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Establishment of Hematological and Serum Biochemical Parameters in 4,834 Healthy Cynomolgus Monkeys (Macaca fascicularis)

Xueying Ling^1,a, Ting Luo^1,a, Qingyu Huang², Ying He², Longbao Lv³, Yun Wang³, Ronghua Ma⁴, Jiewen Chen², Jicun He², Jinrong Zhang², Hongyu Lu⁴, Jian He¹, Danrong Lin¹, Siyu Chen¹, Taiqi Liu¹, Yangqing Guo¹, Zhuhong Liu¹, Jiaqi Feng¹, Yudan Mao¹, Zhiying Chen¹, Ren Huang^1,a,* and Wen De Li^1,a,*

¹Guangdong Laboratory Animals Monitoring Institute, Key Laboratory of Guangdong Laboratory Animals, Guangzhou 510663, China

²Guangdong Blooming-spring Biological Technology Development Co., Ltd., Guangzhou 510940, China

³Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China

⁴Huazheng Laboratory Animal Breeding Centre, Guangzhou 510920, China

^aThese authors contributed equally to this work.

*Corresponding to: Ren Huang, E-mail: 1649405216@qq.com; Wen De Li, E-mail: 113587350@qq.com

Received: March 23 2022; Revised: May 28 2022; Accepted: July 11 2022; Published Online: July 26 2022

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Cite this paper:

Xueying Ling, Ting Luo, Qingyu Huang, Ying He, Longbao Lv, Yun Wang, Ronghua Ma, Jiewen Chen, Jicun He, Jinrong Zhang, Hongyu Lu, Jian He, Danrong Lin, Siyu Chen, Taiqi Liu, Yangqing Guo, Zhuhong Liu, Jiaqi Feng, Yudan Mao, Zhiying Chen, Ren Huang and Wen De Li. Establishment of Hematological and Serum Biochemical Parameters in 4,834 Healthy Cynomolgus Monkeys (Macaca fascicularis). BIO Integration 2022; 3(4): 151–160.

DOI: 10.15212/bioi-2022-0011. Available at: https://bio-integration.org/

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© 2022 The Authors. This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See https://bio-integration.org/copyright-and-permissions/

Abstract

Background: In China and other countries, cynomolgus monkeys are commonly used experimental animal models in biomedical research. Reference hematologic and biochemical parameters must be established to evaluate healthy cynomolgus monkeys and investigate drug safety in non-clinical research. In the present study, data from 4,834 healthy cynomolgus monkeys were used to establish a reference for various age groups.

Methods: A total of 4834 healthy cynomolgus monkeys (649 males and 4185 females) were selected and divided into six groups according to age and sex. Twenty hematological parameters and 17 serum biochemical parameters were detected, and the effects of age and sex were analyzed.

Results: A reference database of hematological and biochemical parameters was established according to age (six groups) and sex (male and female). Significant differences were observed in White blood cell, Red blood cell, Hemoglobin, Hematocrit, Total protein, Albumin, Total bilirubin, Direct bilirubin, Alkaline phosphatase, Glucose, Blood urea nitrogen, Creatinine, Calcium, Total cholesterol, Triglyceride, and Lactate dehydrogenase values according to age in the juvenile and young groups (P<0.05). Significant differences between males and females were observed in Red blood cell, Hemoglobin, Hematocrit, Mean corpuscular hemoglobin concentration, White blood cell, Lymphocyte, Lymphocyte percentage, Monocyte, Monocyte percentage, Basophil, and Basophil percentage in most age groups (P<0.05).

Conclusion: Reference ranges for healthy cynomolgus monkeys were established in different age and sex groups in this study. The findings may be useful in clinical care and non-human-primate research.

Keywords

Biochemistry, cynomolgus monkeys, hematology, Macaca fascicularis.

Introduction

Abundant non-human-primate resources exist in China. Cynomolgus monkeys (Macaca fascicularis) are highly genetically similar to humans, thus making them important experimental animal models in biomedical research fields [1], including cerebral diseases, traditional Chinese medicine, molecular genetics, pharmacokinetics, and toxicology [2–6]. Given the important roles of these monkeys in research, reference hematologic and biochemical parameters must be established to aid in the assessment of animal health status, and investigation of the effects induced by drugs or treatments.

A prior study has reported the hematological and biochemical parameters of cynomolgus monkeys according to age and sex [7]. Reference clinical pathology data have been reported in another study [8]. A previous report has also investigated the reference values of hematological and biochemical parameters, and compared the differences between two species (cynomolgus monkeys and Rhesus monkeys) [9]. However, although many studies have reported hematologic and biochemical parameters of non-human primates [7, 9–14], only several studies have provided reference data from an extensive number of animals, ranging in age from juvenile to old.

Therefore, his study was aimed at establishing a reference database of hematologic and biochemical parameters of cynomolgus monkeys according to age and sex, to provide a basis for selecting healthy animals for constructing disease models, and for improving the effectiveness of disease diagnosis and treatment.

Materials and methods

Animals

A total of 4,834 healthy cynomolgus monkeys (649 males and 4,185 females) 1–22 years of age (Y), were obtained from three monkey farms at Guangdong Blooming-Spring Biological Technology Development Co., Ltd.; the Kunming Institute of Zoology; and the Huazheng Laboratory Animal Breeding Centre. We divided the animals into six age groups: animals 1–3 Y were in the juvenile group, those 4–6 Y were in the young group, those 7–9 Y were in the adult group, those 10–12 Y were in the middle-age group, those 13–15 Y were in the old group, and those older than 15 Y were in the very old group. All cynomolgus monkeys collected from the three monkey farms were of Chinese origin and were determined to be healthy in annual examinations including their hair, behavior, spirit, activity, skin, joints, excretion, blood parameters, and microbial parameters. All animals had tested negative for tuberculosis and Herpes B virus. In addition, all female monkeys were non-pregnant. The present study and all procedures were approved by the animal care and use committee of Kunming Institute of Zoology, Chinese Academy of Sciences (approval No: IACUC 16006). In addition, all experiments in monkeys from the three monkey farms were conducted in accordance with the Guide for the Care and Use of Laboratory Animals [15]. Hematological and biochemical data were gathered during routine annual quarantine and health monitoring from 2015 to 2017. No animals were sacrificed in this study.

Blood sample collection and preparation

All monkeys were fasted overnight for at least 8 hours before blood was drawn. The males were anesthetized with ketamine hydrochloride, and the females were conscious; 3-mL blood samples were drawn from cephalic veins with syringes. First, the 1-mL blood sample was transferred into a tube containing ethylenediamine tetraacetic acid dipotassium tube, and the blood was adequately mixed by inversion of the tube at least five times. Then 2-mL of the remaining blood sample was transferred into a vacuum drying tube, and kept at room temperature for 30 min. The serum was separated by centrifugation at 1600 g for 15 min.

Hematological analysis

Hematological analysis was performed with a Sysmex XT-2000iv automated hematology analyzer. The following parameters were analyzed: white blood cell count (WBC, 10⁹/L), red blood cell count (RBC, 10¹²/L), hemoglobin (HGB, g/L), hematocrit (HCT, %), mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin mass (MCH, pg), mean corpuscular hemoglobin concentration (MCHC, g/L), platelets (PLT, 10⁹/L), mean platelet volume (MPV, fL), plateletcrit (PCT, %), neutrophils (NEUT, 10⁹/L), lymphocytes (LYMPH, 10⁹/L), monocytes (MONO, 10⁹/L), eosinophils (EO, 10⁹/L), basophils (BASO, 10⁹/L), neutrophil percentage (NEUT%, %), lymphocyte percentage (LYMPH%, %), monocyte percentage (MONO%, %), eosinophil percentage (EO%, %), and basophil percentage (BASO%, %).

Biochemical analysis

Biochemical analysis was performed with a HITACHI7020 automated biochemical analyzer. The following parameters were analyzed: alanine aminotransferase (ALT, U/L), aspartate aminotransferase (AST, U/L), total protein (TP, g/L), albumin (ALB, g/L), total bilirubin (TBIL, μmol/L), direct bilirubin (DBIL, μmol/L), alkaline phosphatase (ALP, U/L), blood urea nitrogen (BUN, mmol/L), creatinine (CREA, μmol/L), glucose (GLU, mmol/L), total cholesterol (CHOL, mmol/L), triglyceride (TG, mmol/L), high-density-lipoprotein cholesterol (HDL-C, mmol/L), low-density-lipoprotein cholesterol (LDL-C, mmol/L), lactate dehydrogenase (LDH, U/L), calcium (Ca, mmol/L), and phosphorus (P, mmol/L).

Statistical analysis

The hematological and biochemical results were statistically analyzed in SPSS version 22.0, with two-tailed Student’s t-tests and Kruskal-Wallis tests to detect significant differences between age groups for each sex, and between sexes in each age group. The data are reported as mean ± SD, and a p-value less than 0.05 was considered statistically significant.

Results

Hematological and biochemical values

The values of hematological parameters are presented in Table 1. In the juvenile and young groups, age had a statistically significant effect on RBC, HGB, HCT, MCV, MCH, NEUT%, LYMPH%, and BASO% (P<0.05). Compared with the adult and old groups, the WBC, RBC, HGB, and HCT values were significantly higher in the juvenile and young age groups (P<0.05). Significant differences between males and females were observed for RBC, HGB, HCT, MCHC, WBC, LYMPH, LYMPH%, MONO, MONO%, BASO, and BASO% in most age groups (P<0.05).

The biochemical values are presented in Table 2. Significant differences were observed in TP, ALB, TBIL, DBIL, ALP, GLU, BUN, CREA, Ca, CHOL, TG, and LDH according to age in the juvenile and young groups (P<0.05). ALP values were significantly higher in the juvenile age group (1–3 Y) than the adult and old groups. TP values increased with age in the young age groups (1–6 Y) and became stable after adult age. Significant differences between males and females were observed for ALP and CREA in most age groups (P<0.05).

Effects of geographical location, breeding conditions and climatic factors on hematological and biochemical values in three monkey farms

All samples originated from three monkey farms: Guangdong Blooming-Spring Biological Technology Development Co., Ltd. (farm 1), the Kunming Institute of Zoology (farm 2), and the Huazheng Laboratory Animal Breeding Centre (farm 3). Because of the variations in the animals’ age distribution among the three farms, we present a comparison of hematological and biochemical values in two age groups (4–6 Y and 7–9 Y) among the three farms (Table 3 and Table 4). A significant difference in RBC, HGB, and HCT values was observed among the three farms in the 4–6 Y group (P<0.05; Table 3). In the 7–9 Y group, no significant differences were observed in MCHC, MPV, NEUT, EO, MONO%, and EO% values among the three farms (P>0.05). No statistically significant difference was found in ALT values among the three farms in both age groups (P>0.05). In the 4–6 Y age group, no significant difference was observed in Ca values in females among the three farms (P>0.05), whereas a significant difference was found in males (P<0.05). In the 7–9 Y age group, no significant differences were observed in the Ca and P values in males and females among the three farms (P>0.05).

Correlation of hematological and biochemical parameters with age

The correlation analysis of hematological and biochemical parameters with age is presented in Table 5. WBC, RBC, HGB, HCT, LYMPH, LYMPH%, ALP, GLU, BUN, TG, and LDH values were significantly higher in the 1–3 Y group than the other age groups. RBC and ALP levels were generally higher in males than in females, and the difference gradually decreased with age (Figure 1). The serum TP and NEUT% increased with age in the 1–3 Y and 4–6 Y groups, and remained stable thereafter. Moreover, LYMPH% showed an opposite trend from NEUT%.

Discussion

Our study provided a reference database of hematologic and biochemical parameters in 4,834 healthy cynomolgus monkeys of a range of ages, and determined the effects of age, sex, and colony conditions on these parameters. Cynomolgus monkeys, the most commonly used non-human primate experimental animals, substantially contribute to many biomedical research fields [12]. The goal of our study was to establish the reference ranges of hematologic and biochemical parameters in healthy cynomolgus monkeys for evaluating healthy individuals, constructing disease models, improving the effectiveness of disease diagnosis and treatment, and evaluating drug safety in non-clinical research. Moreover, we provide data on old age groups not reported in previous studies. However, the results were affected by factors including housing [16], feeding, fasting, and sedation.

Animals in our study were housed indoors in mixed-sex social groups in large cages, but under laboratory conditions, they were usually transferred into individual cages providing a relatively small space. This change in living environment might have caused stress, which consequently directly influenced the sympathetic adrenal medulla system and potentially led to the production of stress hormones, such as epinephrine, that increased blood parameters (e.g., WBC).

Previous research has reported the differences in blood hematologic and biochemical measurements according to age and sex [7]. For example, hematologic and biochemical parameters including RBC, HGB, HCT, ALP, and CREA have shown significant effects dependent on both age and sex, in agreement with our statistical results. In addition, WBC, HGB, and MCHC displayed high variability within the age range from 1 to 5 Y, and the values were significantly higher in this group than in other groups. Some concentrations of serum biochemical parameters were significantly lower within the age range from 1 to 5 Y, such as ALT, ALP, LDH, and TP, whereas the concentration of TBIL was significantly higher within the age range from 3 to 5 Y, possibly because of the environmental and breeding conditions.

Because geographical location, breeding conditions, and climatic factors may affect hematological and biochemical values, we compared and analyzed the variations in the results among the three monkey farms, and found significant differences in hematological parameters, including RBC, HGB, and HCT values. The three monkey farms in our study are located in Kunming and Guangzhou in China. Kunming, in a temperate climatic zone, is located in the central Yunnan-Guizhou Plateau, southwest China (102°10’ to 103°40’ E, 24°23’ to 26°22’ N), where altitudes range from 1500 m to 2800 m. Guangzhou, in a subtropical climate zone, is located in south China (112°57’ to 114°3’ E, 22°26’ to 23°56’ N), where altitudes range from 0 m to 1210 m. In the present study, the results (Table 3) demonstrated that the WBC, RBC, HGB, LYMPH, and LYMPH% values were significantly higher, whereas the NEUT% values were lower, in the Kunming animals than the Guangzhou animals, thus supporting the hypothesis that adaptation to lower oxygen at high altitude results in a physiological increase in RBC and hemoglobin concentration [17].

A comparison of the ALP and LDH values among age groups indicated that the concentrations of ALP and LDH in juveniles were significantly higher than those in other age groups. The ALP level decreased with age, then reached a stable level in adults. ALP is produced in the liver, bones, small intestine, and placenta, and usually reflects bone anabolism [18, 19]. It may be associated with elevated osteoblast activity in juvenile monkeys. A similar change in ALP level has also been reported by Ogawa [20]. Previous studies have indicated that ALP is significantly higher in patients with metastatic prostatic cancer, and may serve as a prognostic marker of bone metastases and multiple osteoblastic lesions. [21] Therefore, detecting ALP levels is meaningful in diagnosing and monitoring bone diseases, as well as evaluating drug efficacy. Although serum LDH levels are known to increase under myocardial injury and liver injury, we observed significantly higher LDH in the juvenile monkeys than in the other age groups, in agreement with a report by Koga et al. [22]. Whether this finding was associated with high levels of aerobic metabolism in juvenile monkeys remains to be further studied.

In conclusion, reference ranges for healthy cynomolgus monkeys were established according to age and sex in this study. Researchers and laboratory technicians routinely need to identify etiological factors, improve clinical care, and establish human disease models and new drug screening. However, multiple uncontrollable factors, including differences between individuals and breeding environments, affect hematologic and biochemical values, thus resulting in differences in data between laboratories. The above factors should therefore be considered in practical applications.

Financial support

This work was supported by the National key research and development program of China (2021YFF0702600), and the Guangdong Provincial Platform Base and Science and Technology Infrastructure Construction Project (No. 2021B1212060002).