Introduction

Neotropical primates (NP; suborder Platyrrhini) have a wide geographical distribution, that includes the Southern part of Mexico, Central and South America, and are represented by a huge variety of species, with a remarkable diversification in their biology, life history and behavior (^{Rosenberger & Hartwig, 2001}). In Costa Rica, four different species of NP are distributed, including mantled howler monkey (Alouatta palliata), white-faced capuchin monkey (Cebus imitator), spider monkey (Ateles geoffroyi) and squirrel monkey (Saimiri oerstedii). Three of these are at risk of extinction (mantled howler, spider and squirrel monkeys), and one (white-faced capuchin monkey) is a threatened or vulnerable species, according to the Costa Rican legislation (Decree No. 2635) and the International Union for Conservation of Nature (IUCN) (^{Cuarón et al., 2008}; ^{MINAE, 1997}; ^{Wong et al., 2008}). At the same time, these three species are included in appendix 1 of the Convention on International Trade of Endangered Species of Wild Fauna and Flora (^{CITES, 2021}) and the white-faced capuchin monkey has yet to be classified.

Costa Rica is considered one of the countries with the greatest diversity in the world and has a wide variety of microclimates and life zones that favor its notable levels of biodiversity. However, the biological diversity of the country is currently threatened by the illegal extraction of wood, the conversion of forests for agricultural activities or by tourist and real estate development, which strongly affect the connectivity between forest patches (^{Calvo-Obando & Ortis-Malavassi, 2012}). As natural environments are transformed, through anthropogenic activities, the natural habitat of the NP is either reduced in size or fragmented, directly affecting them. Furthermore, as monkey' populations concentrate, troops decrease in number of individuals, causing a reduction of the genetic pool, an increase in stress, promoting immunosuppression and the eventual transmission of diseases (^{Gilbert & Dodds, 1987}).

Given this, counting on reference data about health status, genetic variety, biological characteristics, among other variables, are a priority in conservation programs. Hematological reference values are a valuable tool to help determine the health status of wild populations (^{Takeshita et al., 2011}). This data may be used as comparison patterns between populations that are distributed in landscapes of different forest types or captive environments. And, in addition, may also be useful for reintroduction and management programs (^{Sánchez-Sarmiento et al., 2014}).

Health studies and the establishment of normal blood values research have increased over the last decades, providing an important amount of information on normal parameters for several species and are valuable tools for assessing and monitoring the health and resilience of wild populations (^{Maceda-Veiga et al., 2015}). This information helps identify potential effects of diseases, pollutants, or other changing environmental conditions that would be difficult to understand without knowledge of normal species variations in blood variables. These kinds of studies usually include variables that are used in veterinary medicine and are assumed to have the same utility in wild populations (^{Arguedas et al., 2021}).

Health status evaluations, taking in consideration hematological parameters, have been done for several species of NP and are used as a reference for the comparisons between or within species (^{García-Feria et al., 2017}). Nevertheless, there are few studies published on the hematology of NP in natural environments from Central America, due to lack of project financing or even research interest. In this study, we evaluate some hematological analytes of mantled howler monkeys (Alouatta palliata) and white-faced capuchin monkeys (Cebus imitator) in the wild coming from natural environments from Costa Rica, by analyzing such parameters and comparing the results obtained between genders and the populations sampled.

Materials and methods

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page. All procedures for trapping and handling NP were approved by bioethics and welfare permits given by the University of Costa Rica and the capture permits given by SINAC (Resolution No. 27 2013 SINAC).

Animal Sampling: During 2014, mantled howler and white-faced capuchin monkeys from Costa Rica were captured (Fig. 1). Capture was done using 3.3-1.1 mg/kg Zoletil 50® (Virbac) or Ketamine 10 % (Bremer Pharma GmhH, 5-20 mg/kg) with Xylazine 2 % (Virbac, 0.5-2.0 mg/kg) (^{Glander et al., 1991}; ^{Varela, 2006}; ^{West et al., 2007}) in darts (PneuDart. Inc, Type P, 1cc) with a compressed gas rifle (PneuDart. Inc, model X-Caliber Gauged CO2). Lateral faces of triceps and femoral quadriceps were the target of injection (^{Glander et al., 1991}). Vital signs, including heart rate, respiratory rate, body temperature, oximetry, and mucous membrane color were observed during immobilization. A physical examination was performed on the animals looking for abnormalities that could indicate illness. When animals were fully recovered from anesthesia, they were released. General body condition of the individual was evaluated using a five-point scale.

Fig. 1 Location of field sites in Costa Rica: A. Hermosa Beach. B. Nicoya. C. Curú. D. San Lucas Island. E. Manuel Antonio. 

Laboratory Analysis: Once the individual was anesthetized, 2-4 ml blood samples were extracted from the femoral vein using the Vacutainer® system. Samples were collected in EDTA tubes and transported to the laboratory at 4 ºC. At the lab, the following hematological parameters were evaluated using Sysmex KX 21 equipment, (Sysmex Corporation, Japan): red blood cell count (RBC, 10⁶/mm³), hemoglobin (Hb, g/dl), hematocrit (Hct, %), mean corpuscular volume (MCV, fl), mean corpuscular hemoglobin (MCH, pg), mean corpuscular hemoglobin concentration (MCCH, g/l), platelet count (PLT, 10³/ mm³), and white blood cell count (WBC, 10³/ mm³).

Statistical analysis: For each parameter measured, Mann-Whitney test was performed, depending on the normality of data a Wilcoxon and t student tests were performed in order to determine if there were significant differences between females and males from each species sampled. Also, for mantled howler monkeys, an ANOVA tests was performed to determine if there are significant differences between sampled populations. Statistical analysis was done in R program (^{R Core Team, 2016}) with a < 0.05 significance level.

Results

A total of 35 (17 females and 18 males) mantled howler monkeys and 12 (5 females and 7 males) white-faced capuchin monkeys were captured in seven localities from Costa Rica, five localities for mantled howler monkeys, including: San Lucas island (9°56'24.28'' N & 84°54'15.24'' W), Nicoya (10°08'40'' N & 85°27'22'' W), Hermosa beach (4°46'00'' N & 85°16'00'' W), Manuel Antonio (9°22'32'' N & 84°08'09'' W), Curú (9°47'35.55'' N & 84°55'32.49'' W), and 2 localities for white-faced capuchin monkeys, including Manuel Antonio (9°22'32'' N & 84°08'09'' W) and Curú (9°47'35.55'' N & 84°55'32.49'' W) (Fig. 1), (Table 1). All individuals appeared to be healthy and did not present any sign of disease.

For mantled howler monkeys, the measures of Hemoglobin (Hb), Hematocrit (Htc), red blood cells count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Platelets (PLT) and White blood cell count (WBC) were higher in males than females. For white-faced capuchin monkeys, values of Hb, Htc, RBC, PLT and WBC were also higher for males than females, but MCV, and MCH values were similar for both. Nevertheless, no significant differences were found between males and females for any of the parameters analyzed in both species (Table 2).

Significant differences for mantled howler monkeys and in all the parameters evaluated were determined, except for PLT. Individuals from the Nicoya locality were the individuals that presented the highest scored hematological values. At Curú just one individual was captured, so it was not included in the analysis (Table 3).

For the other hematological parameters evaluated, 3/35 (8.6 %) of mantled howler monkeys and 3/12 (25 %) of white-faced capuchin monkeys presented WBC counts above 10 400/mm³. In the same way, 16/35 (45.7 %) of mantled howler monkeys and 2/12 (16.7 %) of white-faced capuchin monkeys presented PLT values above 250 000/mm³, and one howler monkey presented an abnormally low value of platelets (17 300/mm³).

Discussion

Actual information related to the biology, ecology, health and diseases supports and facilitates programs related to the management, medicine and conservation of wildlife populations (^{Varela, 2006}). Changes in landscape, habitat fragmentation and the anthropogenic activities directly affect the wellness of NP, and this may be reflected in the hematological parameters. In order to obtain and compare the reference patterns of hematological parameters for mantled howler and white-faced capuchin monkeys from Costa Rica, free-living individuals of both species were captured at different geographical regions of Costa Rica.

The results of this study are the first hematological parameters reported for mantled howler monkeys (A. palliata) in Costa Rica. When these results are compared with values published for free living A palliata individuals from Tabasco, Mexico (^{Canales-Espinosa et al., 2015}), and for A. pigra individuals from Balancán, México (^{García-Feria et al., 2017}), differences in the averages of parameters measured are evident. Nevertheless, when our results are compared with the ones reported for Mexican howler monkeys, values obtained are within the maximal and minimal ranges described (^{Canales-Espinosa et al., 2015}).

For white-faced capuchin monkeys, the hematological parameters obtained in this work are similar to those reported by ^{Bernal-Valle et al (2020}), where 16 out of 25 individuals also from Costa Rica. The median value for HCT reported by ^{Bernal-Valle et al (2020}) is 40.5 % for males and 37.5 % for females, compared with the values of 43.5 and 38.6 % obtained in this work. In the same way, the hemoglobin values obtained for males and females in both studies are similar (12.7 g/dl and 10.5 g/dl compared with 12 g/dl and 11.4 g/dl respectively). An important difference between both studies lies in the values obtained for white blood cells, where ^{Bernal-Valle (2020}) reports median concentrations of 5 700 WBC/ mm³ in females, compared with the values of 8 900 WBC/mm³ obtained in the present study. It is important to highlight those average values obtained in this study for white-faced capuchin monkeys are similar to the ones reported by ^{Larsson et al (1999}) in Brazil. Nonetheless, since different hematological analyzers were used in all the studies mentioned, it is important to always take this bias in account when comparing hematological values.

There are no significant differences between genders for any of the parameters evaluated in both species of monkeys studied. However, this data contrasts with those reported by other researchers, where values observed for males are greater than those reported for females in most of the parameters analyzed (^{Flaiban et al., 2009}; ^{García-Feria et al., 2017}; ^{Kagira et al., 2007}; ^{Larsson et al., 1999}; ^{Moore, 2000}; ^{Sánchez-Sarmiento et al., 2014}; ^{Takeshita et al., 2011}; ^{Wirz et al., 2008}). This might be due, principally, to hormonal influence, since testosterone stimulates erythropoiesis, while estrogen inhibits it (^{Kagira et al., 2007}; ^{Wirz et al., 2008}) or to effects of the menstrual cycle, pregnancy period and breastfeeding.

Differences between population may have occurred because of the excitement and tension that wild animals suffer during its capture, the individual may show signs of acute stress, known as “alarm reaction,'' which may be the cause of an increase in the values of various parameters, including RBC, Hb, HCT, MCV, MCH, PLT and WBC (^{Thrall, 2004}). In normal conditions, stress is an adaptive stimulus for a changing environment (^{Fowler, 1978}) and causes beneficial metabolic changes for the protection against external aggressions (^{Cubas, 2007}). On the other hand, other factors may cause differences in some hematological parameters, for example, some degree of dehydration can be a reason to have higher HCT, Hb and RBC, which can be suspected from animal coming from drier regions such as dry forest environments, like Nicoya.

Some of the individuals that had WBC higher than 10 000/mm³, can be because of an inflammatory process, bacterial infection or a severe stress reaction during capture (^{Thrall, 2004}), further studies evaluating leukocyte response in these species have to be done in order to confirm these changes.

Apparent thrombocytosis (>250 000/mm³) here found in 16 howler monkeys and 2 capuchin monkeys, one cause of this, is reactive thrombocytosis, which is driven by elevated endogenous levels of thrombopoietin, interleukin-6, other cytokines, or catecholamines that may be produced in inflammatory, infectious, or neoplastic conditions or in situations of stress (^{Schafer, 2004}). Another finding was that one individual of howler monkey, presented an abnormally low platelet count, this may be, it can be due several reasons, for example, infectious agents or immune-mediated disorders (^{Thrall, 2004}), or incidental, like platelet clumping due to venipuncture, which can be this case, since this particular individual, showed values within the normal ranges in the sample.

The difference in the hematological parameters evaluated might also be associated with other factors, including the type of feeding, diet and environmental factors among others. Health evaluations of free-ranging primates provides important information about the incidence and prevalence of infectious diseases, as well as their general health status and allows elucidating variations both within and between populations (^{Riviello & Wirz, 2001}; ^{Takeshita et al., 2011}).

Health evaluations should not be limited to epidemiological surveillance and should also consider animals' clinical and physiological parameters (^{Sánchez-Sarmiento et al., 2014}). For example, normal hematological values are of special value in Costa Rican primates, since they frequently suffer from car accidents and electrocutions, Several species of Costa Rican wildlife are suffering terrible injuries along the power lines and transformers in rural areas of the country., and hematological parameters can be a way to predict (up to a point) the possible outcome of the patients and to be able to treat the animal in a more comprehensive way (^{Sánchez-Murillo & Arguedas, 2021}). Besides, nonhuman primates exhibit a pronounced stress when they are confined (^{Suleman et al., 2000}), and require a long period of time for adapting to captivity. Thus, hematological parameters may take even months to stabilize (^{Kagira et al., 2007}).

Hematological parameters allow researchers and wildlife managers to predict and understand different disease processes, stress levels and hydration status of wild populations and thus, determine whether a population faces any stress related to environmental changes or anthropogenic causes. At the same time, these results can be used as a baseline when NP must be managed in captivity or during rehabilitation.

Ethical statement: the authors declare that they all agree with this publication and made significant contributions; that there is no conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements. All financial sources are fully and clearly stated in the acknowledgements section. A signed document has been filed in the journal archives.