Hematological profile of captive bearded capuchin monkeys ( Sapajus libidinosus ) from Northeastern Brazil Perfil hematológico de macacos-prego ( Sapajus libidinosus ) do nordeste do Brasil

Bearded Capuchin or Black-striped Capuchin monkeys (Sapajus lidibinosus) are New World robust capuchin monkeys widely used in medical research. Few data are available concerning hematological reference values for these species, with no studies available from the Northeast region in Brazil. The aim of this study was to determine the hematological reference values for healthy bearded capuchin monkeys and to analyze the influence of sex and age factors. Blood samples were collected from 50 healthy bearded capuchin monkeys housed in captivity. These were analysed for total erythrocyte, hemoglobin, leukocyte and platelet count, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). When considering the age factor, significant differences were reported for total erythrocyte count, PCV, hemoglobin, total leucocytes, band neutrophils, eosinophils and lymphocytes (higher in juveniles). Significant sex-associated differences were noted for total erythrocyte count, PCV, hemoglobin (higher in males) and number of lymphocytes (higher in females).We have reported for the first time the hematological profile of bearded capuchin monkeys in captivity in the state of Paraíba, Brazil. These results can contribute for a better understanding of the normal physiology of capuchin monkeys, while demonstrating that factors such as sex and age influence hematological parameters should be taken into consideration in the hematological evaluation of this species.

ABSTRACT: Bearded Capuchin or Black-striped Capuchin monkeys (Sapajus lidibinosus) are New World robust capuchin monkeys widely used in medical research.Few data are available concerning hematological reference values for these species, with no studies available from the Northeast region in Brazil.The aim of this study was to determine the hematological reference values for healthy bearded capuchin monkeys and to analyze the influence of sex and age factors.Blood samples were collected from 50 healthy bearded capuchin monkeys housed in captivity.These were analysed for total erythrocyte, hemoglobin, leukocyte and platelet count, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).When considering the age factor, significant differences were reported for total erythrocyte count, PCV, hemoglobin, total leucocytes, band neutrophils, eosinophils and lymphocytes (higher in juveniles).Significant sex-associated differences were noted for total erythrocyte count, PCV, hemoglobin (higher in males) and number of lymphocytes (higher in females).We have reported for the first time the hematological profile of bearded capuchin monkeys in captivity in the state of Paraíba, Brazil.These results can contribute for a better understanding of the normal physiology of capuchin monkeys, while demonstrating that factors such as sex and age influence hematological parameters should be taken into consideration in the hematological evaluation of this species.Key words: Sapajus libidinosus, hematology, blood parameters, Reference interval.
Despite the scientific community's interest in working with non-human primates as experimental models, information about the reference values is scarce, and, mostly concerning Old World monkeys, such as rhesus monkeys, as well as chimpanzees (BUCHL, HOWARD, 1997;IHRIG et al., 2001).There are a few studies in the literature involving bearded capuchin monkeys, such as ROSNER and colleagues (1986), in Paraguay, RIVIELLO & WIRZ (2001), in Italy and NUNES and collaborators (2008), in Chile.However, to the best of our knowledge, there are no studies from the Northeastern region of Brazil.The one research concerning bearded capuchin monkeys in Brazil was carried out in the Southern region, more than 3000km from the Northeast (FLAIBAN, 2008).Considering regional differences related to climate (temperature and humidity), values obtained in that region may not be applicable to the reality of the Northeastern region.Thus, the aim of the present study was to determine the hematological profile of bearded capuchin monkeys maintained in captivity in the state of Paraiba, located in the Northeastern region of Brazil, and to search for possible variations in hematological values related to sex and age.

Animals
Blood samples were collected from 50 bearded capuchin monkeys (Sapajus lidibinosus) maintained in captivity in the Screening Center for Wild Animals of the Brazilian Institute of Environment and Renewable Natural Resources in the Town of Cabedelo/State of Paraíba, Brazil.Animals were considered healthy on the basis of a physical examination, body temperature, and hair condition.In addition, they lived under similar sanitary, nutritional and environmental conditions.Animals were grouphoused in clean indoor-outdoor primate facilities and fed twice a day with local fruits and vegetables, and a freely available water supply.
Total erythrocytes and leukocytes counts were made in a Neubauer chamber.Each one was counted twice, and then, the mean value was calculated.The PCV was obtained by micro centrifugation at 12,000rpm and the hemoglobin concentration by using the cyanometa hemoglobin method (THEML, 2004).Values for the MCV and MCHC indexes were then calculated.Blood smears were prepared, air dried and stained with a modified Romanowsky stain (Rapi-Diff II Stain Kit, Atom Scientific, Manchester), a staining protocol for the differential leukocytes count.After that, two blood smears were analyzed, with 100 cells were counted in each one of them, and the mean value was calculated.Platelet count was performed using the Fonio's method (FONIO, 1992).

Statistical analysis
The calculations of basic descriptive statistics, RI and their confidence interval limits (90%) were performed with the Reference Value Advisor V 2.1 software, a free set of macroinstructions to calculate RI with Microsoft Excel (GEFFRÉ et al., 2011).The RI were estimated following the ASVCP guidelines (FRIEDRICHS et al., 2012), namely those recommended for samples size (x) included in the interval 40 ≤ x < 120.
The SPSS software (Statistical Package for the Social Sciences, Chicago, IL, USA) version 20.0 was also used for statistical analysis.Data were subjected to the analysis of variance (univariate ANOVA) and the Fisher test was used to evaluate whether sex differences existed within each age category and whether age differences existed within each sex category.All values were expressed as mean and standard deviation (SD).In all statistical comparisons, P<0.05 was accepted as denoting a significant difference.

RESULTS
The hematological profile of 50 healthy bearded capuchin monkeys (Sapajus libidinosus), are present in table 1.In order to examine the impact of sex and age, we compared subgroups.Parameters with statistically significant differences between subgroups are reported in table 2.
Significant sex-associated differences were noted for the total erythrocyte count, PCV, hemoglobin (higher in males) and the number of lymphocytes (higher in females).The same differences were reported comparing adult males and adult females.Significant differences for hematological variables between juvenile males and juvenile females were reported for hemoglobin concentration (higher in juvenile males) (Table 2).We also found a tendency for higher values of RBC in juvenile males (P=0.06),compared to juvenile females.
Significant differences related to age were also reported for the total erythrocyte count, PCV, hemoglobin, total leucocytes, band neutrophils, eosinophils and lymphocytes (higher in juveniles).The same significant differences were seen between juvenile males and adult males (higher values in juvenile males) and between juvenile females and adult females (higher values in juvenile females) (Table 2).

Sex-associated differences
Regarding sex, differences were found for PCV, hemoglobin (higher in males) and number of lymphocytes (higher in females).Higher values of the total erythrocyte count in males were in accordance with previous studies performed in capuchin monkeys (ROSNER et al., 1986;WIRZ et al., 2008), as in other nonhuman primates (RAHAMAN et al., 1975;JAIN, 1993;VIDEAN et al., 2008).The lower erythrocyte value obtained in females can be due to the hormonal influence of estrogens.These hormones can have an inhibitory action on the erythropoiesis (BLOBEL, ORKIN, 1996;WIRZ et al., 2008).Menstrual cycle can also have an additional effect, as this species has a relatively short menstruating cycle (20 days) (CAROSI, et al., 1999).Higher hemoglobin values observed in males can be attributed to the fact that males have a greater muscle mass than females, thus a greater demand for hemoglobin to carry out the adequate supply of oxygen to the tissues (IHRIG, 2001)..20-406.00 164.93-376.51 143.82-186.25 353.33-397.93PCV -packed cell volume; SD-Standard deviations; Min-minimum; Max -Maximum; RI-Reference interval; LRL -lower reference limit; URL -upper reference limit; 90% CI -confidence interval around the limit.
Ferreira et al.
The higher number of circulating lymphocytes in females has not been previously described in these species (FLAIBAN et al., 2008;WIRZ et al., 2008).NUNN and collaborators (2000) observed that white blood cell counts were significantly higher in nonhuman primates whose females have more mating partners.These authors hypothesized that the high risk of sexually transmitted diseases is probably a major factor leading to systematic differences in the primate immune system (NUNN et al, 2000).Although, female capuchin monkeys do not exhibit a truly polygamic behavior, a female can occasionally accept other males during the fertile period.However, we could not rule out the possible effect of climatic environment, food and even local antigen stimulation, requiring further studies to confirm this fact.

Age-related differences
The total erythrocyte count, PCV, hemoglobin, total leucocytes, band neutrophils, eosinophils and lymphocytes were higher in juveniles than in adults.According to MANGRUM (1975), young animals have higher red cell values because the bones have greater erythropoietic activity.Regarding leukocytes, it was reported that young animals are more prone to excitement than adults, which may increase mobilization of lymphocytes by epinephrine released.This phenomenon may be accompanied by a slight to moderate increase of eosinophils too (POITOUT- BELISSENT, et al., 2000).WU et al. (2014) reported identical differences between juvenile and adult Tibetan monkeys.In 2008, studies performed in capuchin monkeys presented dissimilar results without obtaining statistical differences for these variables (FLAIBAN et al., 2008;WIRZ et al., 2008).Concerning the differences observed for band neutrophils, no similar results were reported in the literature.Given the fact that segmented neutrophils did not showed statistical difference between ages, the authors equate the possibility of differences observed for band neutrophils being a consequence of the statistical analysis done in a small sample with a 100 cell differential accounts and probably not clinically relevant.
Controversial results presented by different reports from different parts of the world support our hypothesis that local geographic specificities can influence hematological values and reinforce the idea that it is extremely important to make regional studies to better understand normal ranges, in order to contribute to the local improvement of species at risk.Furthermore, the differences between methods and laboratories also can influence the results.
In this study an anesthetic ketaminebased protocol was used for blood collection, consequently we cannot exclude its possible effect on the hematological values.Nevertheless, ketamine has been the drug of choice for anesthesia of primates due to its rapid effect, wide margin of safety, and relatively short duration effect (WINTERBORN, et al., 2008); therefore, the majority of studies that report hematological values on this species have also used the same drug for anesthetic purposes (WIRZ, 2008;TEIXEIRA, 2013;MOTA, 2016).
One of the limitations of the present study was the limited number of animals, which prevented a more specific approach, namely calculating hematologic RI by age and sex.Further studies with larger samples sizes are needed in order to completely characterize the bearded monkey hematological RI.
This research reported the first hematological profile of bearded capuchin monkeys (S. lidibinosus) in captivity in the state of Paraíba, Brazil.Regional climatic and dietary variations can possibly influence the hematologic profile.Consequently, it seems to be of considerable importance to carry out local studies in different regions.In conclusion, our results revealed that sex and age influence the hematological profile in bearded capuchin monkeys; therefore, such factors must be taken into account when interpreting a blood test on this species.