minutes and the clean urine runs directly into cryotubes.
Conclusions:
(1) The apparatus and method described, allow a timed
and individual urine sampling of all members of marmoset groups without
disturbing the normal daily life of the group in any respect.
(2) Matched
urine samples- from entire marmoset groups open up the possibility of closely
monitoring complex social dynamics in a non-invasive way.
(3) The method is
highly reliable with respect to possible contamination's of the urine, because
the urine is voided almost directly into the cryo tube.
(4) The apparatus
and method allow one to collect fecal samples as well and this is done for
regular parasitological and bacteriological checkups. In this case, the monkeys
remain confined after having urinated, and they typically defecate soon
afterwards.
(5) The system presented seems to be adaptable for the use in
other facilities as well as with other primate species.
Electroretinography in the non-human primate - a non-invasive method for determination of ocular toxicity.
W.H. Bee, R. Korte & F. Vogel
CORNING Hazleton GmbH
Muenster, Germany
Electroretinography (ERG) serves as an objective measure of retinal function in humans. The "Standard for Clinical Electroretinography" (SCE), published by the International Society for the Clinical Electrophysiology of Vision, proposes five types of measurements as a minimum requirement in order to obtain international standardization for investigations in man. On the basis of this guideline, a standardized method for ERG investigations was developed for the non-human primate. ERG traces including the five standard responses were recorded from 100 cynomolgus monkeys (Macaca fascicularis) under general anesthesia using a full-field ("Ganzfeld") dome.
After 30 minutes of dark adaptation, the scotopic ERG was recorded by seven measurements with increasing flash intensity up to the maximal response to the standard flash (SF) of 2.6 cds/m2. A standard rod response was elicited with flashes 2.6 log units below the SF. The oscillatory potentials were then measured at 0.4 log units above the SF. At the onset of light adaptation and 10 minutes thereafter, 30 Hz flicker responses were obtained with the SF in the presence of background illumination (150 cd/m2). For the following photopic ERG, the red flash cone responses were recorded with increasing flash intensity. The final photopic trace to be recorded was the single flash cone response with the white SF.
The results of the recordings performed in accordance with the SCE correspond very well with the results obtained in humans. Thus, the cynomolgus monkey has been proved to be an excellent animal model for testing of retinal function in toxicity assessment.
Is saliva cortisol a reliable indicator for perturbations in the squirrel monkey?
*D. Benisch, C. Kirschbaum , A.Bieser, E. Fuchs
Division of
Neurobiology, German Primate Center, Goettingen
*Department of Psychology,
University of Trier
Activation of the hypothalamus-pituitary-adrenal(HPA)-axis by physical, chemical, and psychological perturbations is known to result in elevated levels of serum corticosteroid hormones. Corticosteroids are principal effectors in the stress response and are thought to be responsible for both adaptational and maladaptational processes to perturbing situations. They have profound effects on mood and behavior and affect neurochemical transmission and neuroendocrine control. Cortisol, the predominant corticosteroid in primates, is often regarded as the "stress hormone" and consequently serves as a marker of stress. Cortisol can be measured in blood, urine, and saliva. Determination of plasma/serum cortisol is used in many laboratories. It requires capture and handling of the animals along with venepuncture, a procedure which can be stressful by itself. In contrast, analysis of urinary cortisol is a non-invasive approach. However, this method is unsatisfactory when information of cortisol levels within short time intervals are needed. Recently, the stress-free assessment of cortisol in saliva has become a valuable alternative to both blood and urine analysis (Kirschbaum and Helihammer, 1 994). To examine the feasibility of sampling saliva samples from squirrel monkeys (Saimiri sciureus), and to test whether saliva cortisol is a reliable indicator for perturbations in this species, the present pilot study was designed. Saliva samples were collected from animals with a cotton-wool tip and cortisol was quantified by an DELFIA assay kit. Baseline levels were in the range of 7 + 3 pmol/ml cortisol . Significant increases of salivary cortisol (60 pmol/ml) were registered after disturbances in the animal facilities such as unusual high levels of noise unfamiliar persons or fluctuation in the animal caretaker staff. Furthermore, a clear activation of the pH-axis was observed during behavioral discrimination task in particular when the tests were new and/or complex to the animals.
These findings are indicative that the assessment of salivary cortisol is an easy to handle and stress-free method for the documentation of physiological variations of the HPA-axis activity in squirrel monkeys.
Reference: C. Kirschbaum & D.H. Helihammer, Psychoneuroendocrinology 19: 313-333, 1994
Immunopathogenensis of EAE in the common marmoset: analysis of the desaese course with non-invasive methods.
A. Bert , T. Hartl, B. Groen*, B. Melchers* & L.
Massacesi**
Dept. Immunobiology, BPRC, Lange Kleiweg 151, 2288 GJ
Rijswijk, Holland
*Dept. Pharmacology, TNO-DO, Lange Kleiweg 137, 2288 GJ
Rijswijk, Holland
**Dept. of Neurological and Psychiatric Sciences,
University of Florence, Italy
Experimental autoimmune encephalomyelitis (EAE) is the best accepted animal model for human MS. The disease is characterized by inflammation and demyelination of white matter in the central nervous system. Much information on the disease pathogenesis has been obtained in small rodent species, but these have their limitations. There is now a need for a good disease model in non-human primates, a.o. for efficacy testing of human-specific therapy strategies. EAE has been evoked in macaques, but appeared poorly representative of human MS. Only recently, a new EAE model has been described, evoked in the common marmoset (Callithrix jacchus). The model was also introduced in the BPRC. The main question we wish to address is to find a relation between pathological events in the CNS and clinical expression of the disease.
Longitudinal monitoring of the course of EAE is done with daily macroscopic observation, computerized tests for sensory and motor-functions, whereas events in the CNS is monitored with in vivo NMR imaging techniques. We are now working on biochemical markers which can be measured in the urine. The results which will be presented are not fully conclusive, but will serve to illustrate the informative value of the applied methods in the studied disease.
This project is subsidized by the Human Capital and Mobility Program of the European Union.
Approaches to home-cage behavioural monitoring.
A.P. Bowditch, P.C. Pearce, H. Crofts*, C.A. Dickson & L. Scott
Chemical and Biological Defence Establishment, Porton Down, Salisbury,
SP4 OJQ, UK *Psychopharmacology Unit, University of Bristol School of Medical
Sciences
University Walk, Bristol, BS8 1TD, UK
Monitoring and testing trained behaviours in non-human primates has traditionally involved removing animals from their home environment and placing them in speciaily designed test chambers. A different approach that has been successfully applied in our laboratories is to conduct testing in the home cage. We believe there are many benefits to be gained from such an approach.
A number of different tests of both trained and spontaneous behaviours have been developed or adapted for use in marmosets (Callithrix jacchus) and/or rhesus monkeys (Macaca mulatta). The techniques and equipment needed for home-cage testing of spontaneous activity, foraging, visually guided reaching, differential reinforcement of low rate responding (DRL), reaction time and complex discrimination will be described.
The relatively simple apparatus is attached to the home cage, causing minimal disturbance to the test animal and others housed in the same room. For each of the tests, baseline performance data and, where approriate, the effects of pharmacological intervention, will be presented. The advantages and disadvantages of the home-cage approach will be discussed.
The Role of Primate Urine and Faeces in New Drug Development
Colin Brown
Inveresk Research International
Tranent EH33, 2NE,
Scotland
Regulatory toxicology studies are generally conducted in accordance with national and international guidelines, determining in detail most of the required features of the study. Typically these will include detailed observations, body weight and food consumption measurement, ophthalmoscopy, ECG/BP measurement, clinical pathology, necropsy and histopathology data; parallel studies will provide information on the metabolism and kinetics of the novel material. Urine and faeces collections are normally undertaken routinely at pre-determined intervals, are used in clinical pathology assessments, and frequently contribute useful data on metabolism and kinetics.
The use of non-human primates in hazard evaluation is increasingly under scrutiny. Fundamental justification for use; species preference; the numbers of animals per experiment; allowable techniques; severity limits; environmental enrichment - all are current issues to the extent that, in the contract research environment at the present time, each must be addressed before every experiment. Of the many forms of new chemical entity - therapeutic agent, agrochemical bulk or speciality chemical, cosmetic, consumer product - non-human primate use is restricted to therapeutic agents. Non-invasive techniques have always had a place in the research lab, but are perhaps worthy of re-appraisal in the current climate. In this poster presentation, the value of data obtained from urine and faeces collected non-invasively from non-human primates will be examined.
The collection of high quality urine samples from primates is difficult. There are many potential contaminants, and the preservation of enzymic activity is at risk due to the general urinary environment, and specific factors such as temperature regulation and bacterial contamination. Urinary enzymic analysis has the potential to assist in the early identification of renal damage during toxicology studies, but is rarely used to advantage. Histopathology remains the main indicator of renal damage in primates, despite the time lag before such data is available.
It cannot be taken for granted that the non-human primate is, for any given novel therapeutic, the ideal model for man, and therefore the most suitable non-rodent species for preclinical toxicology studies. Drug metabolism studies undertaken using radiolabelled material provide a useful and necessary contribution towards identifying species differences. Urine and faecal samples can provide important data on clearance of a test material, and on the production and elimination of metabolites.
With current trends in animal welfare, it is not inconceivable that a metabolism study may become an essential prerequisite in justifying the use of the primate in toxicology studies .
On cage monitoring of a complex discrimination task in marmosets and rhesus monkeys.
H.S. Crofts*, A.P. Bowditch, N.G. Muggleton, P.C. Pearce, D.J. Nutt* &
E.A.M. Scott
Medical Countermeasures, CBDE, Porton Down, Salisbury, U.K.
* Psychopharmacology Unit, University of Bristol, U.K.
Tests from the Cambridge Neuropsychological Test Automated Battery (CANTAB) have been used extensively in humans (Sahakian, B.J. and Coull, J.T. 1994 Drug Dev Res 31:80-88) and marmosets (Roberts, A.C. et al 1988 Q.J. Exptal Psychol 40:321341 ) to examine neuropsychological fianctioning. This comparative approach facilitates cross species extrapolation, particularly in the study of neurodegenerative disorders and the effects of pharmacological intervention.
Such tests are traditionally presented to animals in sound attenuated remote testing chambers. In this laboratory an alternative approach has been adopted such that elements from the CANTAB tests are presented to both marmosets and rhesus monkeys in their home cages.
To date, 13 marmosets and 4 rhesus monkeys have completed several sequences of an 8 stage discrimination task involving simple discriminations7 compound discriminations and reversals. The techniques developed can be applied to other species and to the presentation of other behavioural tests
Behavioral and morphological indices of reproductive status.
Alan Dixson, D.Sc.
MRC Group, University of Cambridge,
Sub-Department of Animal Behaviour
Madingley, Cambridge CB3 8AA, U.K.
This lecture will provide examples of how measurements of behaviour and reproductive anatomy, which are relatively simple to make and non-invasive in nature, can contribute to knowledge of reproductive physiology and evolutionary biology.
Firstly, measurements of testicular length and breadth allow calculation of testes volumes and (by use of an appropriate correction factor) testes weights. Double logarithmic plots of testes versus body weights allow predictions to be made about the nature of mating systems and prevalence of sperm competition. Examples drawn from the anthropoid primates and the nocturnal prosimians will be used to illustrate the argument that larger relative testes sizes and greater sperm competition occur in forms having multimale/multifemale and dispersed mating systems. Further analyses of genital structure (e.g., sperm length and penile morphology) and behaviour (e.g., copulatory patterns and ejaculatory frequencies) will then be presented and related to the effects of sexual selection and occurrence of sperm competition in mammals.
Next, the androgen-dependent, secondary sexual traits of male primates will be considered. In certain species, development of some traits may be delayed during puberty and in adulthood. Inter-male competition may be responsible for such effects and competition is also linked to lowered plasma testosterone, reduced mating success and reproductive success. Some recent work on semi-free ranging mandrills (Mandrillus sphinx) in West Africa will be presented in order to illustrate these arguments.
Finally, the question of the "sexual skin", ovarian cyclicity and sexual behaviour in female catarrhines will be discussed. In some species measurements of swelling size and turgidity, as well as the timing of detumescence (sex skin "breakdown") provide useful indices of the occurrence of ovulation. Increases in females' sexual invitations to males (proceptivity) may also occur during the periovulatory period. There is also the interesting question of how inter-female competition may influence the ovarian cycle and sexual skin morphology in baboons, mandrills and other catarrhines.
Haemodynamic parameters in cynomolgus monkeys.
C. Duserre
Biologie Servier
France
It is vital that cardiovascular investigations record haemodynamic and electrical parameters without disturbing the animals involved, since stress often affects the validity of the measurements obtained. Preclinical cardiovascular safety studies require conscious animals so that all the physiological control systems are functional. All experimental studies using animals must be carried out under optimum conditions for these animals so as to decrease the stress caused by handling. These 3 deciding factors lead us to use the remote monitoring technique for cardiovascular investigations on monkeys.
By surgery, a miniature device is implanted subcutaneously in a ventral position. It is linked to 1 or 2 catheters to record systemic arterial pressure or left ventricular pressure and to 2 electrodes to record the electrocardiogram. The pressure and ECG signals can be recorded as soon as the catheters or electrodes are in place. When the animal is awake, the recordings may be taken while it is moving freely. Recordings over a 24-hour period make it possible to detect spontaneous variations in pressure and heart rate. The most significant variation is that between day and night. During the diurnal period, the heart rate is about 1 20 beats/mn and the average arterial pressure about 90 mmHg, while during the nocturnal period, the heart rate decreases to 85 beats/mn and the average arterial pressure goes down to 75 mmHg. All compound cardiovascular actions after dosing can be observed precisely at the beginning of parameter modifications, at their peak and at the return to normal values. Handling the animals causes the heart rate and average arterial pressure to increase to 250 beats/mn and 150 mmHg, respectively. When cardiovascular recordings are performed on restrained animals, handling interferences are continuous and lead to a loss of accuracy in recorded values. With remote monitoring, the highest quality data are obtained because many types of stress-induced artifact are avoided.
Cardiovascular remote monitoring in non-human primates meets two important requirements:
Application of radiotelemetry on the rhesus monkey (Macaca mulatta) to cardiovascular studies in research field and in safety pharmacology.
Y. Fradin
Cardiovascular department, Roussel Uclaf
102 route de
Noisy, 93235 Romainville, Cedex, France
The use of monkeys as subjects in cardiovascular pharmacological experiments is difficult and has been limited. For instance, pharmacological effects can be hidden by anaesthesia and the use of intravascular catheters imposes significant restrictions on the classical measurements of haemodynamic parameters. Accurate measurement of cardiovascular parameters like blood pressure in conscious and unrestrained monkeys by telemetry is an old and continuing quest for biomedical researchers. Despite the fact that this technology began in the 1950's, it is only recently that the evolution of the electronic, packaging, sensor and battery technology has made it feasible. In our laboratory, the telemetric system employed (D.S.I.) enables both to measure blood pressure and to collect an electrocardiogram on conscious rhesus macaques (Macaca mulatta).
We have established a primate laboratory specializing in telemetric monitoring with facilities for eight monkeys. The cages, equipped with telemetry devices, are connected to each other by means of sliding doors. The monkeys are habituated to their new environment and the transmitters then surgically implanted under strict asepsis. After recovery, the animals go back into their respective cages and the experiments could start two or three weeks later. All the animals are under video control during the experiments to correlate the data obtained by telemetric recording and their behaviour. All the individual values after calculation in the microcomputer are tranferred through a network to a Vax computer.
The use of this new technology enables us to work intermittently but regularly on this experimental monkeys. As a matter of fact, in our laboratory, this species is not the most used for our pharmacological studies but remains essential for specific questions. Over the last two years, with a small staff and a minimum care for the animals we performed a significant number of pharmacological experiments and, time to time, studies in safety pharmacology. For instance, in cadiovascular field, we determined a full pharmacological profile for an angiotensin II antagonist. And moreover, on the same eight animals, after to have change the transmitters and after washout periods, two others studies have been carried out in safety pharmacology. The benefits of telemetry for both the scientists and the monkeys are unquestionable. The use of telemetry on large animals is a form of technical progress which reconcilies ethics and animal experimentation and which leads to results which are more reliable with respect to their potential predictive value.
Activity monitoring: an overview.
E. Fuchs, S. Prowse* & L. Scott*
German Primate Center,
Kellnerweg 4, D-37077, Goettingen
*CBDE, Porton Down, Salisbury, U.K.
Motor activity represents a class of spontaneous behaviour which involves 'the co-ordinated participation of sensory, motor and integrative processes' (MacPhail et al, J Amer Coll Toxicol (1989) 8, 117-25). Whilst the investigation of spontaneous activity is sometimes considered simplistic and uninformative, it is now generally acknowledged that changes in activity reflect alterations in physiological and/or pharmacological status. In addition, studies of spontaneous activity make important contributions to fundamental research in a wide range of disciplines, including behavioural genetics and preclinical psychopharmacology.
Because of its sensitivity to manipulations of enviromnental, physiological and pharmacological factors, spontaneous activity is frequently quantified in animal experiments. Studies, mainly conducted in rodents, have demonstrated the importance of careful experimental design in generating reliable and reproducible results. Many methods for automated recording of activity have been developed, but are of limited use for species other than rodents as they generally register movement in only one plane. Many of the deficiencies in such systems have been overcome by recent advances in videotracking technologies. It is now possible to record 3-dimensional spontaneous activity of animals of varying size in the home cage. This will enable inforrnation on patterns of activity to be determined more readily and applied more widely.
On the sociophysiology of the pair bond in common marmosets (Callithrix jacchus).
Patricia Gerber1,2, Christian R. Schnell2 &
Gustl Anzenberger1
1Anthropologisches Institut und Museum der
Universitat Zuerich
Winterthurerstrasse 190, 8057 Zuerich, Switzerland
2Cardiovascular Research Department, CIBA, 4002 Basel,
Switzerland
Rationale of the study: Under captive conditions common marmosets (Callithrix jacchus) exhibit a monogamous lifestyle. Although several authors have experimentally investigated the pair-bond of marmosets, information on socio-physiological and/or socio-endocrinological correlates of this motivational construct is currently lacking. On the assumption that species-specific social grouping is the result of selective forces operating at different levels, we may conclude that an established pair-bond corresponds to behavioural and physiological homeostasis for any monogamously living female or male. It follows that major challenges to the integrity of the pair-bond, such as separation from the mate and/or the presence of a third conspecific, will trigger coping mechanisms at different proximate levels of behaviour. Accordingly, in this study the pair-bond of marmosets will be investigated at three different levels of behaviour (ethological, sociophysiological and socioendocrinological). The responses to separation from the pairmate and to confrontations with an unfamiliar opposite-sexed conspecific will be quantified. Data will be recorded simultaneously at all levels, thus allowing monitoring of behaviour in parallel with changes in both physiological control systems, the sympathetic-adrenal-medullary axis and the hypothalamic-pituitary-adrenal-cortical axis.
Animals and methods: Pair-mates of 6 established pairs will be tested. Behavioural data will be videotaped by a remote-controlled system installed within the cage. Cardiophysiological data (heart rate and blood pressure) as well as general activity will be recorded by an implanted remote-controlled telemetry system adapted for marmosets [Schnell CR & Wood JM. Am J Physiol 264: H1509-H1516, 1993]. Socioendocrinological data will be determined from urine samples collected under stress-free conditions. Urinary cortisol will be determined for all individuals as an indicator of stress response. In addition, urinary estrone (E1C) will be measured in females in order to determine follicular and luteal phases of their ovarian cycle, respectively. The following four types of experiments will be conducted: (1) Separation of pair-mates. (2) Separation of pair-mates and subsequent confrontation of one of them with an unfamiliar opposite-sexed conspecific while the other pair-mate is absent. (3) Separation of pair-mates and subsequent confrontation of one of them with an unfamiliar opposite-sexed conspecific while the other pairmate is visually present. (4) Separation of pair-mates and subsequent confrontation of one of them with an unfamiliar opposite-sexed conspecific while the other pair-mate is behind a one-way screen. [Note that the interacting pair is not aware of the presence of the observing pair-mate.] Here, only the responses of the observing pair-mate are measured. Every experimental trial is divided into 3 segments of 10 min duration: (1) baseline; (2) separation (with or without confrontation); (3) reunion. During the first and third segments, cardiophysiological data can be sampled only from one pair-mate, whereas during separation this will be possible for both. Every individual will be tested in three replicates of all types of experiments.
Conclusions: If the study can be successfully conducted as designed it will provide: (1) A thorough causal analysis of social dynamics at three different levels of behaviour for the first time in a primate species. (2) A method of stress-free investigation of sociophysiological and -endocrinological correlates of such a complex behavioural syndrome as the pair-bond. (3) A more comprehensive understanding of proximate causes of primate monogamy. (4) Possible new insights into handling and maintenance of marmosets, which may contribute to an improvement of captive.conditions.
Molecular analyses for kinship studies of a freeliving community of bonobos (Pan paniscus) employing non-invasive sampling methods.
U. Gerloff
Zoologisches Institut (Abt. Tautz)
Universitat
Muenchen, Luisenstr.14, 80333 Muenchen
Over a period of four years, faeces samples of a free living group of bonobos (Pan paniscus), the Eyengo community, were collected by Barbara Fruth and Gortfried Hohmann (Max-Planck-Institut fuer Verhaltensphysiologie, Seewiesen). Nuclear DNA extracted from these samples was used to amplify hypervariable simple sequence repeats (microsatellites) via PCR, which are used for paternity analysis and kinship studies (GERLOFF et al., 1995). Five loci have so far been analyzed. Analyses of DNA extraction's from 90 faeces samples of 40 animals revealed that multiple extraction's are sometimes necessary to obtain DNA suitable for PCR. It was observed that occasionally only one of the two alleles present at a given locus of an individual was amplified. This is possibly due to the limited amount of DNA in the samples. Repeated PCRs were used to verify homozygosity found at a given locus.
Preliminary allele frequencies computed for each locus showed a high degree of polymorphism and heterozygosity, making these loci highly suitable for kinship studies. In the preliminary results for fourteen mother-child pairs and eight prospective fathers, the adult males of the community, up to seven males could be excluded as potential fathers per pair. Allele frequencies compared between all males and all females of the community show tendencies (statistically not significant) which support field observations that males in one community are more closely related to each other than are the females.
Reference: U. Gerloff, C.Schlotterer, K. Rassmann, I. Rambold, G. Hohmann, B. Fruth, & D. Tautz: Amplification of hypervariable simple sequence repeats (microsatellites) from excremental DNA of wild living bonobos (Pan paniscus), Mol. Ecol . 4: 515-518,1995
Eye growth and myopia in the marmoset (Callithrix jacchus jacchus).
B. Graham & S.J. Judge
University Laboratory of
Physiology
Parks Road, Oxford, OX1 3PT, England
The common marmoset is a small primate that can be bred readily in captivity. In our colony triplets are usually born at an average interval of c. 155 days and two infants are generally successfully reared. Average birth weight is c. 25-30gm. Weight doubles in c. 4 weeks and animals reach 90% of their adult weight by c. 40 weeks. Adult eyes are about 11mm long and visual acuity is high (up to 30 cycles/degree, Troilo, Howland &: Judge, 1993). The marmoset is therefore an attractive species for studies of primate visual development. We have used standard optometric methods (retinoscopy and A-scan ultrasonography) to study the normal postnatal growth of the marmoset eye, and the effects on ocular growth of visual deprivation and the wearing of spectacles.
Early in life marmosets, like most other species studied (including Homo sapiens), are hyperopic (long-sighted) with the degree of hyperopia rapidly decreasing so that by the age of 6 weeks animals are emmetropic (have little or no refractive error). It is important that we understand the visual and non-visual mechanisms responsible for this change and the subsequent close matching of the optics and the focal length of the eye (see Wallman, 1993). Monocular visual deprivation disrupts the normal pattern of eye growth and produces eyes that are myopic (short-sighted), largely due to an increase in the depth of the vitreous chamber (Troilo & Judge, 1993). Lens rearing produces effects that depend on the paradigm used: a lens worn over one eye from the age of four to eight weeks has little effect (at least the two eyes develop similarly) but if lenses of opposite powers are worn over the two eyes and each is alternately occluded (so that the animal has to use both eyes) the two eyes grow differentially to compensate (partially) for the lenses (Judge & Graham, 1995).
These observations, along with a very recently reported study of eye growth in macaques (Hung, Crawford & Smith, 1995) raise the question whether human ocular growth might also be affected by ocular demand.
References:
Hung, L.-F., Crawford, M.L.J. &Smith, E.L., Nature
Medicine 1: 761-765, 1995
Judge, S.J. and Graham, B., Journal of
Physiology 485.P: 27P, 1995
Troilo, D.B. and Judge, S.J., Vision Research 33:
1311-1324, 1993
Troilo, D.B., Howland, H.C. & Judge, S.J., Vision
Research 33: 1301-1310, 1993
Wallman, J., Progress in Retinal Research 12:
133-153, 1993
Evidence of health status of non-human primates by the analysis of urine, faeces and saliva.
Barbara Gregory, John Stewart, Alma Gower & Peter McAnulty
Pharmaco LSR, Eye
Suffolk IP23 7PX, UK
Safety evaluation studies with novel pharmaceuticals, agrochemical and other test materials are conducted in a variety of animal species. Among the non-rodents used are both old and new world primates. During the in-life phase of a toxicology study in primates, organ damage is routinely assessed by analysis of blood and urine for various biochemical markers. However, there are occasions when an invasive method such as blood sampling is not suitable, and non-invasive methods have to be considered. In the case of the marmoset, the animals small size precludes taking large blood samples, and therefore non-invasive techniques may be the only option.
Urine sampling is the main option for non-invasive investigations. This provides good information on kidney function and some aspects of nephrotoxicity, and there are also a number of markers available which provide evidence of damage to other organs and tissues. Analysis of urine samples also provides information on the route and rate of clearance of materials from the body and can also help to identify metabolites.
Analysis of faeces is largely limited to determination of the presence of occult blood which may indicate damage to the gastrointestinal tract. If infectious and parasitic diseases are a concern, then microscopic examination of faeces is of value. As with urine sampling, the clearance and metabolism of compounds from the body can often be determined from faecal analyses. Other techniques are seldom used. Saliva is an ultra filtrate of blood and contains a number of small molecules that can be analyzed. These include various steroids, and in particular progesterone can be analyzed in some species. Exhaled air can also be analyzed, but this is limited to detection of radioactive carbon dioxide in metabolic mass balance studies.
Endocrine approaches to non-invasive reproductive assessment based on urinary and faecal hormone analysis.
John Keith Hodges & Michael Heistermann
Department of
Reproductive Biology, German Primate Center
Kellnerweg 4, D-37077
Goettingen
Reliable information on reproductive status is necessary in many aspects of physiological and behavioural research as well as for effective animal management. Among captive primates, scientific and animal welfare, as well as practical considerations emphasise the importance of a non-invasive approach to reproductive assessment, whilst for field-based studies, there are no reasonable alternatives. Of the various possibilities, endocrine methods based on the analysis of hormones in urine and/or faeces are the most useful in that animal contact is avoided, yet accurate and reliable information on reproductive status can be provided. Until recently most studies were carried out with urine analysis, although there is now rapidly growing interest in the application of techniques based on measurement of hormones in faeces. Although at the moment faecal analysis is technically the more difficult, it has the advantage that samples can be relatively easily collected from animals in group situations and in the wild.
Given that sample collection itself is reasonably straightforward, the main factors determining the reliability and practicality of hormone analysis in excreta relate to validation of laboratory procedures and interpretation of data. Thus, an awareness of species differences in reproductive patterns and hormone metabolism together with knowledge of the temporal relationships between hormone excretion patterns and reproductive events is necessary for generation of meaningful results. Hormone assay procedures must also be relatively rapid and simple if they are to provide information of practical value and, where possible, should also be suitable for cross-species application.
The present paper provides an overview of currently available methodologies for urinary and faecal hormone analysis and discusses their application to monitoring reproductive status in both captive and free-ranging primates.
Report on a UK survey of housing husbandry and welfare provision for animals used in toxicology studies by the Toxicology and Welfare Working Group.
R Hubrecht
UFAl2V, 8 Hamilton Close, South Mimms
Potters Bar,
Herts, EN6 3QD
Background: The Toxicology and Welfare Working Group was formed on 15 December 1993 to address and promote welfare issues in regulatory toxicology. The Group consists of representatives from the British Toxicology Society (BTS), the Universities Federation for Animal Welfare (UFAW), the British Laboratory Animal Veterinary Association (BLAVA), the Association of British Pharmaceutical Industries (ABPI), the Laboratory Animal Science Association (LASA), the Institute of Animal Technology (IAT) and an observer from the Home Office. Regulatory Toxicology uses laboratory species in well-established study designs, and one of the objects of the Group is to review housing, husbandry and welfare provisions and to seek ways of improving animal welfare during the studies. To do this the group produced a questionnaire in May 1994 to be circulated to laboratories, either known or thought to be, carrying out toxicological studies. The Group hoped to document current housing and husbandry practice, and to determine the extent to which welfare innovations were being explored or implemented. The Group also wanted to find out whether specific innovations were successes or failures and to discover what improvements are possible under existing regulations.
Methods and Results: The Group identified 32 establishments known or thought to be carrying out regulatory toxicology studies; to each of them UFAW sent out questionnaires. Additionally some questionnaires were forwarded through professional bodies, so as to maintain the confidentiality of their mailing lists. The questionnaire asked for information on the types of animals used, cage/pen design and structure, the environment, and handling and the animals' response to it. The form also had sections for reports on the animal's behaviour, including incidence of aggressiveness and of specific behaviour categories for each species, which rnight indicate a poor ability to cope with the laboratory environment. There was also a 'catch all' question asking about any 'other abnormal behaviour. An open question asked wether any attempts had been made to reduce stress/distress and/or to provide enrichment. The respondents were asked whether they considered these initiatives to be either successes or failures.
The typical primate in regulatory toxicology is captive bred, and lives in solid walled cages with a mesh or barred front providing some visibility to animals in neighbouring cages. The most frequently used species are the cynomolgus macaque, an Old World monkey, and the common marmoset, a New World monkey. Old World primates apparently receive a different quality of housing than marrnosets. Old World primates are less likely to be given visual retreats, whilst marmosets are often provided with structures such as nest boxes. Old World primates are much more likely to be single-housed than marmosets, although there is a trend to use or assess group-housing or the use of connecting cages. (Members of the group have reported that since the questionnaire was completed there has been a significant move towards group-housinb Old World Primates.) Sites with single-housed Old World primates were more likely to report an incidence of abnormal behaviour, and the only site that housed marmosets singly was also the only one to report behavioural abnormalities. The feed of primates can broadly be split into three categories: the standard diet (provided daily, and usually readily accessible), supplementary foods (provided as an addition to the daily diet and which may vary from day to day), and forage (primarily provided to occupy the primates over an extended period). Marmosets receive a more varied standard diet than Old World primates and the diet is more likely to be given ad lib. There is not, however, much difference in the provision of supplementary foods, and over 50% of the respondents gave some form of foraging provision. Old World primates showed a low incidence of biting the wire or shell of the cage, self mutilation, cercling and weaving, while marmosets showed no biting of the wire/shell or self mutilation, and a very low incidence of cercling and weaving (when weaving did occur it was associated with single housing). A wide variety of procedures was used to improve the welfare Of both Old World monkeys and marmosets, and these met with a considerable degree of success. Primates used in regulatory toxicology are usually not handled for enrichment purposes, and most sites reported that their animals were either cautious of being caught or attempted to avoid capture. Some considered, however, that the response of the animals improved with time and that there was considerable variation between animals The animals' response to strangers was thought to be wary or curious in Old World monkeys and curious in marmosets. Aggression was an occasional problem. The importance of good cage design aras emphasised by the response to the question on injuries due to the cage. There were substantial differences between different designs, but unfortunately, none of the respondents specified the type of injury, the severity; or whether it was caused by the cage itself or by the type of social housing adopted within the cage.
Monitoring of uterine activity by telemetry in pregnant cynomolgus monkeys.
INRA/INSERM and Maternite Cochin-Port-Royal*
Paris, France
The macaque monkey has proved in the past to be an outstanding animal model for the study of uterine motility, essentially to understand its regulation and its dysfunction as occurs during the human pregnancy. The reasons why we monitor the uterine activity are several. We want to understand how the activation of excitable smooth muscle cells of the uterus, represented by their electromyograms, is converted into a contractile force and thereby into variations in intraamniotic pressure. We also want to know which specific pattern of uterine activity is expressed during the reproductive cycle. Finally, we want to evaluate the effect of molecules which have a potential interest to enhance or to inhibit the uterine activity. The major drawback of these studies was that up to now we had to restraint our animals under experience, to secure our catheters and electrodes implanted into the uterus. The restraining chair had been the solution for us but the discomfort was evident, so that chronic recordings could hardly extend more than 2 weeks. In the past 3 years, we started using a telemetric system to record the intra-uterine pressure. We present the result of a study that we completed two months ago, in monkey s implanted with a pressure transducer manufactured by DSI.
Protocol of the study: We wanted to know if the nomegestrol acetate progestomimetic treatment given in the last third of gestation, from day 120 and onward, had a protective effect against an uterotonic stimulus represented by an IM injection of 25 Fg of the PGE2 analog sulprostone. 3 animals were enrolled in the progestomimetic group, and 3 others in the placebo group. They were implanted, on day 120 of gestation and returned to the breeding colony with a tocolytic treatment for one week. On the next week, they were transferred to the recording room. They were monitored for 3 control days, they received on the fourth day a sulprostone injection, and they were monitored again for an additional round of 24 hours. They were then returned to their breeding cage and re-operated on day 140 to take off the pressure transducer.
Results of the study: Contractions recorded at high speed paper demonstrated that the telemetric system was sensitive enough to record uterine activity. In the placebo group, we identified clear nyctohemeral rhythms for uterine activity, with the lowest activity occurring at around midday. A single injection of sulprostone produced an increase in uterine activity lasting for about 12 hours. By contrast, in animals which were given progestomimetic treatment, the same dose of sulprostone produced only a limited increase of uterine activity that lasted at most 6 hours. It was therefore clear that progestomimetics have a protective effect on the responses of the uterus to an uterotonic challenge test. Comparison of the conventional and the telemetric methods of recording: In terms of measurement accuracy, the two methods are equivalent. The total duration in days during which we can record data is limited in the conventional method because of the discomfort of the animal on a chair. By contrast the telemetry system has an theoretical autonomy of 6 months, which is largely enough for the full gestation of a macaque. However, it is clear that if one want to place an indwelling catheter to inject drugs in parallel to the measurement of amniotic pressure, he looses the benefit of telemetry because he has to use again either a restraining device or the jacket system to secure the catheter. Higher cost was found with the telemetry system because one have to send back the pressure implant to the factory for refurbishment. Regarding animal welfare, the telemetry system was good and only in animals under 3.5 kg the pressure implant was less well tolerated as in larger animals. Regarding scientist welfare, we have been slaving for years for monkeys, to attenuate their discomfort while they were on a restraining chair, and to preserve our experiments at the same time. If today we have the possibility to carry out simultaneously lab experiments at the bench and in vivo recordings with monkeys, it is because telemetry has liberated us from some nasty occupations. From this very point of view, we rate as outstanding the telemetry system.
Vocal behaviour in marmoset family groups: remote monitoring with a microphone.
Bidda S. Jones
Departments of Psychology and Biology, Royal
Holloway
University of London, Egham, UK.
Present mailing address:
Research Animals Department, RSPCA, Causeway, Horsham, RH12 1HG, UK
An indication of the function of a particular vocalization is often apparent from the context in which the call is given. However, many of the vocalizations of New World monkeys do not, on first examination, appear to be associated with any specific behavioural context. Calls may be given regularly in many contexts throughout daily activity when callers are in relatively close range of other individuals. These contextually-generalised calls are often referred to as ' contact' or ' monitoring' calls, implying that they are involved in establishing and/or maintaining contact within and between social groups.
According to previous literature, the phee, twitter and trill calls of the common marmoset are given at a relatively high tate by all animals, in a variety of contexts without any obvious behavioural change other than antiphonal calling. The present investigation uses two different approaches to examine the use of these calls within marmoset family groups. The first approach looks at the extent to which the physical and social environment affects vocal behaviour by comparing general rates of calling across age classes and between two housing environments. The second relates the use of vocalizations to non-vocal behaviour and the spacing position of the vocalizer relative to other group members. This second method is made possible by the use of continuous audio and behavioural monitoring to provide information on the frequency, duration and sequence of both vocal and non-vocal behaviour.
In both housing environments the rate of phee and trill calling increased with increasing distance of the vocalizer from other group members. This pattern was especially pronounced for trill calls: as individuals moved away from other group members they appeared to compensate for the decrease in physical contact by an increase in vocal contact using trills. In contrast, twitter calls were given at their highest rate when the vocalizer was within 20 cm of another group member. Analysis of the behavioural context of calling indicated that phee calls were given predominantly by parents and subadults, and were not associated with any specific behavioural context. Trill calls were given at a high rate by all age classes across all behavioural categories. Twitter calls were given at their highest rate by adolescents and juveniles during social play. These results indicate that phee, twitter and trill calls are given in distinctly different circumstances and at differing rates according to the status of the vocalizer and the social and physical environment in which the call was given. These contextual differences provide valuable information about the function of particular vacalizations in the common marmoset.
Tracking primates in space and time: examples from nocturnal lemurs.
Peter M. Kappeler
AG Verhaltensforschung und Oekologie, Deutsches
Primatenzentrum
Kellnerweg 4, D-37077 Goettingen
In this paper, I review reasons and methods for tracking primates in space and time, focusing on questions and problems of field research. Such tracking of individually marked primates can yield detailed information on social organization, growth and development, population dynamics, social behaviour and entire life histories rarely obtained from anonymous animals. Because variation in reproductive success is ultimately crucial for many of these studies, tracking of genes across generations should also be included in these investigations. The present discussion focuses on pros and cons of other methods of individual tracking, however, including natural variation, manipulations of animals and the use of various gadgets, such as radio-collars and transponders. The best method for marking and recognizing individuals varies across species? primarily as a function of size, making general recommendations difficult. The simultaneous use of more than one method seems generally advisable, however. The potential payoffs of capturing and marking wild primates are illustrated with three examples from lemurs. By marking local populations of three species in Madagascar, it was possible to initiate several new studies that were previously not feasible, including the first systematic investigation of the behavioural ecology of inactivity in two nocturnal solitary species.
Experiences in the application of long-term heart rate telemetry in common marmosets kept undisturbed in their social environment.
J Kerl
Institut fur Anthropologie der Universitat
Goettingen
37130 Gleichen-Sennickerode, Germany
A data-collecting-system for simultaneous collecting of heart rate and behavioural data of up to eight small laboratory mammals is presented. All data are stored on a cartridge hard disk of an ATARI® PC for further processing. The data-logging program guarantees the storage of the heart rate data together with the corresponding behavioural data for each animal. During a preliminary study (from January 1995 to April 1995) with a couple of common marmosets (Callithrix jacchus) on the influence of two parameters (cage-size (3 stages) and cage-furnishing (2 stages)) of their environment on their heart rate and behaviour, the developed system proved to be a powerful tool. The influence of cage-size and cage furniture was reflected in the collected data and the first results will be presented.
Ultrasonographic documentation of uterine and fetal growth in a New World primate (Saguinus fuscicollis, Callitrichidae).
Irmgard Kuederling & Michael Heistermann
Department of
Reproductive Biology, German Primate Center
Kellnerweg 4, D-37077
Goettingen
For callitrichids a precise and immediate information on the stage of pregnancy and embryonic/fetal development is often required for research as well as colony management purposes. The classic procedures to diagnose and monitor pregnancy in callitrichids have been transabdominal palpation and determination of hormonal changes in plasma or urine. Neither approach, however, permits an assessment of embryonic development or viability of fetuses. In contrast ultrasonography can provide this information non-invasively and immediately as has been shown for the common marmoset (Callithrix jacchus, C.j.). The present study aimed at i) evaluating the suitability of ultrasonography to document the progress of pregnancy in another callitrichid species, the saddle back tamarin (Saguinus fuscicollis, S.f.) and i-i) examining whether embryonic loss is an explanation for the smaller litter size in S.f. compared to C.j. (predominantly twins in S.f., triplets in C.j.). Six S.f. females served as subjects. Uterine and fetal head dimensions were monitored once weekly in the unsedated, handled monkeys using an ESAOTE AU 530 real time ultrasound scanner with two probes of 7,5 and 10 MHz. Circulating and urinary progesterone concentrations were determined simultaneously to establish the day of conception and to monitor the hormonal changes throughout gestation. Pregnancies could reliably be diagnosed by ultrasound 2 to 3 weeks after conception by the appearance of a lumen in the middle of the uterus. Gestation sacs could be distinguished from day 35, the accurate number of embryos could be determined from day 50 and individual embryonic/fetal heartbeats were visible from day 58 of pregnancy. The width of the fetal skulls was measurable from day 80 of pregnancy. The average pregnancy length was 150 days, 5 females gave birth to twins, one to a singleton. Loss of single embryos was not observed in the study. These findings indicate that ultrasonography is a reliable non-invasive method to diagnose and monitor pregnancy also for S.f., suggesting that this method will be widely useful for small primates. Embryonic and fetal development was similar to that reported for C.j.. The reason for the difference in gestation length (150 days in S.f., 142 days in C.j.) may in part be due to a prolonged embryonic period in S.f. (heartbeats visible at day 58 in S.f., day 54 in C.j.). The lower number of offspring per litter in S.f. compared to C.j. is probably not caused by embryonic loss.
Semen collection for experimental use after natural ejaculation in the common marmoset, Callithrix jacchus.
Irmgard Kuederling, Jane M. Morrell & Penelope L. Nayudu
Department of Reproductive Biology, German Primate Center
Kellnerweg
4, D-37077 Goettingen
An important contribution to the conservation of endangered primate species is the development of assisted reproductive techniques such as gamete cryopreservation, in-vitro fertilization or artificial insemination. In our laboratory the marmoset monkey, Callithrix jacchus (C.j.) is used as a model species to develop these techniques for New World primates. A prerequisite for these studies is the reliable collection of functional sperm repeatedly from an individual. The collection of semen by vaginal washing after natural copulation's has the advantage of minimal stress to the animals and of exposure of the sperm to normally produced secretions from the male's accessory glands and the female's vagina. The purpose of the present study was to adapt and improve the procedure of vaginal washing in C.j. to allow routine semen collection at specific times. The first objective was to identify an easily discernible behavioural pattern which reliably indicated an ejaculation, the second objective was to test the influence of separation of the male for 1 to 6 days on the quality of the ejaculate. Six adult ovarectomized and five adult intact females were used for vaginal washings, the males of these 11 females served as donors of ejaculates. Mating at specific times were stimulated by separating the males from their females for a certain time period and subsequently either bringing together the mates again or introducing unfamiliar males to the females. Vaginal washings were performed on the unsedated females immediately after each observed mating. The seminal samples were analyzed for sperm concentration, the proportion of motile and the proportion of live sperm. The results have shown a) that the behaviour of the female who terminates a copulation by characteristic movements is indicative of an ejaculation with a reliability of 86.1%, b) that semen quality is not affected by separation of the male for several days, c) that the sperm samples are of high quality. The findings indicate that vaginal washing after natural copulation is a reliable, non-stressing and practical method for routinely collecting sperm samples from marmoset monkeys and possibly other New World primates for assisted reproductive techniques.
Non-invasive reproductive assessment in the female bonobo (Pan paniscus) by measurement of urinary and faecal steroids.
U. Moehle, M. Heistermann, H. Vervaecke*, L. van Elsacker* & J.K. Hodges
Department of Reproductive Biology, German Primate Center, Kellnerweg 4,
D-37077 Goettingen * Royal Zoological Society of Antwerp, Koningin Astridplein
26, B-2018 Antwerpen
The pygmy chimpanzee or bonobo has attracted considerable interest in a wide range of research areas due to its close phylogenetic relation to man. With an estimated population size of 10.000 individuals in the wild and only 100 animals in captivity the species is listed as endangered under Appendix I of CITES. Since 1988 bonobos have been included in an internationally coordinated breeding program which aims to promote a self-sustaining captive population. Despite extensive efforts in captive propagation, however, practically no information exists on tile reproductive endocrinology of the species and a reliable method for monitoring reproductive status has hitherto not been described. Knowledge in this area could be extremely valuable for improving natural breeding as well as providing the basis for artificial breeding by application of assisted reproductive technologies. Furthermore the ability to assess female reproductive status is a prerequisite for detailed studies into the reproductive biology of the species. Therefore the aims of this study were i) to describe hormonal changes throughout the menstrual cycle and pregnancy by measurement of steroid metabolites in urine ii) to demonstrate the reliability of faecal hormone analysis as an alternative non-invasive method to reproductive assessment suitable for application in the field and iii) to examine the temporal relationship between changes in perineal sex skin swelling and endocrine events.
Matched urine and faecal samples were collected daily along with menstrual records and swelling scores during 6 non-conception cycles (length: 3 1 -5 1 days) in four females. Patterns of excretion of urinary hormones revealed a 3-5 fold increase in oestrone conjugates (EIC) during the follicular phase (length: 17-40 days) reaching pre-ovulatory peak concentrations 0-4 days before a defined rise in levels of pregnanediol glucuronide (PdG) at the onset of the luteal phase (length: 11-15 days). Measurements of EIC, pregnanediol (Pd) and progesterone (P4) immunoreactivity in faeces revealed profiles qualitatively similar to their urinary counterparts, although however, there was higher day-to-day variability, particularly for EIC. Despite a time lag (0-1 days for E1C, 1-4 days for Pd and P4), length of the cycle and its component phases and occurrence of endocrine events can nevertheless be reliably monitored by quantifying faecal hormones. The periods of perineal swelling were relatively long (average: 68 % of cycle length) and their patterns differed considerably between individuals, with durations of maximal swelling ranging from 4-23 days (average: 40,5 % of cycle length). Onset of maximal tumescence occurred 1-16 days before the urinary E1C peak, while timing of detumescence was less variable, occuring 0-7 days after tlae E1C peak and 0-3 days following the PdG rise.
Steroid excretion was also monitored throughout three full-term pregnancies. While measurement of both urinary and faecal EIC was useful for early pregnancy detection, oestriol which was the major oestrogen in late pregnancy showed the most pronounced increase in hormone levels throughout gestation. Compared to oestrogens, progesterone metabolites in both urine and faeces showed a more modest increase. The present results demonstrate the reliability of urinary and faecal steroid analysis for reproductive assessment in female bonobos. Apart from its value in captive propagation, the availability of this technique offers new opportunities for investigating the relationship between perineal swelling patterns, mating behaviour and reproductive events. This in turn may help to better understand the evolution of human socio-sexuality.
Primate research: the view from the psychiatrists couch.
David Nutt
University of Bristol, Psychopharmacology Unit
School
of Medical Sciences, University Walk, Bristol BS8 1TD, U.K.
Primate research has proved one of the founding plattorms of biological psychiatry. The work of Harlow and colleagues provided the scientific underpinning for the development of a discipline of child psychiatry that emphasised care and nurturing. It also focused attention to the developmental processes that might go wrong and so contribute to adult psychopathology. Although the traumas produced in these early experiments are more extreme than those experienced by most psychiatric patients sadly even today psychiatrists still meet some patients in whom similarly severe chronic childhood trauma has resulted in permanent disability that normally proves very hard if not impossible to treat. Preliminary investigations of ours into the physiology of these patients has suggested patterns of excess cardiovascular reactivity and sleep disruption that may explain their excessive lability of mood and behaviour.
Primate work can be used to address psychiatric disorders other than those relating to deprivation stress. Some anxiety disordeors especially simple (specific) phobias may not exist in captive bred animals until learnt from parents; these offer a great opportunity to discover the biological mechanisms of conditioned anxiety which is a central feature of most anxiety disorders. Social phobia is another major psychiatric condition that contributes to alcohol and drug abuse as well; presumably the essential features are also displayed in primate groups.
The study of depression and drug abuse are already well established by primate researchers. The pioneering work of Sapolsky and others on the behavioural and endocrine correlates of social position in wild baboon colonies has excited clinicians by its parallels with human depression and the implications that the elevation of corticosteroids in this condition may lead to hippocampal damage. Drug abuse is a growing problem in which primate studies may have role wider than their present one of determining abuse liability by self administration rates. The work of Suomi and colleagues has begun to explore the genetic basis of impulsive behaviour and drinking by selectively breeding rhesus monkeys with high and low brain serotonin levels. Those with low serotonin show increased aggression; they have shorter life expectancies and more facial scars. In addition they show increased propensity to consume alcohol often drinking into unconsciousness when it is freely available. It would be of great interest to explore the role of other neurotransmitters in brain function using this approach. The other major psychiatric conditions are schizophrenia and the dementias. The former is hard to address using animal models although chronic amphetamine use has been shown to produce some behaviours that mimic aspects of the illness. Dementia is being studied using old animals and cholinergic lesions but alternative models would be helpful. Almost all psychiatric disorders are long lasting and have to be studied in a longitudinal fashion. Technical developments especially sophisticated telemetry appeal to biological psychiatrists as they offer the means of characterising the longitudinal course of interventions in primates. The potential to explore variables such as cognitive development sleep and other physiological measures along with behaviour and the added advantage of postmortem analysis of brain neurochemistry means primate research has a central role in biological psychiatric research.
Ultrasoumd monitoring of the ovarian cycle and pregnancy in the common marmoset (Callithrix jacchus).
Ann-Kathrin Oerke
Department of Reproductive Biology, German
Primate Center
Kellnerweg 4, D-37077 Goettingen
Ultrasonography is a non-invasive method for monitoring reproductive status, providing immediate results by direct visualization of reproductive organs. In captive primates ultrasound has been mainly applied to detection and monitoring of pregnancy, whilst its use for determining of ovarian changes throughout the cycle is limited. Furthermore, the studies reported to date have been restricted to Old World monkeys and virtually no information exists for New World primates. A project was therefore carried out to investigate the use of ultrasonography for monitoring the ovarian cycle and pregnancy in the common marmoset (Callithrix jacchus). Scans were performed transabdominally on non-sedated females using a 7.5 MHz probe for studies of pregnancy and a 10 MHz probe for examinations of ovaries during the cycle. Ovarian cycles were monitored by plasma progesterone and controlled by application of prostaglandin (PGF = day 0) to induce luteolysis.
In ultrasound images, the uterus of the non-pregnant marmoset appears as an ovoid organ measuring 6 mm x 8 mm in cross section. It is characterized by the central bright echo of the endometrium. Sonographic detection of pregnancy is possible between day 14 and 15 after ovulation by the appearance of a double endometrial echo. Diagnosis of pregnancy by ultrasound can be achieved earlier than by measurement of bioactive chorionic gonadotropin (CG). Embryonic development is indicated by an accumulation of intrauterine fluid causing an expansion of the uterine lumen and, after the first month of gestation, also in the external dimensions of the uterus. Detection of heart beat confirms embryonic viability by day 52, whilst visualization of heads around day 82 indicates the onset of the fetal period. Measurements of fetal skulls provide information on fetal age and expected time of birth.
Ovaries are usually located at the left and right cranial aspect of the uterus measuring approximately 5 mm x 6 mm in diameter. Follicles are seen as anechoic round structures and can be reliably measured when diameters exceed 1.5 mm. Corpora lutea (CLs) are represented by echogenic areas within the ovary and are difficult to measure. Monitoring of the ovarian cycle is possible by tracking growth of individual follicles between day 6 and 7 after PGF until the day before ovulation (follicle diameter 2.7 - 4.0 mm) when the preovulatory LH-surge is detected. Ovulation, which usually occurs on day 10 - 12 after PGF, is indicated by either complete disappearance of follicles or marked increase in internal follicular echogenicity. The post-ovulatory progesterone-rise confirms the development of CLs one day later. In the present study all pregnancies went to term and all cycles except an anovulatory one showed normal follicle and luteal phases. The results presented here indicate, that ultrasound is a suitable non-invasive method for monitoring reproductive status in female marmoset monkeys.
Monitoring EEG and sleep in marmosets by means of telemetry.
P.C. Pearce, H. Crofts*, S. Wilson*, E.A.M. Scott
Behaviour Group,
Medical Countermeasures, CBDE, Salisbury, UK
*Psychopharmacology Unit,
University of Bristol, UK
A method will be described which enables single channel EEG to be monitored by radiotelemetry in freely moving marmosets over long periods in their home cage environment. Without telemetry, restraint chairs, skull caps and trailing wires, all with associated stress factors, are necessary. Such remote monitoring of EEG enables concurrent testing of cognitive performance using a touch screen assembly, previously described (Crofts et al. J Psychopharmacol Suppl 9 1995 A12).
Techniques have been developed for the implantation of single channel biopotential transmitters (Data Sciences International) and to date 14 animals have undergone such procedures. This has enabled the recording of EEG to be collected during performance testing. These data were stored on optical disk and analysed by Fast Fourier Transform analysis at 1000Hz in order to produce a frequency spectrum. Methods have also been devised for the monitoring of EEG during sleep. Human studies indicate that sleep patterns are an important indicator of well-being and they can often be disrupted in conditions such as depression.
The advantages and disadvantages of the methods used will be discussed.
Activity monitoring in marmosets and tree shrews: a preliminary study.
S. Prowse, E. Fuchs*, A.P. Bowditch, L. Spreadbury, I.R. Scott & L.
Scott
CBDE, Porton Down, Salisbury, UK
*German Primate Center,
Kellnerweg 4, D-37077 Goettingen
A recently developed system has been used to monitor the activity of rats in their home cages over 24 hours (Scott and Hollands, Proceedings of the Fifth FELASA Symposium: 364-6, 1993). The aim of the present study was to validate the equipment for monitorinq activity in a wider variety of species.
In this preliminary study, spatial and temporal patterns of activity have been determined in pair-housed marmosets and singly housed tree shrews, using this novel, automated activity monitorinq device (Octec, Bracknell, Berks, UK). The system uses retrospective analysls of video tapes, and unlike many other activity monitoring systems does not necessitate subjects being maintained in strictly controlled environments. In the tree shrew study the system has been used to investigate changes in cage position related to inter-individual differences.
In the marmoset study, automated assessments of spontaneous activity and cage position correlated well with data on cage position collected by observation. High levels of activity were observed in both horizontal extension with foraging provision (A) and a vertical extension (C). Less activlty occurred in a low level caqe extension (B).
Non-invasive monitoring technologies and field studies.
Carel van Schaik
Biological Anthropology and Anatomy
Duke
University, Durham, USA
Because of their different aims and methods, field and laboratory studies of behavior have remained separate traditions. Field work usually tries to elucidate the adaptive significance of behavior, requires pristine environments, and generally takes a descriptive or comparative rather than an experimental approach. Laboratory work usually aims at manipulation of the conditions. Yet, the two approaches provide complementar,v information on behaviour and physiology of animals, which can be of great mutual benefit.
Laboratory work can improve the quality of field work. At a practical level, it can quickly provide information that would be all but impossible to collect observationally. At a conceptual level, it helps to determine the constraints on adaptation, which should greatly enrich behavioural ecology. Field work can also improve the quality of laboratory work. At a practical level, it helps to create optimum husbandry conditions, is essential for conservation, and helps to interpret the results of laboratory studies. At a conceptual level, a diversity of functions implies a diversity of mechanisms, so comparative field studies can provide the physiologist with search images. In this way, field studies can help in selecting the right question or the right animal model. The development of non-invasive monitoring techniques should enrich these interactions. Now, field work can help to determine the range of values of physiological parameters that could serve as a baseline values in the laboratory, and so settle long-standing questions (e.g. is chronic stress a pathological phenomenon or an adaptation to certain social or ecological conditions?).
It is not always easy to collect the relevant parameters non-invasively under field conditions. Techniques for collection and storage will be discussed. The need for exploratory pilot studies is stressed.
Marmoset telemetry: present applications and future highlights.
C.R. Schnell
Ciba-Pharma
Basel, Switzerland
Biotelemetry is the transmission of biologically-important information from an animal to a receiver without the use of connecting wires. The signals used are generally carried by radio waves.
The miniaturization of fully implantable telemetry devices has evolved considerably in the last 10 years. It is now possible to obtain information about marmosets freely moving in their home cages without disturbing the animal or influencing the results by the measuring procedure. Under such conditions it can be postulated that "true" physiological data can be obtained, which was not possible with more conventional methods. The list of biological parameters that have been telemetered in marmosets includes body temperature (CT), bio-potentials (ECG, EEG), blood pressure (BP), heart rate (HR) and body movement (ACT).
Consistent and stable diurnal rhythms were observed for all these parameters under standard conditions. However, BP, HR, CT, ECG and ACT were shown to be very sensitive to environmental changes as well as to social and non-social stress events. More recently EEG could be monitored during task performance testing. In addition, cardiovascular changes during pregnancy, parturition and postnatally were measured in marmosets for the first time. As we move into the future, telemetric devices will become more powerful and easier to use. Transmitters will be reduced in size with improved stability and greater use of multiplexing will be made. These new technological developments will certainly position telemetry solidly in the mainstream of biomedical research in the future.
Non-invasive methods of DNA analysis in conservation genetics and husbandry.
Franziska von Segesser & Robert Martin
University of
Zuerich
Studies of primate reproduction and behavior both in captivity and in the field are being increasingly combined with genetic investigation, for example to address issues in sociobiology and/or conservation. Genetic methods, including those directly focused on DNA, have traditionally employed blood samples, with the advantage that this yields abundant material permitting application of classical protein electrophoresis and mini satellite DNA-fingerprinting. These techniques are relatively unspecific and can be applied to a wide range of primate species. However, there are numerous drawbacks, particularly when studies of free-ranging primates are involved. Elaborate capture programs are needed to take blood samples, hence disrupting behavior, and collected samples must be quickly processed and refrigerated.
The polymerase chain reaction (PCR) has opened up possibilities for non-invasive sampling, although many studies continue to use blood samples. Specific DNA sequences can be amplified from traces of biological material, and it is (for example) possible to conduct analyses using a few hair follicles. DNA typing can then be conducted using either micro satellites (short, single-copy sequences of simple tandem repeats) or mitochondrial DNA. Several studies have been carried out using hair follicles from humans, apes, other non-human primates and certain other mammals. A more practical (but technically demanding) method for field studies is PCR amplification of specific DNA sequences from fecal samples, although this has so far been applied only to bears, baboons and bonobos. Because of the great specificity of the primers used to amplify specific DNA sequences with the PCR method, analyses have been successfully conducted with fecal samples despite the potentially disruptive presence of DNA from a wide variety of other sources in the faces of a given study species.
DNA amplification with the PCR technique has several advantages in addition to the potential for non-invasive sample collection (e.g. direct association of samples with observed individuals, possible avoidance of the need for export/import permits for samples, simplicity of extraction in the case of hair follicles), but it also has drawbacks. Most importantly, the specificity of the primers used for DNA amplification limits direct extension of the method from one species to another and the development of new primers is a laborious procedure.
DNA typing following non-invasive sampling will be illustrated with preliminary results from an ongoing conservation-oriented study of the Barbary macaque (Macca sylvanus).
Electroencephalography in the rhesus monkey - a non-invasive validation study.
F. Vogel, W.H. Bee, R. Korte, F. Schober* & W. Dimpfel*
CORNING
Hazleton GmbH, Muenster, Germany
*Pro Science Private Research Institute
GmbH, Linden, Germany
The brain function relates to electrical changes which can be monitored by recording the electroencephalogram (EEG). Quantitative EEG is one of the most sensitive non-invasive approaches for the detection of drug effects on the brain and modern computer assisted methods allow the continuous monitoring of drug action on the central nervous system. This study was designed to validate the EEG system CATEEM® in the Rhesus monkey (Macaca mulatta) using the well known reference compound diazepam. 6 monkeys (3M/3F) were used, sitting in a restraining chair and observed through a window. Diazepam was administered intravenously at a dosage of 0.2 mg/kg. Measurements were performed using 4 subcutaneous needle electrodes placed above the anterior region and the posterior region of the head with a reference electrode in the center of these electrodes. Data acquisition was performed using a 4 channel amplifier/digitizer unit (512 Hz/12 bit resolution) of the Pro Science CATEEM® system. Both the raw EEG signals and the calculated power spectra were displayed on-line on 2 screens in order to enable visual control. The experiments took place in a tandem room unit consisting of a darkened laboratory and a control room. Data were processed by Fast Fourier Transformation and absolute power values were obtained for 6 frequency bands: Delta (1.25 to 4.5 Hz), Theta (4.75 to 6.75 Hz), Alpha1 (7 to 9.5 Hz), Alpha2 (9.75 to 12.5 Hz), Beta1 (12.75 to 18.5 Hz) and Beta2 (18.75 to 35 Hz). EEGs were recorded over 15 minutes before drug administration and, with a delay of 1 minute, for 35 minutes following administration. With respect to the pre-drug reference period (100%), the following changes were observed: Delta increase, 242% anterior (a), 212% posterior (p), over 35 min; Beta1 increase, 132% a, 143% p; Beta2 increase, 123% a, 111% p; for 6 min; Alpha2 decrease, 78% a, 68% p, over 21 min. These results are in concordance with earlier findings in other animals and humans and indicate that the quantitative EEG measurement using the CATEEM® system is a reliable method for toxicity and pharmacodynamic studies in monkeys.
Determination of excreted corticosteroids as an indicator of adrenal function: application to monitoring stress.
Samuel K. Wasser
Division of Reproductive Endocrinology,
University of Washington
School of Medicine and Center for Wildlife
Conservation, Seattle WA, USA
Indicators of stress can contribute to basic research, animal management and wildlife conservation. They are pivotal to the understanding and improvement of animal well being and reproduction, as well as to monitoring impacts of social and ecological stressors, including habitat disturbance, on health and reproduction of wildlife species. However, unlike reproductive hormones, stress hormone secretion is relatively rapid in response to external stressors. This can confound stress hormone measures from blood owing to the potential stressfulness of blood withdrawal. Urine can provide an excellent alternative vehicle for measurement of stress hormones. Urine has been widely used to non-invasively monitor adrenal glucocorticoids in captive primates. Catecholamines (norepinephrine, epinephrine, dopamine) also can be measured in urine, but the latter use is less common. However, use of urinary stress measures has been limited by difficulties in collection, particularly in social and/or free-ranging environments.
Feces provides another non-invasive alternative for measuring glucocorticoids. No animal material is easier to collect than feces. Moreover, the long lag-time between secretion in blood and excretion in feces makes fecal hormone measures relatively unaffected by stressful conditions surrounding their acquisition. This lag-time may also make fecal stress hormone measures ideal indices of chronic stress.
Measurement of fecal stress hormones has been slowed by unique features of glucocorticoid excretion in feces. Fecal glucocorticoids often are more heavily metabolized than other fecal steroids. This makes them more difficult to extract and also less likely than other fecal steroids to cross-react with available assays for its respective secretagogue in blood. The magnitude of these constraints also appear to vary across species. I describe techniques to help overcome these problems, along with applications of fecal stress hormones in the field.
Cooperation of trained group-living crab-eating monkeys (Macaca fascicularis) in invasive and non-invasive research work without stress.
C. Welker, A. Klaiber
Primatenethologie
Universitat Kassel,
D-34109 Kassel
At the Primate Station of Kassel University crab-eating monkeys are housed in breeding groups. Caused by the strong hierarchical structure of the groups a controlled individual treatment seems to be impossible. Depending on the position in the rank hierarchy some animals get a higher portion of any offered food, others only limited amounts or nothing. This fact may be one important argument for individual housing of macaques for controlled medical treatment (in the case of diseases) or for pharmacological research. We already demonstrated that crab-eating monkeys cooperate in venipuncture, and that the need of invasive treatments are no arguments for single housing or for immobilizing the monkeys connected with stress caused either by the catching procedure, the chemical or physical immobilization, or the post-anaesthesia suffering.l
In addition, we demonstrated the possibility to treat group-living crab-eating monkeys individually with non-invasive methods. The animals were trained to enter a small cage one in a row, to eat a controlled amount of mixture, and to return afterwards to the rest of the group. Crab-eating monkeys cooperate in individual treatment. Without any hazard for the life of the infant even mothers with neonates participate in this procedure.2
We discuss the advantages of training and ask for the benefits of non-invasive versus invasive research work for the non-human and human primates participating in the studies. Beside these general discussions we report the high importance of individual differences and species differences for the success of training efforts.
1 Welker, C., Schafer-Witt, C., Voigt, K., Folia Primatol. 58:
112-117, 1992
2 Klaiber, A., Welker, C., Primate Report 1995: in
press