Exp. Anim. 35(4) 443-447 1986 Normal Hematologic Values in the Cynomolgus Monkeys Aged from 1 to 18 Years Yasuko SUGIMOTO Kohji HANARI Hayato NARITA and Shigeo HONJO* The Corporation for Production and Research of Laboratory Primates and *Tsukicba Primate Center for Medical Science The National Institute of Health. Hachiman-dai Yatabe-machi Tsukuba-gun Ibaragi-ken 305 Japan. (Received 12 March 1986/Accepted 25 April 1986) Normal hematological values including erythrocyte count (RBC) hematocrit value (Ht) hemoglobin concentration (Hb) total leucocyte count (WBC) and differential leucocyte count were determined with 206 healthy cynomolgus monkeys (Macaca fascicularis) aged in 1 to 18 years. These animals were born and reared at Tsukuba Primate Center for Medical Science NIH Japan. Additionally 32 cynomolgus monkeys of wild origin estimated to be 5 or more years old were examined for their hematology. The presence or absence of sex- and age-related differences in the values was statistically analysed. As regards infant and juvenile monkeys there were no significant differences in RBC Ht and Hb between males and females. However these values became significantly larger in males than in females after sexual maturation. Lymphocyte count was larger than neutrophil count until 3 to 4 years of age after birth but this relation was inverted in adult monkeys aged more than 5 years. Segmentation of the nucleus was a prominent finding in neutrophils. Neither sex- nor age-related difference in the number of nuclear segmentation was noted. A number of papers have been published concerning the normal hematologic values in nonhuman primates. However there has appeared as yet no report dealing with age-related changes in normal hematology in the cynomolgus monkey (Macaca fascicularis). The present paper deals with the determination of some hematological parameters in the cynomolgus monkeys aged from 1 to 18 years. Materials and Methods Animals : Two hundred and six cynomolgus monkeys bred and reared at the Tsukuba Primate Center for Medical Science (TPC) National Institute of Health (NIH) were used for this study [3 4]. In addition to them 32 female cynomolgus monkeys of wild origin estimated to be 5 or more years old were used. All the animals used were apparently healthy. Feeding conditions for these animals were the same as described elsewhere [4]. Hematological Determinations : Blood was withdrawn from the femoral vein under slight anesthesia with 5 to 10 mg/kg B.W. of Ketalar (Sankyo-Park-Davis). The blood taken was mixed with the anticoagulant EDTA-2Na (disodium ethylenediamine tetra-acetate). For the determinations of erythrocyte count (RBC) hematocrit value (Ht) hemoglobin concentration (Hb) and total leucocyte count (WBC) an automated blood cell counter (Toa Medical Electronics Co. Ltd. Type CC-108 and CC- 110) was used. Blood-smears were prepared within 2 hours after blood withdrawal and stained with May-Grunwald-Giemsa staining
444 solution. The smeared specimens were observed for differential leucocyte counts under 1:1 1000 magnification and 100 cells were differentiated. Statistical Analyses : Presence or absence of any sex-related differences in RBC Ht and Hb was analysed by the T test. And the F test was used to determine if there was any age-related difference in the differential leucocyte counts. Results RBC (Table 1) : In young monkey groups aged 1 to 3 or 4 years the average of RBC ranged approximately from 600 ~104/pl to 650 ~104/ There was no sex-related difference in RBC regarding these age groups. The averag RBC in the female monkey groups aged more than 5 years was less than 600 ~104/ pl which was significantly lower than in the male monkey groups of the same age. Ht and Hb (Table 1) : Female monkeys showed no age-related changes in Ht and Hb which ranged from 41 to 44% and from 11 to 12 g/dl respectively. In male monkeys however both Ht and Hb showed a significant increase after 5 years of age ranging from 46 to 5000 and 13 to 15 g/dl. As a result sex-related differences in Ht and Hb were detected in the groups of monkeys more than 5 years old. WBC (Table 2) : Regardless of sex and age the average of WBC was more than 10 ~103/ƒÊl ranging from 10.2 ~103/ƒÊl to 14.1 ~103/ƒÊl. Leucoeyte differential (Table 2) : Generally the combined percentag-incidence of lymphocytes and neutrophils was over 90% in almost all monkeys. In the 1- and 2-year-old animal groups the average count of lymphocytes exceeded that of neutrophils. The difference in average count between lymphocytes and neutrophils became small in 3- to 4-year-old animal groups even though the count of lymphocytes was still larger than that of neutrophils. In adult monkeys aged 5 to 10 years the count of neutrophils exceeded that of lymphocytes but the lymphocyte count again became higher than the neutrophil count in animals aged more than 10 years. There was an especially marked difference in both lymphocyte and neutrophil counts between the 1- to 2-year-old animal groups and the 5- to 7-year-old animal groups. ul. As regards the nuclear segmentation in neutrophils (Table 3) 4 lobes were most frequently observed. The average polymorphnuclear number was 4.0 to 4.7 in all agegroups with neither age- nor sex-related difference. The percentage incidence ranged from 2 to 4% with monocytes and from 1 to 3% with eosinophils and was less than 19o with basophils. There were no age-related and sexrelated differences in these incidences. Discussion Many reports have been published dealing with the normal hematology of nonhuman primates [1 2 5-8 10-16 18]. Most of these reports however have not necessarily been perfect with regard to the description of age-related changes. Some papers were concerned with changes in blood pictures during the period from the new-born to juvenile stage using the rhesus monkey [6 7 10] the baboon [1 2] the patas monkey [13] and the rebus monkey [11]. But in the previous papers no baseline data were presented concerning age-related changes from the infant to adult stage. In this paper we have described age-related hematological findings in cynomolgus monkeys whose ages ranged from 1 to 18 years. As regards RBC Ht and Hb the present study demonstrated that the values in male monkeys exceeded those in females after the period of sexual maturity that is after 3 to 4 years of age. This trend is very similar to that in humans in which sex-related difference in these hematological values becomes noticeable after puberty that is after about 12 years of age [9]. According to the statistics of our breeding colony the female cynomolgus monkey begins menstruation at 2.5 years f 8 months on an average p31. As a whole WBC was to some extent greater in our cynomolgus monkeys than in man. This trend is consistent with findings described previously [5 12]. The most interesting finding in the present study concerns the differential leucocyte counts. In man the number of lymphocytes exceeds that of neutrophils until 4 to 6 years of age
445 Table 1. RBC Ht and Hb by different age groups and sexes. t test *:0.05< **:0.01< *:0.005< Table 2. Total and differential leucocyte counts by different ages and sexes. Table 3. Number of nuclear segmentation in neutrophils by different age groups.
446 after which the numerical relation between the two kinds of leucocytes is inverted [9]. The present study clearly demonstrated that in the cynomolgus monkey the count of lymphocytes was larger than that of neutrophils until 3 to 4 years of age while the count of the former became smaller than that of the latter in mature monkeys aged more than 5 years. Furthermore the present study showed that the average count of lymphocytes was again larger than that of neutrophils in 10 or more years old monkeys. Since there was a marked individual difference in differential leucocyte counts among monkeys aged 10 or more years the physiological significance of the larger count of lymphocytes in this fully adult age group may be different from that in monkeys 4 or less years old. It was previously reported that the count of lymphocytes in baboons who had been kept under sterile feeding conditions was virtually large as compared with that in baboons kept under ordinary feeding conditions [2 14]. The number of nuclear segmentation in neutrophils has been described to be generally larger in nonhuman primates than in man[s]. This was the case with the present data of the cynomolgus monkey. In other words neutrophils with multisegmented nuclei were frequently observed regardless of the age of animal and the count of neutrophils itself. Hematological findings in female monkeys of wild-origin estimated to be more than 5 years old were judged to correspond to the findings in laboratory-bred female monkeys aged 5 to 10 years. Huser[5] described that in nonhuman primates about 200 of the mononuclear cell counts were recorded to be atypical lymphocytes. Most of lymphocytes observed in our cynomolgus monkeys were more than 10 pm in diameter having round or elliptical nuclei. Since EDTA- 2Na which may interfere with the morphology of leucocytes was used as an anticoagulant the present study could not accurately differentiate artif actitious atypical lymphocytes from true atypical ones. In our monkeys however lymphocytes having dark-blue cytoplasm stained with Giemsa stain were observed. In normal humans such lymphocytes are called mononuclear lymphocytes counting 1.3% of all plasmacytoid cells [5 17]. Moreover cells with 2 lobes or kindney-shaped nuclei and cells with nuclei of indented form were also observed in our monkeys. Those were considered to be atypical lymphocytes. The incidence of such cells will be studied further. Acknowledgement We sincerely express our thanks to Dr. T. Nagasawa of the Faculty of Clinical Medicine the University of Tsukuba for his valuable aid and advice to the differential observation of leucocytes in this work. References [1] Berchelmann M. L. Vice T. E. and Kalter S. S.(1971). The hemogram of the maternary-reared neonatal and infant baboon (Papio cynocephlus). Lab. Anim. Sci. 21 564-571. [2] Berchelmann M. L. Eichberg J. W. and Kalter S. S. (1975). Hemograms of gnotobiotic and conventional infant baboons (Papio cynocephalus). Lab. Anim. Sci. 25 621-628. [3] Honjo S. Cho F. and Terao K.(1984). Establishing the cynomolgus monkey as a laboratory animal. In Research on Nonhuman Primates A. G. Hendrickx (edit.) Adv. Vet. & Comp. Med. 28 51-80. Academic Press New York. [4] Honjo S. (1985). The Japanese Tsukuba Primate Center for Medical Science(TPC) : An Outline. J. Med. Primatol. 14 75-89. [5] Huser H. J.(1970). Atlas of Comparative Primate Hematology. Academic Press New York. [6] Lucey J. F.(1963). Physiologic jaundice in newborn rhesus monkey. Am. J of Dis. of Child. 106 60-65. [7] Martin D. P. McGovan. M. J. and Loeb W.F. (1973). Age related changes of hematologic values in infant Macaca mulatta. Lab. Anim. Sci. 23 194-200. [8] McNees D. W. and Panzo B. J. (1982). Hematology of common marmosets -150.. Primates. 23 145 [9] Matsumura T. (1967). Shinseiji no ketsuekigaku (Neonatal Hematology) (in Japanese) IGAKU SHOIN Ltd. Tokyo. [10] Rosenblum I. Y. and Coulson F.(1981). Normal range and longitudinal blood chemistry and hematology values in juvenile and adult rhesus monkey (Macaca mulatta). Ecotoxicology and Environmental Safety. 5 401-411. [11] Samonds K. W. Ausmann Lynne M. and Hegsted D. M. (1974). Hematological development of the cebus monkey. Folia Primatol. 22. 72-79. [12] Seki M. Hirashima K. and Kobayashi K. (1981). Hematology of Experimental Animals (in Japanese). SOFT SCIENCE Ltd. Tokyo. [13] Sly D. Lewis London William T. Palmer
447 Ames E. and Pice Jerry M.(1978). Growth and hematologic development of the patas monkey. J. Med. Primatol. 7 156-164. [14] Tanimoto Y. (1982). HEMATOLOGY : Intersection of Man and Experimental Animals. (in Japanese). SEISHI SHOIN Ltd. Tokyo. [15] Vogin Eugene E. and Oser Felix.(1971). Comparative blood values in several species of nonhuman primates. Lab. Anin. Sci. 21 937-941. [16] Wall Hally S. and Else James G. (1983). Normal hematology values of sykes monkeys (Cercopithecus mitis albogularis). Am. J. Primatol. 5 77-81. [17] Wood Turner A. (1967). The atypical lymphocyte. Am. J. Med. 42 923-936.