Haematological parameters in marmosets

ª Primate Center and Department of Physiological Sciences, Institute of Biology, University of Brasília, DF, 70910-900, Brazil.
b Veterinary Hospital, Department of Agronomy and Veterinary, University of Brasilia, DF, 70910-900, Brazil.

*Corresponding author: Tel.:55 061 3072294; fax: 55 061 2741251.
E-mail address: vanner@unb.br (V. Boere).
Correspondence should be sent to: (Department of Physiological Sciences, Institute of Biology, University of Brasilia, DF, 70910-900, Brazil; vanner@unb.br)

Summary

This study investigated the haematological state of captive marmosets submitted to a chronic psychogenic stress, an environmental enrichment, or a control condition. Blood samples were collected, one prior to and one after treatments, and were analyzed for the total erythrocyte (ERY x 106/ l) and leucocyte count (LEU x 103/ l). Peripheral blood smears were used to determine LEU cells differentiation. For all three groups, during the post-treatment phase, a significant increase in monocyte (MONO) count was observed, while lymphocyte (LYM) concentration decreased. Additionally, a higher MONO count was verified in the control group, compared to the stressed marmosets, suggesting a possible higher mobilization of the marginal immune compartment. Glucocorticoid resistance, common in marmosets, may have provided these primates with a physiological defense against the effects of chronic psychogenic stress. Finally, the enrichment procedures adopted failed to significantly enhance the marmosets' immune response. Specific conditions in which the animals were maintained are thought to have diminished attempts to improve the quality of their housing conditions.

Introduction

Stress, an adaptive mechanism of living organisms, is a highly variable phenomenon (Sapolsky, 1993). Captivity for wild animals characterizes a kind of source of stress due, for instance, to space restrictions, reduced social interactions, and decreased specie-specific behaviors. As a result, significant changes may be observed, including severe physiological disturbances, self-isolation, depression, stereotyped behaviors, and death (Sapolsky, 1993; Boere, 2001). Additionally, various pathologies are known to occur, such as diabetes, consumptive disorders, osteoporosis, arteriosclerosis, gastric-duodenal ulcers (Sapolsky, 1993; Johnson et al., 1996). Diminished immune response is one of the most frequently observed consequence of prolonged or intense stress (Leonard & Song, 1996; Sternberg & Gold, 1997; Schapiro et al., 2000).
In order to reduce these stress effects, spatial and social improvements should be implemented and designed to satisfy an animal's physical and behavioral requirements. Environmental enrichment (Boere, 2001), has been shown to improve various physiological parameters of primates, including weight gain, decreased need of surgical interventions, and increased reproductive turnover (reviewed in Poole, 1991; Boere, 2001), as well as immune response (Coe & Ershler, 2001). Periodical variations in housing conditions are thought to reduce the incidence of boredom due to captivity, hence improving the animals' well-being (Boere, 2001). Enrichment procedures have thus been increasingly implemented in species raised in captive conditions, particularly non-human primates.
Stress-related responses in non-human primates, and their ensuing adaptive value, can be evaluated by means of haematological indicators, namely leucocyte count and traffic (Boccia Et Al., 1992; Dhabhar Et Al., 1995; Leonard & Song, 1996). Leucocytes are intensely mobilized during acute stress via adrenal catecholamines and glucocorticoids. Chronic stress conditions(,) induce changes in leucocyte dynamics, though their mechanisms are not fully established, and have been poorly investigated in non-human primates (Schapiro Et Al., 2000; Coe & Ershler, 2001). The use of haematological indicators in vivo, nonetheless, enables a more comprehensive approach to evaluate the impact of psychogenic stress.
The marmoset, a neotropical anthropoid of the genus Callithrix, has been frequently employed in biomedicine and behavioral investigations (Smith et al., 2001). Callitrichids are known to be resistant to cortisol, although they demonstrate high basal levels of circulating glucocorticoids (Brandon et al., 1989). Indications of a hypercortisolemic condition is also absent in these small primates. Traits such as these are presumed to benefit species selection when susceptible to high risks of predation and living in unstable environments (Ferrari, 1993; Barros et al., 2002). Furthermore, a rapid habituation to changes in the environment typically depicts these primates' stress-related response, such as when confronted with a conspecific intruder (French & Inglett, 1991), in incidents of social subordination (Abbott et al., 1998), and during blood sampling restraints (Johnson et al., 1996). Glucocorticoid turnover is also more pronounced in marmosets, compared to Old World primates (Bahr et al., 2000). In spite of these particularities, marmosets' physiological response to psychogenic stress has remained poorly explored.
Accordingly, this study was designed to investigate the haematological response of captive-raised glucocorticoid resistant-to-glucocorticoid primate - the cerrado`s marmoset (Callithrix penicillata) - to stress and enrichment conditions. For this purpose, marmosets were submitted for nineteen consecutive days to either potential environmental enrichment manipulations or social isolations associated to 'aversive psychogenic' procedures.

Methods and Aims

Twenty-four captive-raised marmosets (C. penicillata), seventeen adults and seven juveniles, distributed into well established groups of heterosexual pairs or groups of three, served as subjects. Animals were maintained in indoor/outdoor enclosures (2 x 2 x 1.3 m) furnished with a suspended wood nest-box (0.3 x 0.3 x 0.6 m), feeding platform 1.5 m from the ground, two natural perches, tree trunk, and natural dry leaves covering the floor. Groups had olfactory, audible and visible contact with each other, although no physical contact was possible. All marmosets were fed once daily, at 6:30/7:30 to 17:30 h, with a mixture of various chopped fresh fruits and puppy dog chow, enriched with vitamins and boiled eggs. Water was available ad libitum.

Maintenance and testing occurred at the Primate Center, University of Brasília, Brazil, conforming to the regulations and with authorization of the Brazilian Institute of the Environment and Renewable Natural Resources - IBAMA (registration number 1/53/1999/000006-2). The study was approved by the Animals Ethics Committee of the Institute of Biology, University of Brasília, Brazil, and handling of marmosets was supervised by a veterinarian (V.B.).

Haematological data were collected during two experimental phases: pre- and post-treatments. During the pre-treatment phase, a blood sample was collected for each subject to determine the basal haematological profile (see below). Following blood sample collection procedures, marmosets were randomly assigned to one of three experimental groups: an environmental enrichment, psychogenic stress, or a control condition. Following treatments (post-treatment phase), a second blood sample was collected for each subject (see below).
The enriched group (group E: two adult males, three adult females, one juvenile male and one juvenile female) received daily permanent or intermittent housing improvements deemed essential for the environmental enrichment of captive primates (Crepeau & Newman, 1991; Poole, 1991; Boere, 2001). Table 1 describes the enrichment treatments and their order of presentation.

Table One: Description of the environmental enrichment procedures and order of presentation.

Marmosets submitted to the psychogenic stress condition (group S: four adult males, four adult females, one juvenile male and juvenile female) were individually housed in cages (1 x 0.5 x 0.7 m) located in a different building than the original colony room, containing a wood nest-box (0.15 x 0.15 x 0.3 m), two parallel wood perches (0.015 x 0.7 m), food and water containers. Cages were covered with an opaque plastic curtain to eliminate visual contact among subjects and with the surrounding environment, however, still allowing proper light and ventilation conditions. Each subject was submitted to a sequence of distinct daily stressor presentations, as psychogenic stress is induced essentially by novelty, lack of control and unpredictability (Sapolsky, 1993). Accordingly, order of presentation, type of stressor and time of day were randomly assigned, occurring within the marmosets' activity period (07:00-18:00 h), except for the 'involuntary awakening' (Table 2). Stressor presentations consisted of raising the curtain that surrounded the cage and exposing the subject to a pre-established stressor for sixty seconds. Sixty seconds interval is thought to be sufficient to induce a state of prolonged stress (Bercovitch et al., 1995). Following the sixty seconds exposure, the curtain was lowered and this same procedure held with other marmosets of the stressed group. Table 2 describes the stressors stimuli employed, some of which were adapted from previous investigations (Clarke, 1991; Crepeau & Newman, 1991; French & Inglett, 1991; Carey et al., 1992; Blanchard et al., 1998), while others were based on observations of an earlier pilot study held in our colony with a different group of marmosets.

Table Two: Description of psychological stress procedures and odrer of presentation

The control group (group C: three adult males, two adult female, two juvenile males) was exposed to a neutral stimulus (a brick discretely placed in one corner of the home cages' entrances) each day of the experiment. The neutral stimulus remained in the home cage throughout the day. It was rapidly substituted by a familiar observer every morning with one of two bricks allocated for each control group.
Blood Sample Collection and Analysis
Each subject was captured in a net, injected with ketamine hydrochloride for immobilization (10 mg/kg, intramuscularly; Vetaset, Baton Rouge, USA), and then placed into a small transportation cage for 1-3 min, until effective anesthesia. Subsequently, the marmoset was placed on a restraining apparatus, situated in a room adjacent to the colony, while 0.3 ml-0.8 ml of blood was collected from femoral vein into a disposable syringe. The sample was then rapidly transferred to an ethylenediaminetetra-acetic acid (EDTA) anticoagulant tube, and analyzed 2-4 h later. Blood was collected between 7:00-10:00 h.
Total cell count of (ERY), (LEU) and leucocyte subpopulations (i.e. lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), and segmented neutrophils (SEG) were evaluated manually using Wright-Giemsa stain. Cell counts were held in an automated electronic veterinarian blood cell counter (CC 550, Celm, São Paulo). Counting was performed by a experienced veterinarian at clinical laboratory diagnosis (G. R. P.).

Statistical analysis
The marmosets' haematological profile of the three experimental conditions were compared using the Kruskal-Wallis test for unrelated samples, followed by the Mann-Whitney test whenever significant values were obtained. Within each group, comparisons between the two experimental phases were held employing the Wilcoxon test. Differences between capture intervals were analyzed by Student's t-test. Analyses were based on two-tailed results, represented by mean standard error and the level of significance was set at P 0.05.

Results

Two female marmosets (one adult and one juvenile) died during psychological stress manipulations (data no showed). The results are the analysis of twenty and two marmosets resting. The captures procedures, prior to venepunctures, lasted 41.28 ± 28.28 seconds for the first sample, and 58.5 ± 40.9 seconds for the second. Capture intervals did not vary significantly between the first and second venepuncture (P = 0.091).

Comparisons among the groups E, S and C, prior to treatments, indicated similar erythrocytes ERY (P 0.56), total leucocytes LEU (P 0.32), lymphocytes LYM (P 0.61), monocytes MONO (P 0.92), segmented neutrophils SEG (P 0.46) and eosinophils EOS (P 0.39) concentrations among them.
Following tre

atments, however, MONO concentrations were shown to significantly differ between groups ( 2 =6.093, d.f. = 2, P 0.05). Post-hoc analysis showed that group S presented less MONO, when compared to group C (nC = 6, nS = 9, Z = -2.659, P 0.008). Such difference was not observed between group C and group E (nC = 6, nE = 7, Z = -1.502, P 0.13), neither between group S and group E (nE = 7, nS = 9, Z = -0.106, P 0.91) (Fig. 1). Concentrations of the remaining haematological parameters after treatments were not shown to significantly differ from those observed during the pre-treatment phase (ERY: P 0.79, total LEU: P 0.41, LYM: P 0.51, and SEG: P 0.86, EOS: P 0.51).
Analysis of the haematological profile after treatments for each group, revealed that subject's MONO concentrations increased significantly, compared to the pre-treatment phase (group C: Z = -2.023, P 0.04; group E: Z = -2.201, P 0.03; group S: Z = -2.666, P 0.01; Fig. 1). Conversely, a significant decrease in LYM concentration was observed following treatments in all three groups (group C: Z = -2.023, P 0.05; group E: Z = -2.366, P 0.02; group S: Z = -2.666, P 0.01; Fig. 1).

The erythrocytes (group C: P 0.68; group E: P 0.73; group S: P 0.76), total LEU (group C: P 0.08; group E: P 0.12; group S: P 0.21), and EOS concentrations (group C: P 0.31; group E: P 0.56; group S: P 0.15) were not found to significantly differ in the three groups after treatments. SEG was not significantly different in the group C and S (group C: P 0.68; group S: P 0.10), but in the group E there was a tendency to increase after treatment (P 0.06).

Discussion

The three experimental conditions (i.e. stress, environmental enrichment, control) were found to alter the cerrado`s marmosets' (C. penicillata) haematological profile. The effects observed following treatments were, in fact, associated to distinct blood cell types that constitute the cell-mediated immune response.

Accordingly, a significant decrease in LYM content, while an increase in MONO concentration was observed in the peripheral blood of all three groups after treatments. Similarly, several studies have been reported that stressed animals demonstrate a reduction in LYM number and proliferation of MONO (Leonard & Song, 1996; Connor et al., 1997).
Nonetheless, marmosets' immune response to the experimental manipulations differed between LYM and MONO. The decrease in LYM availability following treatments did not differ between groups. On the other hand, a smaller MONO mobilization was observed in the peripheral blood of the stress group, compared to the control group. Consequently, MONO redistribution in our subjects may be due to the nature of the experimental manipulation. Isolation and constant exposure to unpredictable novel events (i.e. stress condition) seem to have had a greater impact on the haematological response of cerrado's marmosets. Additionally, MONO are known to be distributed in the blood and other immune compartments (i.e. marginal) in humans. Dhabhar et al. (1995) suggested a marginal compartmentalization in rats, reporting that physical restraint stress in rats, modified the number and percentage of LYM and SEG in the peripheral blood, returning to normal basal levels following a 3 h interval. The existence of a MONO marginal cellular compartment, however, has not been reported in other Neotropical primate species, although glucocorticoid administration was shown to induce monocytosis in dogs (Jain., 2000). Accordingly, a marginal compartment is suggested to also be present in nonhuman primates as marmosets.

Contrarily, total LEU, EOS, and SEG concentration were found not to differ between the pre- and post-treatment phases, nor among groups. In spite of this, we do not have a reasonable explanation for the tendency to increment of SEG in group E after treatment. We can speculate a tendency to higher phagocytosis activity of neutrophils in group E then group C and S, maybe in consequence to high blood lost in the venepuncture. In fact, sometimes subjects of group E presented local haematomes after venepuncture.
The erythrocytes concentration was also not altered by any of the experimental manipulations. A change in MONO proportion was observed, however, probably due to the action of adrenal glucocorticoids. These, in turn, influence mitogenesis, chemical communication and adhesiveness of blood cells involved in the immune response (Leonard & Song, 1996; Turnbull & Rivier, 1999; Bauer et al., 2001). Furthermore, activation of the autonomic nervous system, with a consequent acute release of catecholamines into the peripheral blood, modulates the LEU response via receptors located on blood vessels and lymphatic organs (Bauer et al., 2001). Although many factors are known to alter the immune response, the nature of the stressor seems to influence the level and type of LEU mobilization. For instance, psychogenic stress alters the distribution of immune cell sub-types in humans (Uchakin et al., 2001). An increase in LYM cytolysis has also been shown in rhesus monkeys submitted to psychogenic stress (i.e. social isolation), as opposed to physical stressors (i.e. laparoscopy) (Lemieux et al., 1996).
Importantly, in spite of the decrease in LYM, its concentrations, regardless of treatment, remained above the minimum physiological level of 3 x 109/L reported for common marmosets (Hawkey et al., 1982). Therefore, LYM decrease was not sufficient to induce pathologic lymphopaenia, even in subjects submitted to severe stress. Glucocorticoid resistance, a physiological condition of high basal plasmatic cortisol, without pathological signs (Brandon et al., 1989) commonly observed in genus Callithrix (Johnson et al., 1996), may have influenced this result. Mitosis of cells precursors of LYM and MONO couldn't be inhibited because the receptor hormone attachment is weak in glucocorticoid resistance primates (Scammel et al., 2001). However further studies are necessary to test this assumption.

Enrichment procedures presently adopted failed to significantly enhance the marmosets' immune response, compared to the control group. Specific conditions in which the animals were maintained may have minimized improvements made during this experimental manipulation. Marmosets employed in this study were distributed in socially compatible groups and housed in enclosures furnished with substrates deemed appropriate for an enriched environment for these simians. In addition, the Primate Center is located within an forested protected area. Marmosets were therefore exposed to the same conditions present in their natural habitat, thus creating an added factor to their well-being.
Lastly, blood cell counts observed prior to treatments were similar to those previously reported for cerrado`s marmosets (Diniz, 1997) and common marmosets (C. jacchus) (Hawkey et al., 1982; Yarbrough et al., 1984).

Studies analyzing haematological modifications, incorporating diagnostic and prognostic data, may be useful to increase reproductive rates, survival and well-being of captive-raised animals. Further detailed investigations on the dynamics of LEU and its sub-types, as well as on antibodies and cytokine production, are deemed necessary. This is especially true since, compared to studies on the effects of environmental enrichment procedures, biobehavioral investigations analyzing the effects of psychogenic stress in neotropical primates are presently scarce.

Conclusions

In short, we conclude that the psychological stress diminishes the immune response of LYM and MONO in cerrado's marmoset, but high frequency of innovations in a environmental enrichment program seems not to improve the immune response. Despite the severe stressors, putative glucocorticoid-resistance may confer an adaptive immune advantage of marmosets towards the frequent and unpredictable challenges present in captivity.

Acknowledgments

We are grateful to Mr. R. Oliveira and Mr. W. Vargas for the care of animals. We thank to Dr I. O. Silva and Dr M. Barros to helpful comments on this paper. The investigation was supported CAPES/PICDT fellowship to V.B., and PIBIC/DPP/CNPq fellowship to G. C. and T. P. FINATEC/DF was a partial financial supporter.

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Day*	Enrichment	Description
1	Replacement	A wooden platform for sun bathing and 2 perches were placed at a medium height
2	Polyvinyl chloride tube	Tube (0.3m x 0.10m) was hung from the cage's wire mesh ceiling.
3	Rope	Rope was hung from the cage's wire-mesh ceiling
4 and 11	Food variations	Transparent plastic food container, containing small pieces of fresh fruit (strawberry and peach) unknown to the subjects or pieces of gelatin, was attached outside the cage's front wire mesh.
5	Larvae 1	Transparent plastic food container, containing Tenebrio mollitor larvae was attached outside the cage's front wire mesh.
6	Trapeze	Wood perch (0.3m x 0.03m) was hung from the cage's wire mesh ceiling.
7 and 14	Food bin	Parakeet and parrot food bin (Trill, Waltham Effem, Brazil) were hung from the cage's wire mesh ceiling.
8 and 20	Exploratory apparatus	Open transparent plastic bottle (600 ml) was placed upon the cage's floor, containing Tenebrio mollitor larvae
9 and 17	Cage rearrangement	The 2 perches and the polyvinyl chloride tube were placed at a greater height
10 and 16	Fishing	Transparent plastic bottle (600 ml) with a small window opening and containing water and alevines, was attacehd outside the cage's front wire mesh
12	Cardboard tubes	4 cardboard tubes (0.3m x 0.05m) placed upon the cage's floor
13	Larvae 2	Transparent plastic food container, containing Tenebrio mollitor larvae covered with dry natural leaves, was attacehd outside the cage's wire mesh ceiling.
15	Sugar cane	peeled sugar cane stick was hung from the cage's wire mesh ceiling
18	Bird's nest	Commercial bird's nest was place dinside the cage at height, containing 3 hard boiled quail's eggs
19	Gum tree	Hollow piece of bamboo, containing honey, placed vertcially in the cage.
		* Order of presentation for each day of the enrichment procedures

Day*	Stress	Description
1	Redistribution	Subjects were placed individually in cages with small nest box, perches and opaque plastic curtain around the cage
2 and 14	Gloves	Exposure to a pair of leather gloves normally used for restraint about 0.2 m from the front of the cage.
3	Lab coat	Exposure to white lab coat on back of a chair about 0.2 m from the front of the cage
4 and 16	Unfamiliar Observer	Unfamiliar human observer stood before the cage, staring at the subject
5	Involuntary awakening	Subject awakened during nightly rest at 22.00 h by turning on lights for 10 minutes
6	Black curtain	Black curtain placed over subject's cage
7 and 17	Net	Exposure to net usually used for capturing marmosets about 0.2 m from the front of the cage.
8	Alarm clock	Exposure to alarm clock about 0.2 m from the front of the cage until the end of the alarm sound
9 and 18	Dragging	Cage was dragged to another place and subsequently moved back to original position
10	Striking	Striking of the window and door of the isolation room
11	Light spot	Light spot from lantern shone through opaque plastic curtain surrounding cage
12 and 20	Fire	Exposure to metal tray with 10 lighted candles on bench 0.5 m from the front of the cage.
13	Observer in lab coat	Exposure to unfamiliar human observer wearing a human lab coat
15	Light flash	Intermittent flashing red light from a bicycle at 0.2 m from cage
19	Sham feeding	Food container placed at usual hour without any food in
		* Order of presentation for each day of the stress procedures

Effects of novelty, isolation stress, and environmental enrichment on some haematological parameters in marmosets (Callithrix penicillata).