Journal of the American Association for Laboratory Science Vol 53, No 1 Copyright 2014 January 2014 by the American Association for Laboratory Animal Science Pages 12–17

Effects of Age and Sex on the Hematology and Blood Chemistry of Tibetan (Macaca thibetana)

Di Wu,1 Yong Yi,4 Fei Sun,2 Liang Zhou,4 Feng Yang,4 Hongxing Wang,4 Guodong Zhang,5 Yu Alex Zhang,1 and Feng Yue3,*

Tibetan macaques (Macaca thibetana), also known as Chinese stump-tailed macaques, are a threatened species. Although Tibetan macaques are Old World monkeys in the genus of Macaca, limited age- and sex-related physiologic data are available for this particular species. We used 69 apparently healthy Tibetan male and female macaques to explore the effect of age and sex on physiologic parameters. Somatometric measurements, biochemistry, and hematologic parameters were analyzed. Significant age-related differences were found for weight, BMI, RBC count, Hgb, Hct, neutrophils, eosinophil count, ALT, AST, ALP, GGT, creatine kinase (muscle and brain subtypes), LDH, α-amylase, creatinine, apolipoprotein A1, total protein, albumin, cholesterol, HDL, and potassium. Significant differences by sex were noted for weight, BMI, ALT, total bilirubin, and indirect bilirubin. An interaction between age and sex accounted for statistically significant differences in the values for weight, BMI, and lymphocyte and eosinophil counts. These physiologic data will provide veterinarians and researchers with important age- and sex-specific reference ranges for evaluating experimental results from Tibetan macaques.

Hematologic and biochemical values are of great importance in modest correlations between age and various hematologic and evaluating the wellbeing or pathologic conditions of nonhuman biochemical parameters in Tibetan macaques younger than 10 y.33 . However, many factors including but not limited to However, statistical correlation in this relatively small group did not handling stress, relocation, fasting, and anesthesia can affect he- necessarily demonstrate the effect of age and sex on the physiologic matologic and biochemical parameters in marmosets, cynomolgus parameters in Tibetan macaques. and rhesus macaques, and chimpanzees.11,13,17,35 Changes in many The purpose of this study was to determine the effects of hematologic and biochemistry parameters have been reported age and sex on the hematologic and serum biochemical param- during normal development and maturation in various nonhuman eters of a laboratory colony of 69 apparently healthy Tibetan primates. A previous study reported that the albumin:globulin macaques and to establish clinically relevant reference ranges ratio, BUN, BUN:creatinine ratio, and triglyceride concentration for all ages of adults of both sexes. changed most with age in rhesus macaques, according to data from the Primate Aging Database.25 In addition, significant dif- Materials and Methods ferences by sex have been noted in many New World and Old . In the current study, we used 69 apparently healthy 20,22,31 World primates. Understanding the effect of age and sex on Tibetan macaques that originated from wild macaques from south- hematologic and serum biochemical parameters will help us to west and that were colony-bred at the Institute of Laboratory distinguish the boundaries between normal changes and diseases- Animal Sciences, Academy of Medical Sciences (Chengdu, related changes and to pave the way to identify other factors or China). All animals were group-housed in clean indoor–outdoor conditions that may affect those parameters. primate facilities and fed commercial monkey chow (Foshan T and Tibetan macaques, an Old World primate belonging to the su- F Pet Food, Foshan City, China) twice daily, fruits daily, and freely perfamily of Cercopithecoidea, range from eastern Tibet east to available purified water supply. The sample population were was Guangdong and north to Shanxi in China and recently have been free of the following pathogenic microorganisms: Salmonella spp., 12 reported as far south as northeastern IndiaIndia. Tibetan macaques Shigella spp., Mycobacterium tuberculosis, pathogenic dermal fungi, are the largest species of and one of the largest nonhuman helminths, ectoparasites, B virus, T lymphotropic virus, and primates known to live in Asia. Several features make these animals simian type D retrovirus. Testing for simian T lymphotropic virus potentially useful for experimental studies and include their long and simian type D retrovirus was done by Xishan Biotechnology lifespan (greater than 20 y), large body size, and calm temperament. (VRL China, Suzhou City, China). Macaques were housed in indi- They have been approved for use in scientific research by the State vidual cages for about 1 wk before blood sampling. All adult female Forestry Administration of the People’s Republic of China and have macaques were considered to be nonpregnant via daily observation been used in studies of intraocular pressure measurement and gene of menses in the morning prior to blood sampling. 29,34 analysis. In a previous report, we found strongly positive cor- All experiments were conducted in compliance with national relations between age and various somatometric parameters and and provincial guidelines and in accordance with the Guide for the Care and Use of Laboratory Animals,8 as sanctioned by the Received: 31 Jan 2013. Revision requested: 16 Mar 2013. Accepted: 24 Jun 2013. Institute of Laboratory Animal Sciences, Sichuan Academy of 1Cell Therapy Center, 2Beijing Geriatric Healthcare Center, and 3Department of Neuro- Medical Sciences. This study was approved by the Animal Use biology, Beijing Institute of Geriatrics, Xuanwu Hospital, Capital Medical University, and Care Board of the Institute of Laboratory Animal Sciences, 4 Beijing, China; Institute of Laboratory Animals of Sichuan Academy of Medical Sciences Sichuan Academy of Medical Sciences. and Sichuan Provincial People’s Hospital, Chengdu, Sichuan Province, China; 5Wincon Laboratory, Wincon TheraCells Biotechnologies, Nanning, Guangxi Province, China. *Corresponding author. Email: [email protected]

12 Physiologic parameters of Tibetan macaques

Table 1. Demographics of adult Tibetan macaques Female Male Young Mature Old Young Mature Old Number 13 15 5 13 11 12 Age (y) 3.62 ± 0.65 10.1 ± 1.5 17.4 ± 2.0 3.62 ± 0.78 12.3 ± 1.0 19.2 ± 4.0 Age range (y) 3–5 8–13 15–20 3–5 10–13 15–28 Weight (kg)a,b 3.95 ± 0.76 11.0 ± 1.5 9.6 ± 1.0 4.46 ± 1.52 18.0 ± 3.8 17.2 ± 3.3 BMIa,b 18.3 ± 2.4 32.3 ± 4.9 27.9 ± 3.6 19.4 ± 4.0 40.6 ± 5.3 38.6 ± 8.0 aSignificant (P < 0.05) difference according to age. bSignificant (P < 0.05) difference according to sex.

One monkey year is approximately equal to 3 human years Results 15 in terms of skeletal changes. However, limited data about de- Demographic information, hematology and biochemistry velopment and maturation was available for Tibetan macaques. values are presented in Tables 1, 2, and 3, respectively. The Based on relevant information for cynomolgus and rhesus results of statistical analyses according to age, sex, and their 9,32 monkeys, Tibetan macaques were divided into 3 age groups interaction are shown in Table 4. in this study: young adults (3 to 5 y old), mature adults (8 to 13 Significant (P < 0.05) age-related and sex-associated differ- y old), and old adults (older than 15 y; Table 1). ences were found for both weight and BMI. For hematologic Somatometric measurements and blood sample collection. parameters, significant (P < 0.05) age-related differences were Somatometric measurements were done while monkeys were found for RBC count, Hgb, Hct, and neutrophil and eosinophil anesthetized with ketamine (10 mg/kg IM). BMI was calcu- counts. For biochemistry parameters, significant (P < 0.05) age- lated according to body weight (in kilograms) divided by related differences were noted for AST, ALT, ALP, GGT, creatine the square of the crown–rump length (in square meters), as kinase (muscle and brain subtypes), LDH, α-amylase, creatinine, 10 done for rhesus macaques. To avoid any potential influence apolipoprotein A1, total protein, albumin, cholesterol, HDL, and of handling-associated stress on chemistry and hematologic K concentrations. Significant (P < 0.05) differences by sex were parameters, blood samples were drawn during somatometric present for ALT and total and indirect bilirubin. An interac- measurements. Blood samples were obtained from the saphen- tion (P < 0.05) between age and sex accounted for significant ous vein of macaques after a 16-h fasting period. Tubes coated differences in the values for weight, BMI, lymphocytes, and with K2-EDTA were used to collect samples for CBC analysis, eosinophils. trisodium citrate tubes were used to assess coagulation, and Compared with young adults, mature and old Tibetan serum separation tubes were used for chemistry parameters, macaques had higher (P < 0.05) values for weight, BMI, neu- electrolytes, and hormones. trophils, and creatinine and lower (P < 0.05) values for RBC, Analysis of biochemistry and hematologic parameters. Bio- Hgb, Hct, AST, ALT, ALP, GGT, creatine kinase (muscle and chemistry measurements were performed on serum samples brain subtypes), LDH, and albumin. Young and mature adult by using an autoanalyzer (model BS 420, Mindray DS, Shen- Tibetan macaques had more (P < 0.05) eosinophils values than zhen, China) and included total bilirubin, direct bilirubin, did old macaques. Cholesterol, HDL, α-amylase, and apolipo- indirect bilirubin, ALT, AST, ALP, GGT, LDH, creatine kinase, protein A1 values were highest in young adults and lowest in old creatine kinase (muscle and brain subtypes), total protein, macaques. Female Tibetan macaques had higher (P < 0.05) total albumin, globulin, albumin:globulin ratio, creatinine, BUN, and indirect bilirubin and lower (P < 0.05) values for weight, triglycerides, cholesterol, HDL, glucose, LDL, apolipoprotein BMI, and ALT than did male macaques. A1, apolipoprotein B, and α-amylase. Na, K, chloride, normal- ized calcium (that is, the ionized calcium level when pH = 7.40), and total calcium in serum were measured by using a Discussion portable electrolyte analyzer (AFT500, Meizhou Cornley Hi- Many physiologic variables change with age or by sex in Tech, Meizhou, China). nonhuman primates. Our report may be first to give hematologic CBC examinations were done on whole blood by using an au- and serum chemistry parameters for Tibetan macaques accord- tomated hematology analyzer (model LH750, Beckman Coulter, ing to their age and sex. Age-specific and sex-based reference Brea, CA). Analyses included RBC count, Hgb concentration, ranges will help scientists to distinguish normal physiologic Hct, MCV, MCH, MCHC, platelet count, mean platelet volume, changes from age-related diseases in Tibetan macaques. WBC count, neutrophil count and percentage, lymphocyte count ALP, as a byproduct of osteoblast activity, is generally higher and percentage, monocyte count and percentage, eosinophil in children due to active bone formation. The increased ALP count and percentage, and basophil count and percentage. levels in our young adult Tibetan macaques implied rapid bone Statistical analysis. Statistical analysis was performed by us- metabolism and growth. A similar effect of age on ALP levels ing SPSS software (version 17.0, IBM, Chicago, IL).Values that has been found in bonnet, vervet, rhesus, and cynomolgus were 3 SD above or below the means were considered outliers macaques.14,15,19,22 These data indicate that Tibetan macaques and were not included. Data are expressed as mean ± 1 SD. A do not reach full body size and maturity until they are at least 2-way ANVOA was undertaken to analyze the effect of age 8 y old, and they undergo active bone formation at 3 to 5 y of and sex on parameters; this analysis not only determines the age. In humans, ALP is present in all tissues of the body and main effect of the contributions of each independent variable is particularly concentrated in liver, bile duct, kidney, intes- but also identifies any significant interaction effect between tines, bone, and placenta.22 However, the halflife of ALP in the independent variables. gastrointestinal tract and placenta is generally less than 6 min, which is too brief to result in increased serum levels.29 The ref-

13 Vol 53, No 1 Journal of the American Association for Laboratory Animal Science January 2014

Table 2. Hematologic parameters in adult Tibetan macaques Female Male Young Mature Old Young Mature Old RBC (×1012/L)a 5.39 ± 0.39 5.02 ± 0.27 5.26 ± 0.67 5.37 ± 0.52 5.09 ± 0.30 5.04 ± 0.28 Hgb (g/dL)a 13 ± 0.7 11.6 ± 0.4 12.2 ± 1.3 12.7 ± 1.3 11.7 ± 0.6 12.0 ± 0.7 Hct (%)a 0.39 ± 0.02 0.36 ± 0.02 0.37 ± 0.04 0.39 ± 0.04 0.36 ± 0.01 0.36 ± 0.02 MCV (fL) 72 ± 2.7 72 ± 2.8 70.4 ± 1.4 72 ± 3.0 72 ± 2.6 72 ± 2.7 MCH (pg) 24 ± 1.0 23 ± 1.1 23 ± 0.6 24 ± 1.0 23 ± 0.8 24 ± 1.2 MCHC (g/dL) 33.1 ± 0.4 32.4 ± 0.7 32.9 ± 0.2 32.9 ± 0.5 32.7 ± 0.4 32.8 ± 0.7 Platelets (×109/L) 334 ± 81 362 ± 64 373 ± 76 357 ± 54 328 ± 49 322 ± 67 WBC (×109/L) 9.5 ± 2.9 14.3 ± 4.0 12.6 ± 3.2 11.0 ± 4.5 11.1 ± 3.5 11.2 ± 3.1 Neutrophils (×109/L)a 3.5 ± 1.8 5.1 ± 2.2 6.9 ± 3.5 3.4 ± 2.6 5.6 ± 2.2 5.8 ± 2.6 Lymphocytes (×109/L) 5.4 ± 1.7 8.0 ± 3.3 5.1 ± 0.9 6.6 ± 2.2 4.7 ± 2.2 4.4 ± 1.7 Monocytes (×109/L) 0.30 ± 0.22 0.59 ± 0.35 0.49 ± 0.19 0.48 ± 0.36 0.47 ± 0.33 0.67 ± 0.25 Eosinophils (×109/L)a 0.22 ± 0.17 0.52 ± 0.33 0.10 ± 0.02 0.41 ± 0.35 0.35 ± 0.20 0.22 ± 0.10 Basophils (×109/L) 0.09 ± 0.07 0.09 ± 0.08 0.05 ± 0.02 0.10 ± 0.04 0.11 ± 0.08 0.12 ± 0.11 aSignificant (P < 0.05) difference according to age.

Table 3. Serum chemistry values in adult Tibetan macaque Female Male Young Mature Old Young Mature Old AST (IU/L)a 52 ± 31 21 ± 4.7 15 ± 3.6 66 ± 44 31 ± 18 21 ± 11 ALT (IU/L)a,b 89 ± 51 71 ± 38 43 ± 16 130 ± 90 100 ± 59 61 ± 47 ALP (IU/L)a 411 ± 168 86 ± 44 62 ± 15 381 ± 195 78 ± 41 66 ± 33 GGT (IU/L)a 90 ± 35 48 ± 21 34 ± 8.2 90 ± 41 45 ± 16 43 ± 14 Creatine kinase (IU/L) 151 ± 115 166 ± 89 161 ± 69 146 ± 72 171 ± 87 141 ± 82 Creatine kinase-MB (IU/L)a 182 ± 182 54 ± 21 39 ± 28 122 ± 82 52 ± 43 57 ± 52 LDH (IU/L)a 236 ± 63 164 ± 86 102 ± 44 223 ± 98 160 ± 83 166 ± 76 α-amylase (IU/L)a 378 ± 165 233 ± 128 120 ± 55 307 ± 109 225 ± 61 161 ± 47 Total bilirubin (mg/dL)b 0.15 ± 0.05 0.14 ± 0.08 0.16 ± 0.12 0.11 ± 0.06 0.12 ± 0.08 0.09 ± 0.03 Direct bilirubin (mg/dL) 0.06 ± 0.03 0.06 ± 0.03 0.07 ± 0.06 0.05 ± 0.03 0.05 ± 0.04 0.05 ± 0.02 Indirect bilirubin (mg/dL)b 0.08 ± 0.04 0.08 ± 0.06 0.08 ± 0.06 0.06 ± 0.04 0.07 ± 0.05 0.04 ± 0.02 Total protein (g/dL)a 7.6 ± 0.43 7.4 ± 0.38 7.3 ± 0.35 7.7 ± 0.41 7.5 ± 0.33 7.3 ± 0.44 Albumin (g/dL)a 4.9 ± 0.22 4.9 ± 0.35 4.5 ± 0.29 5.0 ± 0.22 4.8 ± 0.27 4.6 ± 0.38 Globulin (g/dL) 2.7 ± 0.30 2.6 ± 0.26 2.8 ± 0.17 2.7 ± 0.28 2.6 ± 0.23 2.7 ± 0.44 Albumin:globlin ratio 1.83 ± 0.19 1.90 ± 0.27 1.61 ± 0.14 1.90 ± 0.20 1.84 ± 0.22 1.79 ± 0.34 Glucose (mg/dL) 53 ± 15.3 49 ± 20 53 ± 24 54 ± 21 52 ± 18 41 ± 13 Triglycerides (mg/dL) 42 ± 15 48 ± 19 58 ± 38 44 ± 22 44 ± 17 41 ± 15 Cholesterol (mg/dL)a 125 ± 32 102 ± 44 82 ± 48 108 ± 30 91 ± 24 78 ± 25 HDL (mg/dL)a 74 ± 20 54 ± 22 29 ± 9 60 ± 16 48 ± 12 32 ± 13 LDL (mg/dL) 67 ± 14 69 ± 27 59 ± 24 63 ± 21 60 ± 17 55 ± 19 BUN (mg/dL) 17.4 ± 4.5 16.5 ± 5.6 16.8 ± 7.8 20.1 ± 4.2 17.4 ± 5.9 18.8 ± 7.0 Creatinine (mg/dL)a 0.48 ± 0.11 0.52 ± 0.21 0.59 ± 0.26 0.44 ± 0.09 0.61 ± 0.15 0.61 ± 0.20 Apolipoprotein A1 (mg/dL)a 126 ± 026 94 ± 36 56 ± 21 109 ± 28 89 ± 21 64 ± 22 Apolipoprotein B (mg/dL) 31 ± 8 32 ± 14 24 ± 14 29 ± 10 27 ± 9 24 ± 9 Na (mEq/L) 147 ± 3 149 ± 2 149 ± 3 150 ± 2 148 ± 3 149 ± 2 K (mEq/L)a 4.2 ± 0.6 3.6 ± 0.8 2.9 ± 0.6 4.0 ± 0.8 3.2 ± 0.4 3.2 ± 0.7 Cl (mEq/L) 104 ± 2 104 ± 2 105 ± 2 105 ± 2 105 ± 2 105 ± 2 Normalized Ca (mEq/L) 1.2 ± 0.1 1.1 ± 0.2 1.1 ± 0.2 1.1 ± 0.1 1.1 ± 0.2 1.1 ± 0.2 Total Ca (mEq/L) 2.4 ± 0.3 2.3 ± 0.4 2.1 ± 0.4 2.2 ± 0.2 2.1 ± 0.3 2.3 ± 0.3 MB, muscle and brain subtypes. aSignificant (P < 0.05) difference according to age. bSignificant (P < 0.05) difference according to sex.

14 Physiologic parameters of Tibetan macaques

Table 4. Results from 2-way ANOVA Age Sex Interaction Weight F(2,69) = 144.602, P < 0.01 F(2,69) = 72.026, P < 0.01 F(2,69) = 16.638, P < 0.01 BMI F(2,69) = 83.626, P < 0.01 F(2,69) = 26.014, P < 0.01 F(2,69) = 5.075, P = 0.009 RBC F(2,69) = 4.570, P = 0.014 nonsignificant nonsignificant Hgb F(2,69) = 12.392, P < 0.01 nonsignificant nonsignificant Hct F(2,69) = 9.380, P < 0.01 nonsignificant nonsignificant WBC F(2,69) = 2.947, P = 0.060 nonsignificant F(2,69) = 2.683, P = 0.076 Neutrophils F(2,69) = 7.344, P < 0.01 nonsignificant nonsignificant Lymphocyte nonsignificant nonsignificant F(2,69) = 6.493, P < 0.01 Eosinophils F(2,69) = 5.490, P = 0.006 nonsignificant F(2,69) = 3.150, P = 0.05 AST F(2,69) = 16.712, P < 0.01 nonsignificant nonsignificant ALT F(2,69) = 4.660, P = 0.013 F(2,69)=4.113, P = 0.047 nonsignificant ALP F(2,69) = 59.656, P < 0.01 nonsignificant nonsignificant GGT F(2,69) = 23.130, P < 0.01 nonsignificant nonsignificant Creatinine kinase-MB F(2,69) = 9.402, P < 0.01 nonsignificant nonsignificant LDH F(2,69) = 7.870, P = 0.001 nonsignificant nonsignificant α-amylase F(2,69) = 17.689, P < 0.01 nonsignificant nonsignificant Total bilirubin nonsignificant F(2,68) = 5.503, P = 0.022 nonsignificant Indirect bilirubin nonsignificant F(2,68) = 4.881, P = 0.031 nonsignificant Creatinine F(2,69) = 4.103, P = 0.021 nonsignificant nonsignificant Apolipoprotein A1 F(2,69) = 20.301, P < 0.01 nonsignificant nonsignificant Total protein F(2,69) = 4.159, P = 0.020 nonsignificant nonsignificant Albumin F(2,69) = 8.537, P < 0.01 nonsignificant nonsignificant Cholesterol F(2,69) = 6.672, P < 0.01 nonsignificant nonsignificant HDL F(2,69) = 20.648, P < 0.01 nonsignificant nonsignificant K F(2,69) = 12.670, P < 0.01 nonsignificant nonsignificant MB, muscle and brain subtypes. erence range of ALP in Tibetan macaques that we established ALT scores to those of cynomolgus, rhesus, bonnet, and vervet is similar to those of cynomolgus, rhesus, bonnet, and vervet monkeys.19,22,23,25 Compared with those for humans, ALT refer- macaques.19,22,23,25 Compared with that in nonhuman primates, ence ranges in nonhuman primates were generally higher.18,21 the reference range for ALP in humans is generally lower.1 Age-related decreases in serum albumin were apparent in this ALT and AST are used as general indicators of liver function, population of apparently healthy Tibetan macaques. Albumin with ALT being more liver-specific. Previous reports found no declines with age in rhesus, bonnet, and vervet macaques19,22,23 age-related differences in ALT and AST in rhesus, bonnet, or and in canines, rodents, and humans.3,6,26 Therefore, the de- vervet monkeys.19,22,25 However, young adult Tibetan macaques crease in albumin with age of our animals might be considered had higher ALT and AST than did mature and old macaques in a normal physiologic changes in . The age-associated the current study. There is no scientific consensus on whether decrease in albumin may also imply an increased demand for age itself affects ALT levels in humans. ALT did not correlate carrier proteins and protein metabolism during early develop- with age in a study of apparently healthy Iranian blood donors.18 ment, as reported in bonnet macaques.19 Serum globulin was In contrast, a recent population-based cohort study found that stable with respect to age in the current study, which differs from ALT levels decreased with age, independent of metabolic syn- reports involving bonnet macaques and vervet monkeys.19,22 drome components, adiposity signaling biomarkers, and other Although found in many tissues, α-amylase is most promi- commonly used liver function tests.4 Other authors concluded nent in pancreatic fluid and saliva and breaks down long-chain that younger age was associated with abnormally elevated ALT carbohydrates into glucose and maltose. We found significant according to both the traditional ALT cutoff threshold (43 IU/L) decreases in α-amylase in apparently healthy old Tibetan as well as revised lower thresholds (men, 30 IU/L; women, macaques in this study. Age-related changes in α-amylase have 19 IU/L).21 One possible reason for this discrepancy is that not been reported to occur in rhesus, bonnet, vervet and tufted decreased ALT may have implied a reduction in the mass or capuchin monkeys.19,22,23,31 Another study found that the rate of function of the normal liver. In addition, the fact that albumin positive staining for α-amylase in the parotid gland of normal and GGT levels decreased with age in Tibetan macaques may humans gradually decreased with age.16 The age-associated account for the apparent age-induced decrease in ALT. Another decrease in α-amylase in old Tibetan macaques may simply possible reason may rest in viral infections that regular screening reflect their decreased energy need. and quarantine tests failed to detect. Some authors have recently Creatinine, a break-down product of creatine phosphate in argued for the exclusion of a greater number of viral pathogens muscle, is usually produced at a fairly constant rate by the body to produce SPF nonhuman primates compared with nonSPF depending on muscle mass. Male Tibetan macaques had slightly animals.27 A higher burden of viral infections in young Tibetan but not significantly higher values of creatinine than did female macaques may have led to the higher ALT levels of this group.5 macaques, whereas mature and old Tibetan macaques had In comparison, mature and old Tibetan macaques had similar higher values of creatinine than YA ones. Significant increases

15 Vol 53, No 1 Journal of the American Association for Laboratory Animal Science January 2014 in creatinine due to age and sex have been reported to occur 2. Chapman KM, Ham JO, Pearlman RA. 1996. Longitudinal assess- in bonnet and vervet monkeys, chimpanzees, and humans, ment of the nutritional status of elderly veterans. J Gerontol A Biol particularly in very elderly humans.2,7,19,22 In contrast, a signifi- Sci Med Sci 51:B261–B269. cant age-associated decrease in creatinine was noted in rhesus 3. Ding J, Kopchick JJ. 2011. Plasma biomarkers of mouse aging. monkeys.25 Age-related increases in creatinine may be due to Age (Dordr) 33:291–307. 4. Dong MH, Bettencourt R, Barrett-Connor E, Loomba R. 2010. a gradual loss of muscle mass, decreased renal blood flow, and Alanine aminotransferase decreases with age: the Rancho Bernardo decreased glomerular filtration rate during aging. The reference Study. PLoS ONE 5:e14254. range of creatinine for Tibetan macaques was generally similar 5. Dong MH, Bettencourt R, Brenner DA, Barrett-Connor E, Loomba to those of humans and other nonhuman primates. R. 2012. Serum levels of alanine aminotransferase decrease with age Many studies report age-related changes in lipid concen- in longitudinal analysis. Clin Gastroenterol Hepatol 10:285–290. trations in nonhuman primates as well as in humans. HDL 6. Gorman LS. 1995. Aging: laboratory testing and theories. Clin Lab tended to decline with the progression of age in normal rhesus Sci 8:24–30. monkeys, whereas serum triglycerides, cholesterol, and LDL 7. Ihrig M, Tassinary LG, Bernacky B, Keeling ME. 2001. He- remained unchanged.28 In another study of rhesus monkeys, matologic and serum biochemical reference intervals for the only triglycerides were found to increase with the rise of age.25 chimpanzee (Pan troglodytes) categorized by age and sex. Comp It has often been reported that increased serum triglycerides and Med 51:30–37. LDL and decreased HDL occur in elderly humans because of 8. Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals, 8th ed. Washington (DC): National obesity. In Tibetan macaques, we found that cholesterol, HDL, Academies Press. and apolipoprotein A1 decreased with age, but triglyceride and 9. Kemnitz JW. 2011. Calorie restriction and aging in nonhuman LDL levels remained the same. Generally, directly comparing primates. ILAR J 52:66–77. lipid levels between aged nonhuman primates and humans 10. Kemnitz JW, Goy RW, Flitsch TJ, Lohmiller JJ, Robinson JA. is difficult, because lipid disorders tend to be complicated by 1989. Obesity in male and female rhesus monkeys: fat distribution, factors of lifestyle, diet, ethnicity, and degree of adiposity.27 For glucoregulation, and serum androgen levels. J Clin Endocrinol example, nonhuman primates generally receive a standardized Metab 69:287–293. low-cholesterol diet, whereas human diets vary greatly.24 11. Kuehnel F, Grohmann J, Buchwald U, Koeller G, Teupser D, In the current study, hematologic analysis revealed that age- Einspanier A. 2012. Parameters of haematology, clinical chemistry, related differences occurred in RBC, Hgb, Hct, neutrophils, and and lipid metabolism in the common marmoset and alterations eosinophils. RBC, Hgb, and Hct decreased from young to mature under stress conditions. J Med Primatol 41:241–250. adults and then stabilized from mature to old Tibetan macaques. 12. Kumar S, Mishra C, Sinha A. 2005. Discovery of the Tibetan macaque Macaca thibetana in Arunachal Pradesh, . Curr Sci The animals showed no sex-related differences in hematologic 88:1387. parameters. However, RBC, Hgb, and Hct increase with age 13. Lambeth SP, Hau J, Perlman JE, Martino M, Schapiro SJ. 2006. 25 in rhesus monkeys. MCV, MCH, and MCHC were reported Positive reinforcement training affects hematologic and serum 22 to increase with age in vervet monkeys. Male rhesus and chemistry values in captive chimpanzees (Pan troglodytes). Am J vervet monkeys generally had higher values than did females Primatol 68:245–256. in RBC, Hgb, and Hct.22,25 The age-related and sex-associated 14. Lee JI, Shin JS, Lee JE, Jung WY, Lee G, Kim MS, Park CG, Kim differences in Tibetan macaques differed from those of other SJ. 2012. Reference values of hematology, chemistry, electrolytes, nonhuman primates. One reason for this difference may have blood gas, coagulation time, and urinalysis in the Chinese rhesus been differences between studies in origin of animals, protocol macaques (Macaca mulatta). Xenotransplantation 19:244–248. design, methods, and implementation. The reference range of 15. Lees CJ, Ramsay H. 1999. Histomorphometry and bone biomar- hematologic parameters in Tibetan macaques was generally kers in cynomolgus females: a study in young, mature, and old similar to that of other nonhuman primates.14,22,23,25 monkeys. Bone 24:25–28. 16. Liu X, Yu G, Gao Y. 2001. [Aging changes of α-amylase and lys- More reliable results may be obtained with a larger sample ozyme in normal parotid gland]. Zhonghua Kou Qiang Yi Xue Za size for each group. In summary, the hematologic and bio- Zhi 36:37–39. [Article in Chinese]. chemistry parameters in this study provide a reference range 17. Lugo-Roman LA, Rico PJ, Sturdivant R, Burks R, Settle TL. for apparently healthy Tibetan macaques according to their age 2010. Effects of serial anesthesia using ketamine or ketamine– and sex. The age-related changes in Tibetan macaques were medetomidine on hematology and serum biochemistry values in generally similar to those published for cynomolgus, rhesus, rhesus macaques (Macaca mulatta). J Med Primatol 39:41–49. and vervet macaques. 18. Mohamadnejad M, Pourshams A, Malekzadeh R, Mohamad- khani A, Rajabiani A, Asgari AA, Alimohamadi SM, Razjooyan H, Mamar-Abadi M. 2003. Healthy ranges of serum alanine ami- Acknowledgments notransferase levels in Iranian blood donors. World J Gastroenterol This work was supported by grants from the State High-Tech 9:2322–2324. Development Plan of the Ministry of Sciences and Technology of 19. Pierre PJ, Sequeira MK, Corcoran CA, Blevins MW, Gee M, China (2012AA020703); the National Basic Research Program of China Laudenslager ML, Bennett AJ. 2011. Hematological and serum (2012CBA01307); the Science Research and Technology Development Project from Bureau of Sciences and Technology of Guangxi China (13- biochemical indices in healthy bonnet macaques (Macaca radiata). 29-06); the High-Level Talent Fund of the Beijing Healthcare System J Med Primatol 40:287–293. (2009-2-14); and the Sichuan Provincial Health Department Scientific 20. 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