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Hematology and serum biochemistry parameters of captive Chinese alligators (Alligator sinensis) during the active and hibernating periods
Tissue and Cell 51 (2018) 8–13
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Hematology and serum biochemistry parameters of captive Chinese alligators (Alligator sinensis) during the active and hibernating periods Fei Penga, Xianxian Chena, Ting Menga, En Lia, Yongkang Zhoub, Shengzhou Zhanga,
T
⁎
a Key Laboratory for Conservation and Use of Important Biological Resources of Anhui Province, College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, China b Alligator Research Center of Anhui Province, Xuancheng, 242000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Chinese alligator Hematology Serum biochemistry Hibernation
The Chinese alligator Alligator sinensis is an endangered freshwater crocodilian species endemic to China. Hematology and serum biochemistry reference range are useful in the assessment and management of animal health condition. In this study, a total of 74 Chinese Alligators (30 males and 44 females) were examined to establish reference range values of hematology and serum biochemistry parameters during the active and hibernating periods. We measured and analyzed 9 hematology and 21 serum biochemistry parameters including 4 serum electrolyte parameters, and described the morphology of different types of blood cells. No statistical differences between the sexes were found for hematology parameter, while significant differences were noted for some serum biochemistry parameters, with males having greater alkaline phosphatase activity level and lower globulin concentration value than females. There were some significant differences between the two different periods with alligators during the active period possessing lower values for mean corpuscular volume, mean corpuscular hemoglobin, total bilirubin and creatine kinase, but higher values for red blood cell and white blood cell counts, monocyte percentage, aspartate aminotransferase, a-amylase, blood urea nitrogen, creatinine, triglycerides, and cholesterol. These baseline data were essential for health condition evaluation and disease diagnosis of this endangered species.
1. Introduction The Chinese alligator Alligator sinensis is one of the most endangered freshwater crocodilian species endemic to eastern China with an important ecological and economic value (Yan et al., 2005). Historically, the Chinese alligator was distributed widely in China, but in recent years, numerous factors, such as habitat destruction, indiscriminate hunting, environment pollution, diseases, have put this species in danger of extinction. Currently, the total population of wild Chinese alligators is probably less than 130 and is declining at an annual rate of 4–6% (Thorbjarnarson et al., 2002). To prevent this species becoming extinct, Anhui Research Center for Chinese Alligator Reproduction (ARCCAR) was established at Xuancheng, Anhui province of China in 1979. In the past two decades, a successful breeding program has generated a captive-bred population of more than 10,000 alligators at ARCCAR (Yan et al., 2005). Meanwhile,
the commercial breeding of Chinese Alligator has been gradually carried out in several provinces of China. Along with the growth of the number of breeding Chinese alligators, the mortality rate from causes of various diseases increased quickly (Chen et al., 2003), which would be a major obstacle to the breeding of this alligator species. The blood examination is the most straightforward and less invasive technique to evaluate the health condition of animals (Artacho et al., 2007). The combination of some parameters is required to identify anemia, infections, inflammatory diseases, hematopoietic disorders, parasitemia, and homeostatic alterations (Campbell and Ellis, 2007; Martins et al., 2008; Vasaruchapong et al., 2014). Erythrocyte counts, HGB and PCV (see abbreviations table) usually reflect the efficiency of oxygen-carrying capacity and nutritional status. Leukocyte counts are often used to evaluate the specific and non-specific immunological conditions, such as infectious diseases (Jamalzadeh et al., 2009), inflammation (Martins et al., 2006), parasitism (Azevedo et al., 2006),
Abbreviations: RBC, red blood cell; WBC, white blood cell; ESR, erythrocyte sedimentation rate; HGB, hemoglobin content; PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; ALP, alkaline phosphatase; Amy, a-amylase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Cho, cholesterol; Cre, creatinine; Glu, glucose; TG, triglycerides; TB, total bilirubin; UA, uric acid; BUN, blood urea nitrogen; GGT, γ-glutam; LDH, lactate dehydrogenase; CK, creatine kinase; TP, total protein; Alb, albumin; Glo, globulin ⁎ Corresponding author. E-mail address: [email protected] (S. Zhang). https://doi.org/10.1016/j.tice.2018.02.002 Received 14 December 2017; Received in revised form 3 February 2018; Accepted 4 February 2018 Available online 06 February 2018 0040-8166/ © 2018 Elsevier Ltd. All rights reserved.
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shown in Table 1. The blood samples were obtained from the caudal vein of alligators, which were restrained manually, none of the alligators were sedated in order to avoid the interference of anesthetic drugs on the blood parameters, and also to prevent additional stress (Brites and Rantin, 2004). Approximately 6 ml blood was taken using a sterile 10 ml syringe with 20 g needles. Tubes coated with K2-EDTA as an anticoagulant were used to collect samples for hematologic analysis, and tubes containing sodium citrate for ESR, and serum separation tubes without anticoagulant were used to collect samples for serum biochemistry parameters and electrolytes. These samples would be processed in less than 24 h in order to avoid the formation of cell fragments (Canfield, 1998).
and stress (Silveira-Coffigni et al., 2004). The serum biochemical parameters such as protein levels, enzymes, and electrolytes could provide information on internal organs, nutritional status, and metabolic state (Newman et al., 1997). Some reference values of hematology and serum biochemistry parameters have been reported for several crocodilian species, including farmed saltwater crocodile(Crocodylus porosus) (Millan et al., 1997), captive mugger crocodiles (Crocodylus palustris) (Stacy and Whitaker, 2000), wild Nile crocodiles (Crocodylus niloticus) (Lovely et al., 2007), Morelet’s crocodiles (Crocodylusmoreletii) (Padilla et al., 2011), and the wild Spectacled Caiman (Caiman crocodilus crocodilus) (Rossini et al., 2011). These studies revealed both conservative properties and some notable population variations in hematology and serum biochemistry parameters between and within species of crocodilians due to inherent genetic differences and various external factors, such as weather, environmental conditions, and diet of populations studied. However, to date, there is still no a comprehensive and complete reference range on hematology and serum biochemistry parameters of Chinese alligators. The aim of this study was to establish baseline hematology and serum biochemistry reference ranges for the breeding Chinese alligators during both the active and hibernating period. To our knowledge, this study may be the first contribution with comparisons of hematology and serum biochemistry parameters related to the active and hibernating periods for a crocodilian species. These baseline data were essential for health condition evaluation and disease diagnosis and useful for conservation and sustainable utilization of this endangered species.
2.3. Analysis of hematology and biochemistry parameters Total number of erythrocytes (RBC) and leukocytes (WBC) were analyzed in Neubauer Chamber, with dilution being performed by standard Hayem's solution for RBCs and Turk's solution for WBCs (Parida et al., 2011). Thrombocytes were identified and counted in the 4 corner squares of the large central square of Neubauer chamber, using Hayem's solution. The HGB concentration was determined using an automated hematology analyzer (BC-3000plus, Mindray, Shenzhen, China). The PCV was examined by using the microhematocrit method (Parida et al., 2011). The tubes were spun in a microhematocrit centrifuge (TDL-50B, Anke, Anting Scientific Instrument, Shanghai, China) for 5 min at 12000 rpm and the PVC was calculated with the total blood level divided by the blood cell level. The MCH, MCV and MCHC were calculated according to standard formulas (Campbell and Ellis, 2007). Erythrocyte sedimentation rate was determined by Westergren. The blood smears were stained with Wright Stain Solution (Tianda Diagnostic Reagents Co., LTD. Hefei, China) for the differential leukocyte count and then examined under a microscope (BM2000, Jiangnan Yongxin Co., LTD. Nanjing, China). The percentages of different leukocytes were determined after counting a total of 1000 cells. Serum biochemistry parameters including activities of CK, ALT, AST, GGT, LDH, Alp, Amy, and concentrations of Cre, UA, Bun, TP, Alb, Glo, GLU, TB, Cho, TG were measured on serum samples by using an autoanalyzer (KHB 450, Shanghai, China) and the concentrations of K, Na, Cl and nCa (standard calcium ion concentration at pH7.4) in serum were measured using a electrolyte analyzer (IMS-972, HORRON, Shenzhen, China).
2. Materials and methods 2.1. Ethics statement This work was conducted as part of a population health assessment approved and supported by the Anhui Research Center for Chinese Alligator Reproduction and college of Life Sciences, Anhui Normal University. All the handing and sampling were performed in compliance with standard vertebrate protocols and veterinary practices, and in accordance with national and provincial Guidelines. 2.2. Animals and sample collection
2.4. Statistical analysis
The Chinese alligators are amphibious poikilotherm whose body temperature and movement change closely as the ambient temperature changes, They would begin to enter the state of hibernation (approximately from late October to late April in the next year) when the environmental temperature continuously falls below 14 °C. In our study, a total of 74 apparently healthy adult Chinese alligators were collected from the Anhui Research Center for Chinese Alligator Reproduction in Xuancheng, Anhui province during the active period (in late May) and the hibernating period (in mid-January), respectively, from January 2014 to May 2016. These alligators were examined for clinical signs of trauma and illness, and the gender was determined by digital palpation of genital organs in the cloacae (Ziegler and Olbort, 2007). Prior to blood collection, sex, length and mass were determined and
Hematology and serum biochemistry data were presented as means and standard deviation (SD) via the software SPSS19.0 for Windows. The results were compared among periods for each sex and among sexes within each period. Significant difference between means was determined using an independent sample t-test model. Results were considered significant at p < 0.05. 3. Results In this study, no hemolysis occurred nor parasites were observed. No chyle serum (having a milky appearance and being abundant in TG
Table 1 Demographics of 74 captive adult Chinese alligators. Group
Number Age range (y) Mass (kg) Body length (cm)
Males
Females
Active period
Hibernation
Active period
Hibernation
9 10–15 13.34 ± 1.28 (9.81–16.86) 145.7 ± 5.72 (132.9 157.4)
21 10–15 15.09 ± 3.14 (9.98 17.23) 147.6 ± 5.91 (132.8 159.3)
22 10–15 12.80 ± 1.47 (9.50 15.70) 145.3 ± 5.39 (132.0 153.1)
22 10–15 13.96 ± 3.01 (9.82 16.12) 147.1 ± 5.97 (132.5 158.8)
9
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Fig. 1. Light micrographs of peripheral blood cells of Chinese alligators with Wright staining (Bar = 10.00 um). A. Erythrocyte B. Thrombocyte C. Heterophil D. Eosinophil E. Basophil F. Large lymphocyte G. Small lymphocyte H. Monocyte
Table 2 Hematology parameters of Chinese alligators during the active and hibernating periods. Hematology Parameters
11
a
RBC (×10 /L) WBC (×109/L) a Thrombocyte (×109/L) HGB (g/L) PCV (%) MCV (fl) a MCH (pg) a MCHC (g/L) Heterophil (%) Lymphocyte (%) Monocyte (%) a Eosinophil (%) Basophil (%) N/L ratio ESR (mm/H)
Males
Females
Active period
Hibernation
Active period
Hibernation
4.91 ± 1.26 (3.02–6.09) 2.15 ± 0.74 (1.37–3.07) 17.70 ± 4.37 (9.62–24.10) 96.72 ± 7.17 (88.00–104.00) 28.24 ± 2.76 (25.37–32.00) 575.13 ± 64.83 (507.01–655.32) 196.99 ± 30.12 (169.48–237.71) 342.43 ± 31.22 (301.43–393.54) 59.54 ± 7.13 (57.01- 68.20) 19.70 ± 5.32 (9.65–26.93) 15.25 ± 4.22 (7.39–20.34) 4.40 ± 1.27 (1.37–6.11) 1.11 ± 0.44 (0–1.96) 4.95 ± 2.33 (2.65–7.62) 3.63 ± 1.10 (2–4)
4.23 ± 1.32 (2.79–5.97) 2.00 ± 0.67 (1.30–2.97) 17.61 ± 3.84 (9.36–20.67) 99.75 ± 7.14 (90.00–107.00) 28.13 ± 2.66 (25.20–31.84) 665.02 ± 70.12 (594.51–767.23) 235.79 ± 30.03 (209.33–273.72) 354.61 ± 32.99 (307.39–398.02) 62.37 ± 2.74 (69.29–70.92) 19.41 ± 5.56 (12.31–23.21) 12.67 ± 2.92 (5.27–16.13) 4.38 ± 1.53 (1.54–6.55) 1.18 ± 0.40 (0.53–1.86) 5.26 ± 2.37 (2.12–8.03) 3.43 ± 0.50 (2–5)
4.97 ± 0.83 (4.00–6.13) 2.12 ± 0.62 (1.45–2.99) 17.66 ± 4.67 (10.17–26.02) 96.26 ± 14.64 (64.00–103.00) 27.70 ± 1.09 (26.48–29.30) 557.38 ± 41.36 (514.10–599.94) 193.67 ± 29.96(158.08–227.43) 347.51 ± 40.07 (302.01–396.12) 59.46 ± 7.35 (56.13–67.08) 19.71 ± 4.91 (9.77–25.12) 15.41 ± 5.47 (7.81–21.03) 4.27 ± 1.54 (1.50–6.66) 1.13 ± 0.39 (0–1.82) 4.94 ± 2.21 (2.69–7.58) 3.67 ± 1.21 (2–4)
4.37 ± 1.43 (3.30–6.04) 1.97 ± 0.89 (1.25–2.94) 17.63 ± 6.55 (9.56–26.57) 98.90 ± 9.76 (83.00–109.00) 27.65 ± 1.89 (23.84–29.27) 632.69 ± 42.81 (565.07–678.75) 226.32 ± 32.72 (175.47–259.61) 357.67 ± 33.26 (316.21–398.72) 62.62 ± 8.42 (60.32–71.18) 19.04 ± 5.93 (8.82–26.96) 13.00 ± 5.41 (7.27–18.05) 4.19 ± 1.52 (1.47–6.01) 1.15 ± 0.38 (0–1.91) 5.43 ± 2.30 (2.10–7.93) 3.44 ± 1.03 (2–5)
agranulocytes. Heterophils were the predominant leucocytes in number, followed by monocytes, lymphocytes, eosinophils, basophils (Table 2). Heterophils were round and large or irregular in shape with an eccentrically located nucleus which was usually kidney-shaped, oval, bilobate or trilobed. Their cytoplasm stained light blue and contained some very small and pink granules (Fig. 1C). Eosinophils were also round in overall appearance with lobed nuclei (Fig. 1D). The nucleus of eosinophils was bilobed or concentrated, located at one end of the cells, the cytoplasm was darkly brown-yellow with abundant strongly eosinophilic granules. Typically, nuclei were completely hidden by the large elongate granules. Basophils were round or oval cells with segmented nuclei (Fig. 1E). The strongly basophilic granules of basophils were large dark atropurpureus granules with which the entire cell was covered, similar to the eosinophils. Based on size, lymphocytes were categorized as large and small lymphocytes (Fig. 1F and G), both of which were rounded or spherical in shape, and possessed eccentric, round, notched nuclei which occupied almost the entire cell leaving a narrow rim of pale or light blue cytoplasm. Normally, monocytes were the largest leucocytes, irregular or rounded in appearance (Fig. 1H), with light blue to gray cytoplasm, and often having
and Cho) appeared during the hibernating period, but some chyle serum appeared during the active period. The morphology of each blood cell type under light microscope for Chinese alligators is shown in Fig. 1. Hematology and biochemistry parameters during the active and hibernating periods are summarized in Tables 2 and 3, respectively.
3.1. Morphology of the blood cells in Chinese alligators The characteristic shape of erythrocytes of Chinese alligators was observed to be elliptical or elongated oval with an oval or round nucleus placed centrally (Fig. 1A). On occasion, some erythrocytes with damaged membranes, spindle-shaped cells and teardrop-shaped cells were seen. Thrombocytes of Chinese alligators presented various morphologies on Wright-stained blood films, often appearing in clusters with 2–10 cells. Generally, they were from fusiform to spindle, elliptical or ovalshaped with a nucleus following the shape of the cell (Fig. 1B). Leucocytes of Chinese alligators could be classified into the following five types: heterophils, basophils, eosinophils, monocytes and lymphocytes; the first three were granulocytes while the rest were 10
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Table 3 Serum biochemistry parameters of Chinese alligators during the active and hibernating periods. Biochemistry Parameters
CK (U/L) a Amy (U/L) a LDH (U/L) ALT (U/L) AST (U/L) a GGT (U/L) ALP (U/L) b TP (g/L) Alb (g/L) Glo (g/L) b A/G TB (umol/L) a BUN (mmol/L) a Cre (umol/L) a UA (umol/L) GLU (mmol/L) TG (mmol/L) a Cho (mmol/L) a K (mmol/L) Na (mmol/L) Cl (mmol/L) NCa (mmol/L) Pb (ug/L)
Males
Females
Active period
Hibernation
Active period
Hibernation
149.22 ± 55.07 (72–218) 727.37 ± 86.97 (620–851) 354.21 ± 77.21 (193–458) 89.07 ± 23.72 (63–128) 193.32 ± 41.64 (147–237) 0 42.10 ± 22.03 (23–91) 73.77 ± 7.39 (67–86.72) 26.47 ± 3.75 (22.73–30.82) 44.41 ± 5.85 (41.01–49.74) 0.56 ± 0.07 (0.51–0.71) 1.20 ± 0.47 (0,72–1.88) 0.44 ± 0.07 (0.35–0.59) 31.19 ± 14.54 (14.54–53.18) 213.90 ± 143.04 (106–479) 6.86 ± 2.01(4.50–9.57) 9.02 ± 1.07 (3.97–16.11) 6.21 ± 0.94 (5.21–7.02) 4.33 ± 0.40 (3.91–4.87) 136.81 ± 3.67(133.17–139.54) 82.74 ± 7.79 (71.93–94.02) 1.20 ± (1.16–1.29) 41.33 ± 14.67 (24.18–57.21)
758.73 ± 99.43(611.00–835.40) 628.00 ± 59.04 (566–687) 306.86 ± 98.24(178.10–421.20) 80.33 ± 16.06 (64–105) 143.00 ± 35.45 (94–199) 11.12 ± 1.21 (2.00–6.11) 41.37 ± 20.25 (25–90) 71.35 ± 5.29 (63–78.6) 25.15 ± 2.97 (22.10–29.40) 44.2 ± 3.00 (40.90–49.20) 0.55 ± 0.08 (0.50–0.70) 2.66 ± 0.80 (1.10–3.49) 0.28 ± 0.10 (0.16–0.50) 6.90 ± 2.97 (3.00–9.46) 183.00 ± 53.32 (104–234) 6.66 ± 2.02 (4.21–9.28) 0.35 ± 0.05 (0.27–0.49) 2.10 ± 0.71 (0.65–2.84) 4.51 ± 0.11(4.51–4.75) 134.89 ± 0.82 (134.03–135.78) 85.98 ± 1.20 (84.30–87.60) 1.25 ± 0.01 (1.24–1.27) 40.76 ± 13.71 (25.23–55.98)
144.80 ± 57.37 (71–210) 725.57 ± 66.36 (623–845) 347.40 ± 78.11 (196–425) 87.00 ± 26.28 (56–140) 194.08 ± 27.41 (153–239) 0 36.67 ± 11.29 (16–50) 78.46 ± 6.95 (70.20–89.80) 28.00 ± 1.66 (26.40–31.60) 50.46 ± 6.20 (43.80–61.10) 0.58 ± 0.07 (0.49–0.71) 1.13 ± 0.32 (0.60–1.54) 0.43 ± 0.06 (0.36–0.56) 31.83 ± 14.36 (17.03–54.92) 217.83 ± 150.25 (100–512) 6.84 ± 2.21 (3.75–9.30) 9.15 ± 4.37 (4.09–15.71) 6.25 ± 0.46 (5.28–6.89) 4.28 ± 0.28 (3.94–4.76) 137.57 ± 4.99 (131.70–147.70) 81.54 ± 7.72 (70.30–93.60) 1.17 ± 0.06 (1.09–1.26) 41.00 ± 15.52(26–57)
729.98 ± 89.23 (603.23–812.13) 643.33 ± 87.29 (571–752) 301.37 ± 94.03 (176.11–417.27) 81.50 ± 24.47 (58–127) 147.83 ± 82.93 (76–296) 12.03 ± 7.01 (2–19) 35.33 ± 10.03 (15–54) 76.05 ± 5.17 (71.21–85.23) 25.05 ± 3.04 (20.50–28.20) 51.00 ± 7.96 (43.30–64.70) 0.52 ± 0.14 (0.30–0.70) 2.29 ± 0.86 (0.91–3.70) 0.26 ± 0.10 (0.18–0.41) 6.40 ± 2.73 (3.91–12.63) 177 ± 55.07 (94–242) 6.99 ± 1.93 (5.22–9.12) 0.36 ± 0.12 (0.29–0.51) 2.21 ± 0.87 (1.40–3.89) 4.59 ± 0.66 (4.18–5.59) 134.27 ± 2.66 (130.9–137.4) 86.03 ± 5.61(79.90–91.90) 1.27 ± 0.08 (1.20–1.39) 43 ± 13.19 (25.32–56.17)
4. Discussion
a few cytoplasmic pseudopodia, with irregularly indented nuclei eccentrically placed.
The health assessments and disease diagnosis are very important for the protection of endangered animal species, so it is essential to establish species-specific reference ranges for hematology and serum biochemistry parameters which could aid in the identification of alterations in animal health condition (Lewbart et al., 2014). Hematology and serum biochemistry parameter values vary under different physiological, ecological, age, sex, and environmental conditions (Sarasola et al., 2004; Seaman et al., 2005; Boily et al., 2006). As we know, for ectothermic vertebrates, two characteristic phases can be distinguished in the annual life cycle, that is, periods of activity and hibernation. In this study, sampling was therefore performed at two fixed time of each year during the active period (in late May, ambient temperature 21–28 °C) and hibernating period (in mid-January, ambient temperature −2–6 °C). Morphologically, the non-mammalian erythrocyte (such as in fishes, amphibians, reptiles, and birds) is nucleated, flattened and ellipsoidal (Rowley and Ratcliffe, 1988). The erythrocyte shape of Chinese alligators was generally similar to that of other non-mammalian species. The mean corpuscular volume of the erythrocyte in Chinese alligators was smaller than that of Dubois’s tree frog (upper range 851.31 fl) (Das and Mahapatra, 2014), while larger than that of mute swans (upper range 196.2 fl) (Dolka et al., 2014). The thrombocyte of lower vertebrates was the equivalent of platelet of mammalian species in the aspect of function, being responsible for clotting and wound healing. The thrombocytes of Chinese alligators were variable in form, but often presented a fusiform, spindle, or long oval shape, which was similar to those of lower vertebrates such as fishes and amphibians, such as Schizothorax prenanti (Fang et al., 2014) and American bullfrog (Bricker et al., 2012); while they differed from previously reported for the giant lizard (Gallotia simonyi) that possessed round or ovoid thrombocytes (Martinez-Silvestre et al., 2005) and are also easily distinguished from mammalian and human platelets which are anuclear and usually, round, stellate, or elliptical. The heterophils of Chinese alligators were the predominant cell type of leucocytes. The shape of heterophils in this species was generally similar to that of human neutrophils, but they contained more basophilic granules and present mostly with a taenia nucleus when
3.2. Hematology parameters of Chinese alligators during the active and hibernating periods Hematology parameters of Chinese alligators are shown in Table 2. Variations in relation to two different sampling periods are noted. The values of RBC and WBC counts, and monocyte percentage were significantly greater during the active period, while the values of MCV and MCH were significantly lower during the active period than those during the hibernating period. Additionally, the ESR value tended to increase during the active period, whereas the values of HGB and heterophil percentage tended to decrease during the active period compared with those during the hibernating period, but the differences were not statistically significant. Meanwhile, some differences in relation to sex were also found, the male alligators tended to have higher PCV, and lower RBC counts than the females, but these differences were not statistically significant.
3.3. Serum biochemistry parameters of Chinese alligators during the active and hibernating periods Serum biochemistry parameters of Chinese alligators are shown in Table 3. Variations in relation to the two sampling periods are noted. The values of Amy, AST, Bun, Cre, TG and Cho were significantly greater during the active period, while the values of CK and TB were statistically lower during the active period than those during the hibernating period. In addition, the values of LDH, ALT, UA tended to increase during the active period, whereas the values of K, Cl and nCa tended to decrease during the active period versus those during the hibernating period, but the differences were not statistically significant. Meanwhile, some differences in relation to sex were also found, the male alligators had significantly higher ALP activity, and significantly lower Glo concentration than the females. Additionally, TP concentrations for males tended to be lower than those for females, but were not statistically different. 11
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increased CK activity was probably associated with heart and skeletal muscle inflammation in animals (Yousaf and Powell, 2012). As CK activity increased, the level of serum TB concentration was also notably increased during the hibernating period. Normally, the high level of TB concentration suggested hepatobiliary disease, which is considered more specific than serum liver enzyme (Webster, 2005). Most poikilothermic animals would hibernate in order to have a greater adaptation to survive in the bitter cold environment, but the deep hibernating animals would probably result in body damage, due to lower metabolic activity, and weak self-regulation and self-healing, which could be explained the higher CK and TB values for Chinese alligators during the hibernating period. According to our information, dozens of Chinese alligators in ARCCAR die during hibernation every year, which was probably related to physical damage in hibernating state. BUN and Cre are very helpful to diagnose kidney disease in animals (Kaneko et al., 2008). There was a significant difference for BUN and Cre levels between the active and hibernating periods with both parameter values from the active period being markedly increased versus those from the hibernating period. This change may be a reflection of feeding and enhanced metabolic activity during the active period. TG and Cho are often used as indexes in evaluation of nutritional status, and hyperlipidemia for obese animals (Kawasumi et al., 2014). The values for the both parameters of Chinese alligators were significantly greater during the active period than those during the hibernating period. TG and Cho levels are directly associated with feeding status (Girling et al., 2015). Elevation in TG and Cho levels during the active period for Chinese alligators may be attributed to consuming additional food to reserve nutrition, in order to copulate in mid-June and lay eggs in July. Previous studies indicated that Cho levels were higher in captive American alligators (Alligator mississippiensis) than in wild American alligators; the Cho levels of Chinese alligators during the active period were higher than that of captive American alligators, with the exception of breeding female American alligators (Lance et al., 2001). In conclusion, this report provided the first comprehensive reference ranges of hematology and serum biochemistry for healthy farmed Chinese alligators during the active and hibernating periods. Most of the hematology and serum biochemistry parameters for Chinese alligators were noted to be similar among the sexes, only two significant differences existed in ALP and Glo values. Some significant differences were found between the two different seasonal periods with alligators during the active period possessing lower values for MCM, MCH, TB and CK, but higher values for RBC and WBC counts, monocyte percentage, AST, Amy, BUN, Cre, TG, and Cho. These baseline data are essential for health monitoring and disease diagnosis and useful for conservation and sustainable utilization of this endangered species.
compared with human and mammalian neutrophils. The eosinophils of Chinese alligators contained abundant, bar-shaped, brown-yellow cytoplasmic granules, which was not consistent with those of lower vertebrates, such as the American bullfrog with globular granules that were light red (Bricker et al., 2012), or those of higher vertebrates, such as human with orange, round or oval, smaller globular granules. In our present study, the basophils and lymphocytes were more similar to other species equivalent cells, except that the basophils contained more and larger basophilic granules than human basophils. Generally, most of the hematology and serum biochemistry parameter values for Chinese alligators were noted to be similar among both sexes, the only two significant differences existed in the ALP and Glo values. Consistently, previous studies also indicated that the vast majority of blood parameter values were equivalent for both sexes in lower vertebrates such as reptiles, amphibians, and fishes, such as Geoffroy's side-necked turtle (Zago et al., 2010), American bullfrogs Rana catesbeiana (Cathers et al., 1997) and Xenopus laevis (Wilson et al., 2011), which may be due to incompletion of gender differences in evolution for lower vertebrates. Seasonality often notably affected some reptilian blood parameter values (Lillywhite and Smits, 1984; Pal et al., 2008). In general, two characteristic phases can be easily distinguished in the annual life cycle for lower vertebrates, that is, periods of activity and hibernation. Up to date, few documents were available on hematology and serum biochemistry parameters in crocodilians during hibernation. RBC counts for Chinese alligators surveyed during the active period were notably greater, while the values of MCV and MCH for the hibernating alligators were significantly higher. Tavares-Dias et al. (2008) have suggested that species with high RBC counts could be well acclimated to a higher metabolic activity. This could account for the elevation of RBC counts for alligators examined during the active period. Prosser (1973) reported a higher MCH value is due to larger size of RBCs. In this study, the hibernating Chinese alligators possessed low numbers of RBCs, but higher MCH levels and larger MCV values, which may contribute to the maintenance of a sufficient oxygen availability to tissues in slow blood flow during hibernation. It is well known that leukocytes in humans and animals are directly associated with immunological response of the organism, such as stress, infection, and inflammation (Swenson, 1984). In this study, the significant increase of WBC counts, especially in monocytes, during the active period possibly due to presence of more risk factors for the active alligators. Similarly, in other reptile species, such as turtles (Wilkinson, 2004), elevation of WBC counts was also noted in summer. More notable differences were found between the two different periods in serum biochemistry parameters for Chinese alligators. Serum α-amylase is mainly secreted by sialaden and pancreas and plays a key role in animal carbohydrate metabolism; it can catalyze the hydrolysis of glycogen, starch, and various other related carbohydrates (Strobl et al., 1998; Franco et al., 2000). The amylase activity levels reported here for Chinese alligators during the active period were strikingly increased, which was likely due to consuming food stimulation during the active period. AST was the other enzyme found to be significantly increased during the active period in this study. AST is a liver-specific enzyme in humans and animals (Jenkins, 2008). Elevation of AST activity for Chinese alligators is probably related to the enhancement of hepatocyte metabolism during the active period. Additionally, we found that the AST activity of Chinese alligators during the active period was significantly greater than that of some crocodilian species such as wild spectacled caiman Caiman crocodilus crocodilus (Rossini et al., 2011), farmed saltwater crocodile Crocodylus porosus (Millan et al., 1997), and wild Nile crocodiles Crocodylus niloticus (Lovely et al., 2007), but lower than that of American alligators Alligator mississippiensis (Hamilton et al., 2016). In this study, CK was the only enzyme demonstrating significantly higher activity in Chinese alligators during hibernation. CK usually presents in skeletal muscle, cardiac muscle, brain, and smooth muscle;
Acknowledgments We would like to thank Xie Yuling for her help in photo processing. We also thank Wuhu Zhengyu medical laboratory for help with determination of serum biochemistry and HGB concentration values. This work was supported by the key program of education bureau of Anhui Province (Grant No. KJ2013A126) and Natural Science Foundation of Anhui Province (Grant No. 11040606M75). References Artacho, P., Soto-Gamboa, M., Verdugo, C., Nespolo, R.F., 2007. Using haematological parameters to infer the health and nutritional status of an endangered black-necked swan population. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 147, 1060–1066. Azevedo, T.M.P., Martins, M.L., Bozzo, F.R., Moraes, F.R., 2006. Hematological and gill responses in parasitized tilapia from valley of Tijucas River, SC, Brazil. Sci. Agric. 63 (2), 115–120. Boily, F., Beaudoin, S., Measures, L.N., 2006. Hematology and serum chemistry of Harp (Phoca groenlandica) and Hooded seals (Cystoohora cristata) during the breeding season in the Gulf of St. Lawrence. Can. J. Wildl. Dis. 42, 115–132. Bricker, N.K., Raskin, R.E., Densmore, C.L., 2012. Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis
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of spleen cells from 2 species of frog, Rana catesbeiana(Aquarana) and Xenopus laevis. Vet. Clin. Pathol. 41 (3), 353–361. Brites, V.L.C., Rantin, F.T., 2004. The influence of agricultural and urban contamination on leech infestation of freshwater turtels, Phrynops geoffroanus, taken from two areas of the Uberabinha River. Environ. Monit. Assess. 96 (1–3), 273–281. Campbell, T.W., Ellis, W.C., 2007. Avian and Exotic Animal Haematology and Cytology, 3rd edition. Blackwell Publishing, USA, pp. 51–81. Canfield, P.J., 1998. Comparative cell morphology in the peripheral blood film from exotic and native animals. Aust. Vet. J. 76, 793–800. Cathers, T., Lewbart, G.A., Correa, M., Stevens, J.B., 1997. Serum chemistry and hematology values for anesthetized American bullfrogs (Rana catesbeiana). J. Zoo Wildl. Med. 28, 171–174. Chen, B.H., Hua, T.M., Wu, X.B., Wang, C.L., 2003. Research on the Chinese Alligator. Shanghai Scientific and Technical Publishers, Shanghai (in Chinese). Das, M., Mahapatra, P.K., 2014. Hematology of wild caught Dubois’s tree frog Polypedates teraiensis, Dubois, 1986 (Anura: rhacophoridae). Sci. World J. 2014, 491415. http:// dx.doi.org/10.1155/2014/491415.eCollection2014. Dolka, B., Wlodarczyk, R., Zbikowski, A., Dolka, I., Szeleszczuk, P., Klucinski, W., 2014. Hematological parameters in relation to age, sex and biochemical values for mute swans (Cygnus olor). Vet. Res. Commun. 38, 93–100. Fang, J., Chen, K., Cui, H.M., Peng, X., Li, T., Zuo, Z.C., 2014. Morphological and cytochemical studies of peripheral blood cells of Schizothorax prenanti. Anat. Histol. Embryol. 43 (5), 386–394. Franco, O.L., Rigden, D.J., Melo, F.R., Bloch C.Jr. Silva, C.P., Grossi de Sa′, M.F., 2000. Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities. Eur. J. Biochem. 267, 2166–2173. Girling, S.J., Campbell-palmer, R., Pizzi, R., Fraser, M.A., Cracknell, J., Arnemo, J., Rosell, F., 2015. Haematology and serum biochemistry parameters and variations in the Eurasian Beaver (Castor fiber). PLoS One 10 (6), e128775. Hamilton, M.T., Kupar, C.A., Kelley, M.D., Finger Jr, J.W., Tuberville, T.D., 2016. Blood and plasma biochemistry reference intervals for wild juvenile American alligators (Alligator mississippiensis). J. Wildl. Dis. 52 (3), 631–635. Jamalzadeh, H.R., Keyvan, A., Ghomi, M.R., Gherardi, F., 2009. Comparison of blood indices in healthy and fungal infected Caspian salmon (Salmo trutta caspius). Afr. J. Biotechnol. 8, 319–322. Jenkins, J.R., 2008. Rodent diagnostic testing. J. Exotic Pet. Med. 17, 16–25. Kaneko, J.J., Harvey, J.W., Bruss, M.L., 2008. Appendices. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, 6th edition. Academic Press, San Diego California, pp. 889–895. Kawasumi, K., Kashiwado, N., Okada, Y., Sawamura, M., Sasaki, Y., Iwazaki, E., et al., 2014. Age effects on plasma cholesterol and triglyceride profiles and metabolite concentrations in dogs. BMC Vet. Res. 10 (1), 1–5. Lance, V.A., Morici, L.A., Elsey, R.M., Land, E.D., Place, A.R., 2001. Hyperlipidemia and reproductive failure in captive-reared alligators: vitamin E vitamin A, plasma lipids, fatty acids, and steroid hormones. Comp. Biochem. Physiol. Part B 128, 285–294. Lewbart, G.A., Hirschfeld, M., Denkinger, J., Vasco, K., Guevara, N., Garc̃a, J., Muñoz, J., Lohmann, K.J., 2014. Blood gases, biochemistry, and hematology of galapagos green turtles (Chelonia mydas). PLoS One 9 (5), e96487. http://dx.doi.org/10.1371/journal. pone.0096487. Lillywhite, H.B., Smits, A.W., 1984. Liability of blood volume in snakes and its relation to activity and hypertension. J. Exp. Biol. 110, 267–274. Lovely, C.J., Pittman, J.M., Leslie, A.J., 2007. Normal haematology and blood biochemistry of wild nile crocodiles (Crocodylus niloticus). J. S. Afr. Vet. Assoc. 78 (3), 137–144. Martinez-Silvestre, A., Marco, I., Rodriguez-Dominguez, M.A., Lavin, S., Cuenca, R., 2005. Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi). Res. Vet. Sci. 78 (2), 127–134. Martins, M.L., Moraes, F.R., Fujimoto, R.Y., Onaka, E.M., Bozzo, F.R., Moraes, J.R.E., 2006. Carrageenin induced inflammation in Piaractus mesopotamicus (Osteichthyes: Characidae) cultured in Brazil. Bol. Inst. Pesca 32, 31–39. Martins, M.L., Mourino, J.L., Amaral, G.V., Vieira, F.N., Dotta, G., Jatoba, A.M., Pedrotti, F.S., Jeronimo, G.T., Buglione-neto, C.C., Pereira, J.G., 2008. Haematological changes in Nile tilapia experimentally infected with Enterococcus. Braz. J. Biol. 68 (3), 631–637. Millan, J.M., Janmaat, A., Richardson, K.C., Chambers, L.K., Fomiattl, K.R., 1997. Reference ranges for biochemical and haematological values in farmed saltwater crocodile (Crocodylus Porosus) yearlings. Aust. Vet. J. 75, 814–817. Newman, S.H., Piatt, J.F., White, J., 1997. Hematological and plasma biochemical
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Hematology and serum biochemistry parameters of captive Chinese alligators (Alligator sinensis) during the active and hibernating periods Fei Penga, Xianxian Chena, Ting Menga, En Lia, Yongkang Zhoub, Shengzhou Zhanga,
T
⁎
a Key Laboratory for Conservation and Use of Important Biological Resources of Anhui Province, College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, China b Alligator Research Center of Anhui Province, Xuancheng, 242000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Chinese alligator Hematology Serum biochemistry Hibernation
The Chinese alligator Alligator sinensis is an endangered freshwater crocodilian species endemic to China. Hematology and serum biochemistry reference range are useful in the assessment and management of animal health condition. In this study, a total of 74 Chinese Alligators (30 males and 44 females) were examined to establish reference range values of hematology and serum biochemistry parameters during the active and hibernating periods. We measured and analyzed 9 hematology and 21 serum biochemistry parameters including 4 serum electrolyte parameters, and described the morphology of different types of blood cells. No statistical differences between the sexes were found for hematology parameter, while significant differences were noted for some serum biochemistry parameters, with males having greater alkaline phosphatase activity level and lower globulin concentration value than females. There were some significant differences between the two different periods with alligators during the active period possessing lower values for mean corpuscular volume, mean corpuscular hemoglobin, total bilirubin and creatine kinase, but higher values for red blood cell and white blood cell counts, monocyte percentage, aspartate aminotransferase, a-amylase, blood urea nitrogen, creatinine, triglycerides, and cholesterol. These baseline data were essential for health condition evaluation and disease diagnosis of this endangered species.
1. Introduction The Chinese alligator Alligator sinensis is one of the most endangered freshwater crocodilian species endemic to eastern China with an important ecological and economic value (Yan et al., 2005). Historically, the Chinese alligator was distributed widely in China, but in recent years, numerous factors, such as habitat destruction, indiscriminate hunting, environment pollution, diseases, have put this species in danger of extinction. Currently, the total population of wild Chinese alligators is probably less than 130 and is declining at an annual rate of 4–6% (Thorbjarnarson et al., 2002). To prevent this species becoming extinct, Anhui Research Center for Chinese Alligator Reproduction (ARCCAR) was established at Xuancheng, Anhui province of China in 1979. In the past two decades, a successful breeding program has generated a captive-bred population of more than 10,000 alligators at ARCCAR (Yan et al., 2005). Meanwhile,
the commercial breeding of Chinese Alligator has been gradually carried out in several provinces of China. Along with the growth of the number of breeding Chinese alligators, the mortality rate from causes of various diseases increased quickly (Chen et al., 2003), which would be a major obstacle to the breeding of this alligator species. The blood examination is the most straightforward and less invasive technique to evaluate the health condition of animals (Artacho et al., 2007). The combination of some parameters is required to identify anemia, infections, inflammatory diseases, hematopoietic disorders, parasitemia, and homeostatic alterations (Campbell and Ellis, 2007; Martins et al., 2008; Vasaruchapong et al., 2014). Erythrocyte counts, HGB and PCV (see abbreviations table) usually reflect the efficiency of oxygen-carrying capacity and nutritional status. Leukocyte counts are often used to evaluate the specific and non-specific immunological conditions, such as infectious diseases (Jamalzadeh et al., 2009), inflammation (Martins et al., 2006), parasitism (Azevedo et al., 2006),
Abbreviations: RBC, red blood cell; WBC, white blood cell; ESR, erythrocyte sedimentation rate; HGB, hemoglobin content; PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; ALP, alkaline phosphatase; Amy, a-amylase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Cho, cholesterol; Cre, creatinine; Glu, glucose; TG, triglycerides; TB, total bilirubin; UA, uric acid; BUN, blood urea nitrogen; GGT, γ-glutam; LDH, lactate dehydrogenase; CK, creatine kinase; TP, total protein; Alb, albumin; Glo, globulin ⁎ Corresponding author. E-mail address: [email protected] (S. Zhang). https://doi.org/10.1016/j.tice.2018.02.002 Received 14 December 2017; Received in revised form 3 February 2018; Accepted 4 February 2018 Available online 06 February 2018 0040-8166/ © 2018 Elsevier Ltd. All rights reserved.
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shown in Table 1. The blood samples were obtained from the caudal vein of alligators, which were restrained manually, none of the alligators were sedated in order to avoid the interference of anesthetic drugs on the blood parameters, and also to prevent additional stress (Brites and Rantin, 2004). Approximately 6 ml blood was taken using a sterile 10 ml syringe with 20 g needles. Tubes coated with K2-EDTA as an anticoagulant were used to collect samples for hematologic analysis, and tubes containing sodium citrate for ESR, and serum separation tubes without anticoagulant were used to collect samples for serum biochemistry parameters and electrolytes. These samples would be processed in less than 24 h in order to avoid the formation of cell fragments (Canfield, 1998).
and stress (Silveira-Coffigni et al., 2004). The serum biochemical parameters such as protein levels, enzymes, and electrolytes could provide information on internal organs, nutritional status, and metabolic state (Newman et al., 1997). Some reference values of hematology and serum biochemistry parameters have been reported for several crocodilian species, including farmed saltwater crocodile(Crocodylus porosus) (Millan et al., 1997), captive mugger crocodiles (Crocodylus palustris) (Stacy and Whitaker, 2000), wild Nile crocodiles (Crocodylus niloticus) (Lovely et al., 2007), Morelet’s crocodiles (Crocodylusmoreletii) (Padilla et al., 2011), and the wild Spectacled Caiman (Caiman crocodilus crocodilus) (Rossini et al., 2011). These studies revealed both conservative properties and some notable population variations in hematology and serum biochemistry parameters between and within species of crocodilians due to inherent genetic differences and various external factors, such as weather, environmental conditions, and diet of populations studied. However, to date, there is still no a comprehensive and complete reference range on hematology and serum biochemistry parameters of Chinese alligators. The aim of this study was to establish baseline hematology and serum biochemistry reference ranges for the breeding Chinese alligators during both the active and hibernating period. To our knowledge, this study may be the first contribution with comparisons of hematology and serum biochemistry parameters related to the active and hibernating periods for a crocodilian species. These baseline data were essential for health condition evaluation and disease diagnosis and useful for conservation and sustainable utilization of this endangered species.
2.3. Analysis of hematology and biochemistry parameters Total number of erythrocytes (RBC) and leukocytes (WBC) were analyzed in Neubauer Chamber, with dilution being performed by standard Hayem's solution for RBCs and Turk's solution for WBCs (Parida et al., 2011). Thrombocytes were identified and counted in the 4 corner squares of the large central square of Neubauer chamber, using Hayem's solution. The HGB concentration was determined using an automated hematology analyzer (BC-3000plus, Mindray, Shenzhen, China). The PCV was examined by using the microhematocrit method (Parida et al., 2011). The tubes were spun in a microhematocrit centrifuge (TDL-50B, Anke, Anting Scientific Instrument, Shanghai, China) for 5 min at 12000 rpm and the PVC was calculated with the total blood level divided by the blood cell level. The MCH, MCV and MCHC were calculated according to standard formulas (Campbell and Ellis, 2007). Erythrocyte sedimentation rate was determined by Westergren. The blood smears were stained with Wright Stain Solution (Tianda Diagnostic Reagents Co., LTD. Hefei, China) for the differential leukocyte count and then examined under a microscope (BM2000, Jiangnan Yongxin Co., LTD. Nanjing, China). The percentages of different leukocytes were determined after counting a total of 1000 cells. Serum biochemistry parameters including activities of CK, ALT, AST, GGT, LDH, Alp, Amy, and concentrations of Cre, UA, Bun, TP, Alb, Glo, GLU, TB, Cho, TG were measured on serum samples by using an autoanalyzer (KHB 450, Shanghai, China) and the concentrations of K, Na, Cl and nCa (standard calcium ion concentration at pH7.4) in serum were measured using a electrolyte analyzer (IMS-972, HORRON, Shenzhen, China).
2. Materials and methods 2.1. Ethics statement This work was conducted as part of a population health assessment approved and supported by the Anhui Research Center for Chinese Alligator Reproduction and college of Life Sciences, Anhui Normal University. All the handing and sampling were performed in compliance with standard vertebrate protocols and veterinary practices, and in accordance with national and provincial Guidelines. 2.2. Animals and sample collection
2.4. Statistical analysis
The Chinese alligators are amphibious poikilotherm whose body temperature and movement change closely as the ambient temperature changes, They would begin to enter the state of hibernation (approximately from late October to late April in the next year) when the environmental temperature continuously falls below 14 °C. In our study, a total of 74 apparently healthy adult Chinese alligators were collected from the Anhui Research Center for Chinese Alligator Reproduction in Xuancheng, Anhui province during the active period (in late May) and the hibernating period (in mid-January), respectively, from January 2014 to May 2016. These alligators were examined for clinical signs of trauma and illness, and the gender was determined by digital palpation of genital organs in the cloacae (Ziegler and Olbort, 2007). Prior to blood collection, sex, length and mass were determined and
Hematology and serum biochemistry data were presented as means and standard deviation (SD) via the software SPSS19.0 for Windows. The results were compared among periods for each sex and among sexes within each period. Significant difference between means was determined using an independent sample t-test model. Results were considered significant at p < 0.05. 3. Results In this study, no hemolysis occurred nor parasites were observed. No chyle serum (having a milky appearance and being abundant in TG
Table 1 Demographics of 74 captive adult Chinese alligators. Group
Number Age range (y) Mass (kg) Body length (cm)
Males
Females
Active period
Hibernation
Active period
Hibernation
9 10–15 13.34 ± 1.28 (9.81–16.86) 145.7 ± 5.72 (132.9 157.4)
21 10–15 15.09 ± 3.14 (9.98 17.23) 147.6 ± 5.91 (132.8 159.3)
22 10–15 12.80 ± 1.47 (9.50 15.70) 145.3 ± 5.39 (132.0 153.1)
22 10–15 13.96 ± 3.01 (9.82 16.12) 147.1 ± 5.97 (132.5 158.8)
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Fig. 1. Light micrographs of peripheral blood cells of Chinese alligators with Wright staining (Bar = 10.00 um). A. Erythrocyte B. Thrombocyte C. Heterophil D. Eosinophil E. Basophil F. Large lymphocyte G. Small lymphocyte H. Monocyte
Table 2 Hematology parameters of Chinese alligators during the active and hibernating periods. Hematology Parameters
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a
RBC (×10 /L) WBC (×109/L) a Thrombocyte (×109/L) HGB (g/L) PCV (%) MCV (fl) a MCH (pg) a MCHC (g/L) Heterophil (%) Lymphocyte (%) Monocyte (%) a Eosinophil (%) Basophil (%) N/L ratio ESR (mm/H)
Males
Females
Active period
Hibernation
Active period
Hibernation
4.91 ± 1.26 (3.02–6.09) 2.15 ± 0.74 (1.37–3.07) 17.70 ± 4.37 (9.62–24.10) 96.72 ± 7.17 (88.00–104.00) 28.24 ± 2.76 (25.37–32.00) 575.13 ± 64.83 (507.01–655.32) 196.99 ± 30.12 (169.48–237.71) 342.43 ± 31.22 (301.43–393.54) 59.54 ± 7.13 (57.01- 68.20) 19.70 ± 5.32 (9.65–26.93) 15.25 ± 4.22 (7.39–20.34) 4.40 ± 1.27 (1.37–6.11) 1.11 ± 0.44 (0–1.96) 4.95 ± 2.33 (2.65–7.62) 3.63 ± 1.10 (2–4)
4.23 ± 1.32 (2.79–5.97) 2.00 ± 0.67 (1.30–2.97) 17.61 ± 3.84 (9.36–20.67) 99.75 ± 7.14 (90.00–107.00) 28.13 ± 2.66 (25.20–31.84) 665.02 ± 70.12 (594.51–767.23) 235.79 ± 30.03 (209.33–273.72) 354.61 ± 32.99 (307.39–398.02) 62.37 ± 2.74 (69.29–70.92) 19.41 ± 5.56 (12.31–23.21) 12.67 ± 2.92 (5.27–16.13) 4.38 ± 1.53 (1.54–6.55) 1.18 ± 0.40 (0.53–1.86) 5.26 ± 2.37 (2.12–8.03) 3.43 ± 0.50 (2–5)
4.97 ± 0.83 (4.00–6.13) 2.12 ± 0.62 (1.45–2.99) 17.66 ± 4.67 (10.17–26.02) 96.26 ± 14.64 (64.00–103.00) 27.70 ± 1.09 (26.48–29.30) 557.38 ± 41.36 (514.10–599.94) 193.67 ± 29.96(158.08–227.43) 347.51 ± 40.07 (302.01–396.12) 59.46 ± 7.35 (56.13–67.08) 19.71 ± 4.91 (9.77–25.12) 15.41 ± 5.47 (7.81–21.03) 4.27 ± 1.54 (1.50–6.66) 1.13 ± 0.39 (0–1.82) 4.94 ± 2.21 (2.69–7.58) 3.67 ± 1.21 (2–4)
4.37 ± 1.43 (3.30–6.04) 1.97 ± 0.89 (1.25–2.94) 17.63 ± 6.55 (9.56–26.57) 98.90 ± 9.76 (83.00–109.00) 27.65 ± 1.89 (23.84–29.27) 632.69 ± 42.81 (565.07–678.75) 226.32 ± 32.72 (175.47–259.61) 357.67 ± 33.26 (316.21–398.72) 62.62 ± 8.42 (60.32–71.18) 19.04 ± 5.93 (8.82–26.96) 13.00 ± 5.41 (7.27–18.05) 4.19 ± 1.52 (1.47–6.01) 1.15 ± 0.38 (0–1.91) 5.43 ± 2.30 (2.10–7.93) 3.44 ± 1.03 (2–5)
agranulocytes. Heterophils were the predominant leucocytes in number, followed by monocytes, lymphocytes, eosinophils, basophils (Table 2). Heterophils were round and large or irregular in shape with an eccentrically located nucleus which was usually kidney-shaped, oval, bilobate or trilobed. Their cytoplasm stained light blue and contained some very small and pink granules (Fig. 1C). Eosinophils were also round in overall appearance with lobed nuclei (Fig. 1D). The nucleus of eosinophils was bilobed or concentrated, located at one end of the cells, the cytoplasm was darkly brown-yellow with abundant strongly eosinophilic granules. Typically, nuclei were completely hidden by the large elongate granules. Basophils were round or oval cells with segmented nuclei (Fig. 1E). The strongly basophilic granules of basophils were large dark atropurpureus granules with which the entire cell was covered, similar to the eosinophils. Based on size, lymphocytes were categorized as large and small lymphocytes (Fig. 1F and G), both of which were rounded or spherical in shape, and possessed eccentric, round, notched nuclei which occupied almost the entire cell leaving a narrow rim of pale or light blue cytoplasm. Normally, monocytes were the largest leucocytes, irregular or rounded in appearance (Fig. 1H), with light blue to gray cytoplasm, and often having
and Cho) appeared during the hibernating period, but some chyle serum appeared during the active period. The morphology of each blood cell type under light microscope for Chinese alligators is shown in Fig. 1. Hematology and biochemistry parameters during the active and hibernating periods are summarized in Tables 2 and 3, respectively.
3.1. Morphology of the blood cells in Chinese alligators The characteristic shape of erythrocytes of Chinese alligators was observed to be elliptical or elongated oval with an oval or round nucleus placed centrally (Fig. 1A). On occasion, some erythrocytes with damaged membranes, spindle-shaped cells and teardrop-shaped cells were seen. Thrombocytes of Chinese alligators presented various morphologies on Wright-stained blood films, often appearing in clusters with 2–10 cells. Generally, they were from fusiform to spindle, elliptical or ovalshaped with a nucleus following the shape of the cell (Fig. 1B). Leucocytes of Chinese alligators could be classified into the following five types: heterophils, basophils, eosinophils, monocytes and lymphocytes; the first three were granulocytes while the rest were 10
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Table 3 Serum biochemistry parameters of Chinese alligators during the active and hibernating periods. Biochemistry Parameters
CK (U/L) a Amy (U/L) a LDH (U/L) ALT (U/L) AST (U/L) a GGT (U/L) ALP (U/L) b TP (g/L) Alb (g/L) Glo (g/L) b A/G TB (umol/L) a BUN (mmol/L) a Cre (umol/L) a UA (umol/L) GLU (mmol/L) TG (mmol/L) a Cho (mmol/L) a K (mmol/L) Na (mmol/L) Cl (mmol/L) NCa (mmol/L) Pb (ug/L)
Males
Females
Active period
Hibernation
Active period
Hibernation
149.22 ± 55.07 (72–218) 727.37 ± 86.97 (620–851) 354.21 ± 77.21 (193–458) 89.07 ± 23.72 (63–128) 193.32 ± 41.64 (147–237) 0 42.10 ± 22.03 (23–91) 73.77 ± 7.39 (67–86.72) 26.47 ± 3.75 (22.73–30.82) 44.41 ± 5.85 (41.01–49.74) 0.56 ± 0.07 (0.51–0.71) 1.20 ± 0.47 (0,72–1.88) 0.44 ± 0.07 (0.35–0.59) 31.19 ± 14.54 (14.54–53.18) 213.90 ± 143.04 (106–479) 6.86 ± 2.01(4.50–9.57) 9.02 ± 1.07 (3.97–16.11) 6.21 ± 0.94 (5.21–7.02) 4.33 ± 0.40 (3.91–4.87) 136.81 ± 3.67(133.17–139.54) 82.74 ± 7.79 (71.93–94.02) 1.20 ± (1.16–1.29) 41.33 ± 14.67 (24.18–57.21)
758.73 ± 99.43(611.00–835.40) 628.00 ± 59.04 (566–687) 306.86 ± 98.24(178.10–421.20) 80.33 ± 16.06 (64–105) 143.00 ± 35.45 (94–199) 11.12 ± 1.21 (2.00–6.11) 41.37 ± 20.25 (25–90) 71.35 ± 5.29 (63–78.6) 25.15 ± 2.97 (22.10–29.40) 44.2 ± 3.00 (40.90–49.20) 0.55 ± 0.08 (0.50–0.70) 2.66 ± 0.80 (1.10–3.49) 0.28 ± 0.10 (0.16–0.50) 6.90 ± 2.97 (3.00–9.46) 183.00 ± 53.32 (104–234) 6.66 ± 2.02 (4.21–9.28) 0.35 ± 0.05 (0.27–0.49) 2.10 ± 0.71 (0.65–2.84) 4.51 ± 0.11(4.51–4.75) 134.89 ± 0.82 (134.03–135.78) 85.98 ± 1.20 (84.30–87.60) 1.25 ± 0.01 (1.24–1.27) 40.76 ± 13.71 (25.23–55.98)
144.80 ± 57.37 (71–210) 725.57 ± 66.36 (623–845) 347.40 ± 78.11 (196–425) 87.00 ± 26.28 (56–140) 194.08 ± 27.41 (153–239) 0 36.67 ± 11.29 (16–50) 78.46 ± 6.95 (70.20–89.80) 28.00 ± 1.66 (26.40–31.60) 50.46 ± 6.20 (43.80–61.10) 0.58 ± 0.07 (0.49–0.71) 1.13 ± 0.32 (0.60–1.54) 0.43 ± 0.06 (0.36–0.56) 31.83 ± 14.36 (17.03–54.92) 217.83 ± 150.25 (100–512) 6.84 ± 2.21 (3.75–9.30) 9.15 ± 4.37 (4.09–15.71) 6.25 ± 0.46 (5.28–6.89) 4.28 ± 0.28 (3.94–4.76) 137.57 ± 4.99 (131.70–147.70) 81.54 ± 7.72 (70.30–93.60) 1.17 ± 0.06 (1.09–1.26) 41.00 ± 15.52(26–57)
729.98 ± 89.23 (603.23–812.13) 643.33 ± 87.29 (571–752) 301.37 ± 94.03 (176.11–417.27) 81.50 ± 24.47 (58–127) 147.83 ± 82.93 (76–296) 12.03 ± 7.01 (2–19) 35.33 ± 10.03 (15–54) 76.05 ± 5.17 (71.21–85.23) 25.05 ± 3.04 (20.50–28.20) 51.00 ± 7.96 (43.30–64.70) 0.52 ± 0.14 (0.30–0.70) 2.29 ± 0.86 (0.91–3.70) 0.26 ± 0.10 (0.18–0.41) 6.40 ± 2.73 (3.91–12.63) 177 ± 55.07 (94–242) 6.99 ± 1.93 (5.22–9.12) 0.36 ± 0.12 (0.29–0.51) 2.21 ± 0.87 (1.40–3.89) 4.59 ± 0.66 (4.18–5.59) 134.27 ± 2.66 (130.9–137.4) 86.03 ± 5.61(79.90–91.90) 1.27 ± 0.08 (1.20–1.39) 43 ± 13.19 (25.32–56.17)
4. Discussion
a few cytoplasmic pseudopodia, with irregularly indented nuclei eccentrically placed.
The health assessments and disease diagnosis are very important for the protection of endangered animal species, so it is essential to establish species-specific reference ranges for hematology and serum biochemistry parameters which could aid in the identification of alterations in animal health condition (Lewbart et al., 2014). Hematology and serum biochemistry parameter values vary under different physiological, ecological, age, sex, and environmental conditions (Sarasola et al., 2004; Seaman et al., 2005; Boily et al., 2006). As we know, for ectothermic vertebrates, two characteristic phases can be distinguished in the annual life cycle, that is, periods of activity and hibernation. In this study, sampling was therefore performed at two fixed time of each year during the active period (in late May, ambient temperature 21–28 °C) and hibernating period (in mid-January, ambient temperature −2–6 °C). Morphologically, the non-mammalian erythrocyte (such as in fishes, amphibians, reptiles, and birds) is nucleated, flattened and ellipsoidal (Rowley and Ratcliffe, 1988). The erythrocyte shape of Chinese alligators was generally similar to that of other non-mammalian species. The mean corpuscular volume of the erythrocyte in Chinese alligators was smaller than that of Dubois’s tree frog (upper range 851.31 fl) (Das and Mahapatra, 2014), while larger than that of mute swans (upper range 196.2 fl) (Dolka et al., 2014). The thrombocyte of lower vertebrates was the equivalent of platelet of mammalian species in the aspect of function, being responsible for clotting and wound healing. The thrombocytes of Chinese alligators were variable in form, but often presented a fusiform, spindle, or long oval shape, which was similar to those of lower vertebrates such as fishes and amphibians, such as Schizothorax prenanti (Fang et al., 2014) and American bullfrog (Bricker et al., 2012); while they differed from previously reported for the giant lizard (Gallotia simonyi) that possessed round or ovoid thrombocytes (Martinez-Silvestre et al., 2005) and are also easily distinguished from mammalian and human platelets which are anuclear and usually, round, stellate, or elliptical. The heterophils of Chinese alligators were the predominant cell type of leucocytes. The shape of heterophils in this species was generally similar to that of human neutrophils, but they contained more basophilic granules and present mostly with a taenia nucleus when
3.2. Hematology parameters of Chinese alligators during the active and hibernating periods Hematology parameters of Chinese alligators are shown in Table 2. Variations in relation to two different sampling periods are noted. The values of RBC and WBC counts, and monocyte percentage were significantly greater during the active period, while the values of MCV and MCH were significantly lower during the active period than those during the hibernating period. Additionally, the ESR value tended to increase during the active period, whereas the values of HGB and heterophil percentage tended to decrease during the active period compared with those during the hibernating period, but the differences were not statistically significant. Meanwhile, some differences in relation to sex were also found, the male alligators tended to have higher PCV, and lower RBC counts than the females, but these differences were not statistically significant.
3.3. Serum biochemistry parameters of Chinese alligators during the active and hibernating periods Serum biochemistry parameters of Chinese alligators are shown in Table 3. Variations in relation to the two sampling periods are noted. The values of Amy, AST, Bun, Cre, TG and Cho were significantly greater during the active period, while the values of CK and TB were statistically lower during the active period than those during the hibernating period. In addition, the values of LDH, ALT, UA tended to increase during the active period, whereas the values of K, Cl and nCa tended to decrease during the active period versus those during the hibernating period, but the differences were not statistically significant. Meanwhile, some differences in relation to sex were also found, the male alligators had significantly higher ALP activity, and significantly lower Glo concentration than the females. Additionally, TP concentrations for males tended to be lower than those for females, but were not statistically different. 11
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increased CK activity was probably associated with heart and skeletal muscle inflammation in animals (Yousaf and Powell, 2012). As CK activity increased, the level of serum TB concentration was also notably increased during the hibernating period. Normally, the high level of TB concentration suggested hepatobiliary disease, which is considered more specific than serum liver enzyme (Webster, 2005). Most poikilothermic animals would hibernate in order to have a greater adaptation to survive in the bitter cold environment, but the deep hibernating animals would probably result in body damage, due to lower metabolic activity, and weak self-regulation and self-healing, which could be explained the higher CK and TB values for Chinese alligators during the hibernating period. According to our information, dozens of Chinese alligators in ARCCAR die during hibernation every year, which was probably related to physical damage in hibernating state. BUN and Cre are very helpful to diagnose kidney disease in animals (Kaneko et al., 2008). There was a significant difference for BUN and Cre levels between the active and hibernating periods with both parameter values from the active period being markedly increased versus those from the hibernating period. This change may be a reflection of feeding and enhanced metabolic activity during the active period. TG and Cho are often used as indexes in evaluation of nutritional status, and hyperlipidemia for obese animals (Kawasumi et al., 2014). The values for the both parameters of Chinese alligators were significantly greater during the active period than those during the hibernating period. TG and Cho levels are directly associated with feeding status (Girling et al., 2015). Elevation in TG and Cho levels during the active period for Chinese alligators may be attributed to consuming additional food to reserve nutrition, in order to copulate in mid-June and lay eggs in July. Previous studies indicated that Cho levels were higher in captive American alligators (Alligator mississippiensis) than in wild American alligators; the Cho levels of Chinese alligators during the active period were higher than that of captive American alligators, with the exception of breeding female American alligators (Lance et al., 2001). In conclusion, this report provided the first comprehensive reference ranges of hematology and serum biochemistry for healthy farmed Chinese alligators during the active and hibernating periods. Most of the hematology and serum biochemistry parameters for Chinese alligators were noted to be similar among the sexes, only two significant differences existed in ALP and Glo values. Some significant differences were found between the two different seasonal periods with alligators during the active period possessing lower values for MCM, MCH, TB and CK, but higher values for RBC and WBC counts, monocyte percentage, AST, Amy, BUN, Cre, TG, and Cho. These baseline data are essential for health monitoring and disease diagnosis and useful for conservation and sustainable utilization of this endangered species.
compared with human and mammalian neutrophils. The eosinophils of Chinese alligators contained abundant, bar-shaped, brown-yellow cytoplasmic granules, which was not consistent with those of lower vertebrates, such as the American bullfrog with globular granules that were light red (Bricker et al., 2012), or those of higher vertebrates, such as human with orange, round or oval, smaller globular granules. In our present study, the basophils and lymphocytes were more similar to other species equivalent cells, except that the basophils contained more and larger basophilic granules than human basophils. Generally, most of the hematology and serum biochemistry parameter values for Chinese alligators were noted to be similar among both sexes, the only two significant differences existed in the ALP and Glo values. Consistently, previous studies also indicated that the vast majority of blood parameter values were equivalent for both sexes in lower vertebrates such as reptiles, amphibians, and fishes, such as Geoffroy's side-necked turtle (Zago et al., 2010), American bullfrogs Rana catesbeiana (Cathers et al., 1997) and Xenopus laevis (Wilson et al., 2011), which may be due to incompletion of gender differences in evolution for lower vertebrates. Seasonality often notably affected some reptilian blood parameter values (Lillywhite and Smits, 1984; Pal et al., 2008). In general, two characteristic phases can be easily distinguished in the annual life cycle for lower vertebrates, that is, periods of activity and hibernation. Up to date, few documents were available on hematology and serum biochemistry parameters in crocodilians during hibernation. RBC counts for Chinese alligators surveyed during the active period were notably greater, while the values of MCV and MCH for the hibernating alligators were significantly higher. Tavares-Dias et al. (2008) have suggested that species with high RBC counts could be well acclimated to a higher metabolic activity. This could account for the elevation of RBC counts for alligators examined during the active period. Prosser (1973) reported a higher MCH value is due to larger size of RBCs. In this study, the hibernating Chinese alligators possessed low numbers of RBCs, but higher MCH levels and larger MCV values, which may contribute to the maintenance of a sufficient oxygen availability to tissues in slow blood flow during hibernation. It is well known that leukocytes in humans and animals are directly associated with immunological response of the organism, such as stress, infection, and inflammation (Swenson, 1984). In this study, the significant increase of WBC counts, especially in monocytes, during the active period possibly due to presence of more risk factors for the active alligators. Similarly, in other reptile species, such as turtles (Wilkinson, 2004), elevation of WBC counts was also noted in summer. More notable differences were found between the two different periods in serum biochemistry parameters for Chinese alligators. Serum α-amylase is mainly secreted by sialaden and pancreas and plays a key role in animal carbohydrate metabolism; it can catalyze the hydrolysis of glycogen, starch, and various other related carbohydrates (Strobl et al., 1998; Franco et al., 2000). The amylase activity levels reported here for Chinese alligators during the active period were strikingly increased, which was likely due to consuming food stimulation during the active period. AST was the other enzyme found to be significantly increased during the active period in this study. AST is a liver-specific enzyme in humans and animals (Jenkins, 2008). Elevation of AST activity for Chinese alligators is probably related to the enhancement of hepatocyte metabolism during the active period. Additionally, we found that the AST activity of Chinese alligators during the active period was significantly greater than that of some crocodilian species such as wild spectacled caiman Caiman crocodilus crocodilus (Rossini et al., 2011), farmed saltwater crocodile Crocodylus porosus (Millan et al., 1997), and wild Nile crocodiles Crocodylus niloticus (Lovely et al., 2007), but lower than that of American alligators Alligator mississippiensis (Hamilton et al., 2016). In this study, CK was the only enzyme demonstrating significantly higher activity in Chinese alligators during hibernation. CK usually presents in skeletal muscle, cardiac muscle, brain, and smooth muscle;
Acknowledgments We would like to thank Xie Yuling for her help in photo processing. We also thank Wuhu Zhengyu medical laboratory for help with determination of serum biochemistry and HGB concentration values. This work was supported by the key program of education bureau of Anhui Province (Grant No. KJ2013A126) and Natural Science Foundation of Anhui Province (Grant No. 11040606M75). References Artacho, P., Soto-Gamboa, M., Verdugo, C., Nespolo, R.F., 2007. Using haematological parameters to infer the health and nutritional status of an endangered black-necked swan population. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 147, 1060–1066. Azevedo, T.M.P., Martins, M.L., Bozzo, F.R., Moraes, F.R., 2006. Hematological and gill responses in parasitized tilapia from valley of Tijucas River, SC, Brazil. Sci. Agric. 63 (2), 115–120. Boily, F., Beaudoin, S., Measures, L.N., 2006. Hematology and serum chemistry of Harp (Phoca groenlandica) and Hooded seals (Cystoohora cristata) during the breeding season in the Gulf of St. Lawrence. Can. J. Wildl. Dis. 42, 115–132. Bricker, N.K., Raskin, R.E., Densmore, C.L., 2012. Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis
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