Seasonal variations in haematological data from Mediterranean tortoises (Testudo graeca and Testudo hermanni) in captivity

Research in Veterinary Science 1986, 40, 225-230

Seasonal variations in haematological data from Mediterranean tortoises (Testudo graeca and Testudo hermanni) in captivity K. LAWRENCE, 23 Woodside Gardens, Chineham, Basingstoke, Hants, RG24 OEU C. HAWKEY, Haematology Unit, Institute of Zoology, Zoological Society of London, Regents Park,

London, NWI 4R Y

Reference values for a variety of haematological parameters in 18 Mediterranean tortoises of two species (Testudo graeca and Testudo hermanni) were determined on six occasions during the year. Statistically significant seasonal variations were demonstrated in all parameters. TORTOISES have been imported into the United Kingdom for over 100 years (Lambert 1980). Even though. the tortoise has been maintained in captivity in large numbers since the 1950s, little basic physiological data has been collected. A number of papers have been published on the haematology of Testudo graeca and T hermanni (Friar 1977), but the results tend to be based on single samples taken from individuals of unknown health status and indeterminate sex. The results quoted provide no evidence for cyclic changes in haernatological values. However, the cyclic nature of these values has been reported by Gilles-Baillien (1973). In view of the interesting physiological implications of

cyclical changes in the blood of hibernating animals, the situation has now been re-examined. The haematological values reported here should also provide normal reference data for captive T graeca and T hermanni for comparison with findings in sick animals at different times of the year. Materials and methods Eighteen healthy, long term captive tortoises, 12 spur-thighed (T graeca) and six Hermann's (T hermannii tortoises ranging in weight from 700 to 2675 g (Table I) were maintained as described by Collins (1980). Blood samples were taken on six occasions as part of a health screening scheme. The tortoises were sampled in January (mid-hibernation), March (on leaving hibernation), June (mid-summer), October (on entering hibernation), November (one month into hibernation) and December (two months into hibernation). Smears were made from blood collected from up to 10of the T'graeca, for differential

TABLE 1: Morphometric data from 18 tortoises in haematological survey Tortoise number

Length

1983 1982 October January

Species

Sex

(rnrn)

80/001 82/002 82/004 82/005 82/006 82/008 83/011 83/013 83/014 83/016 83/017 83/019

T graeca T graeca T graeca T graeca T graeca T graeca T graeca T graeca T graeca T graeca T graeca T graeca

F F M F F F F F F

1475 2675 1000 1425 1475 1600 1075 1375 1550 1475 900 750

1400 2675 850 1400 1425 1600 1075 1350 1525 1450

M M

203 232 174 194 200 214 178 181 189 184 160 151

82/007 82/009 82/010 83/012 83/015 83/018

Thermanni Thermanni Thermanni Thermanni Thermanni T hermanni

F M F F F M

187 168 195 194 172 171

1225

F

900

1300 1425 1250 1000

Weight (g) 1983 March

1983 June

Percentage weight loss 1983 October Oct 82 to Mar 83

1700 2675 1050 1500 1575 1575 1150 1375 1575 1700

1500 2650 1050 1425 1525 1575 1100 1300 1525 1425

900

900

750

1375 2675 800 1350 1400 1525 1075 1350 1525 1425 850 700

750

750

6·8 1·9 20·0 5·3 5·1 4·7 0·0 1·8 1·6 3·4 5·5 6·7

1225 875 1275 1425 1200 975

1200 850 1225 1375 1150 975

1250 1000 1500 1250 1250 975

1175 900 1300 1200 1200 950

2·0 5·5 5·8 3·5 8·0 2·5

900

225

K. Lawrence, C. Hawkey

226

TABLE 2: Comparison of blood samples taken from the dorsal coccygeal vein and a short-clipped nail

Coccygeal vein Mean±SD

Short-clipped nail Mean±SD

9·0 ±0·9 0·81±0·13 0·33±0·08

9·3 ±1·5 0·76±0·16 0·32±0·06

9·9 ±2·0 0·91±0·31 0·35±0·07

10·1 ±1·7 0·83±0·24 0·33±0·07

10·25±1·0 0·86±0·31 0·37±0·09

10·1 ±0·7 0·82±0·15 0·34±0·05

11·3 ±2·2 0·93±0·16 0·34±0·05

11·3 ±1·3 0·96±0·24 0·38±0·06

January

n = 12 Haemoglobin (gdr-I) RBC (1012Iitre- 1) pcv(litreslitre-11 T hermanni n = 6 Haemoglobin (gdl- 11 RBC (1012 litre-I) pcv(Iitres litre-1) T graeca

March

n = 10 Haemoglobin (gdr-I) RBC (1012 litre-I) PCV (litreslitre -1 ) T hermanni n = 6 Haemoglobin (gdl- 1) RBC (1012 litre-II pcv(litreslitre -1) T graeca

white blood cell (WBC) counts and examination of cell morphology in January, March, June and October. Blood samples were initially collected from the dorsal coccygeal vein (Samour et a11984) and by short clipping a nail. Three measurements, red blood cell (RBC) count, packed cell volume (r-ev) and haemoglobin were determined for both samples (Table 2). No significant differences were found between the blood samples collected from the two sites either in January or March and the reported results are based on samples collected by short nail clipping. One hundred and ten samples were collected and analysed. The RBC and WBC counts were performed manually using a stain containing diluent which allowed both

determinations to be carried out on a single sample. The diluent, described by Frye (1981), was the Binder modification of a formula used by Natt and Herrick (1952) in chickens. The formula is: sodium chloride (NaC\), 3· 88 g; sodium sulphate (Na2S04)' 2· 50 g; sodium hydrogen phosphate (Na2HP04.12HP), 2·91 g; potassium hydrogen phosphate (KH 2P04), O' 25 g; formalin (40 per cent), 7' 50 ml; methyl violet, 0'1 g. . These chemicals were dissolved in I litre of distilled water, left overnight and filtered. Immediately before use, O' I ml of a I per cent solution of toluidine blue was added to 10 ml of the diluent. The blood samples were diluted I: 100 and mixed for one minute to ensure an even distribution of the cells. A double haemocytometer (Improved Neubauer, 1/400 mrn-, Hawksley) was filled and the RBC count determined in the usual way, the average of two counts being taken. The WBC count was undertaken using a microscopic magnification of x 450. The blue stairied WBCS were counted in the central I mm square (400 small squares), the average of two counts being multiplied by 1000 to arrive at the final figure. Blood was also collected directly into heparinised microhaematocrit capillary tubes for a rev determination using a Compur MIIOI minicentrifuge. Haemoglobin was measured as oxyhaemoglobin using a haemoglobinometer (BMS, St Petersburg, USA). Blood smears, made at the time of blood collection in January, March, June and October, were stained in Geimsa for differential WBC counts and examination of cell morphology. The mean, standard deviation and range for each parameter was determined and the data was tested for the effect of season using Student's paired t test.

TABLE 3: Haematological data from tortoises. Seasonal variation

January Mean ±SD

Range

March Mean

±SD

Range

June Mean

±SD

Range

n = 12 Hb Ig dl- 11 9·3 1 2 RBC (10 litre-I) 0·76 PCv(litres litre -11 0·32 MCV(fI) 416·0 MCH (pg) 124·0 MCHC (gdl- 1) 29·8 1 WBC (109litre- ) 8'5

± 1·5 ± 0'16 ± 0·06 ±15·0 ±14·2 ± 2·9 ± 2·2

(7'1 - 13'0) (0'54- 0·96) (0·24- 0·451 (350·2 -444·41 (105·2 -148,11 (25·35- 34·4) (5'4 - 12·85)

10·1 0·82 0·34 422·9 126·3 29·8 5·1

± 0·7 ± 0·15 ± 0·05 ±31·9 ±18·1 ± 3'1 ± 2·3

(8'75-- 12-75) (0·65-- 1·08) (0,28- 0,42) 1365·2 -491·8) (89·56-159·8) (24,52- 33·3) (1·5 - 13·0)

9·1 0·67 0·28 427·0 137·3 32·2 7·1

1·4 0·12 0·28 16·3 8·1 2·1 1·6

16·5 - 12·25) (0'46- 0·84) (0'2 - 0·35) (402·7 -462·9) (125'0 -152,7) (29·2 - 35·0) (5'5 - 12'5)

T hermanni n = 6 Hb (gdl- 1) RBC (1012 litre-I) pcv[litres litre- 1) MCV (tI) MCH (pg) MCHC (gdr-I) WBC(109litre- 11

± 1·7 ± 0·24 ± 0'07 ±3O,0 ±13·5 ± 2·4 ± 2·8

(8·6 - 13'0) (0·64- 1·28) (0·25-- 0·45) (350·2 -431-6) (101·2 -137,9) (28·6 - 34'4) (7'5 - 14·0)

11·3 0·96 0·38 408·5 121·6 29·8 7·7

± 1·3 ± 0·24 ± 0·06 ±42·8 ±20·1 ± 2·3 ± 3·6

(9'5 - 12-75) 10,73- 1'36) 10,32- 0·45) (330·9 -441·9) 184·55--143'0) (25·5 - 32·6) (2·0 - 13'0)

9·3 0·67 0·29 425'5 136'9 32·2 9·8

1·3 0·15 0'07 12·1 6·4 1·0 2·3

(6'5 - 12·25) (0,47- 0·871 (0·20- 0·381 (416·7 -444-4) 1127,27-145,8) (30·4 - 33'12) (6·0 - 12·5)

T graeca

Hb Haemoglobin

10·1 0·83 0·33 401·9 124·3 31·1 10·8

Tortoise haematology

227

Results

The haematological findings from clinically normal adult T graeca and T hermanni are listed in Table 3, with the variation in differential WBC count being presented in Fig 1 and Table 4. Fig 2 gives the statistical analysis of the seasonal variations. Seasonal trends were similar in both species with the RBC count, r-ev and haemoglobin levels decreasing between the end of March and June and then remaining stable until October. During November there was a slight increase in values followed by a sudden drop during December with a concurrent rise in the mean corpuscular volume (MCV). These changes occurred very rapidly with normal levels returning within 10 to 14 days (Fig 2). The mean corpuscular haemoglobin concentration (MCHC) fell during hibernation before recovering during the summer. The erythrocytes were therefore hypochromic during hibernation. The total WBC count rose in the autumn, fell during hibernation and increased again during the summer. Differential WBC counts carried out on a limited number of T graeca showed heterophils and lymphocytes to decrease in similar proportions between January and March, whereas there was a relatively large increase in lymphocytes during the active period and an eosinophilic response in October. Statistically significant differences between the two species were limited to higher haemoglobin levels (P
While no significant differences were found

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FIG 1: Seasonal variation in absolute and differential WBC count in 10 T graeca. X - - X Total WBC count, + - - + lymphocytes, e--e heterophils, 0----0 eosinophils

between blood samples collected from the dorsal coccygeal vein or by short-clipping a nail, there is a greater chance that inaccuracies may occur due to the collection technique in the case of nail clipping. This is most noticeable if the sample is obtained after the foot has been squeezed to milk the sample from the

TABLE 3-continued

October Mean ±SD 9·0 0·64 0·27 428·8 140·3 32·8 9·3 9·5 0·69 0·29 422·7 138·1 32·6 13·1

Range

± 0·04 ±26·0 ± 9·6 ± 2·6 ± 2'5

(6·5 - 11·01 (0'47- 0'77) (0·18- 0·33) (378'9 -490·21 (125·0 -156,8) (30'0 - 36·201 (7,0 - 14·01

± 2·1 ± 0·14 ± 0·06 ±13·4 ±23·1 ± 4·3 ± 2·7

(6 - 11-51 (0,48- 0·86) (0'20- 0·36) (412'5 -448·0) (123·1 -184,01 (29,62- 41·1) (9'0 - 16·01

± 1·3

± 0·10

November Mean ±SD n= 14 9-5 0·68 0·29 426·6 140·3 32·6 n=5 10·2

e-n

0·32 423·0 133·1 31-5

Range

± 1·3 ± 0·10 ± 0·04 ±22·3 ±10·9 ± 2·7

(6·0 - 11·31 (0-48- 0·831 (0·24- 0·371 (382·7 -461'5) (123'4 -158,7) (29·7 - 37·01

± 1·5 ± 0·13 ± 0·06 ±26·1 ± 5·9 ± 1·7

(7·5 - 11'5) (0'5&- 0·88) (0,23- 0·401 (385·5 -457·1) (126·5 -141,5) (28·8 _. 32·8)

December Mean ±SD

Range

n=14 <4·0 0·25 0·14 557·3

± 0·09 ± 0·05 ±66·1

(0'11- 0·381 (0'06- 0·201 (500·0 -720,01

n=5 <4·0 0·24 0·13 543·5

(0'13- 0·30) ± 0·007 (0·08- 0·17) ± 0·04 (500·0 -007·01 ±47·6

228

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cut end of the nail. Under these circumstances there may be a reduction in the PCV, RBC count and haemoglobin levels. Rosskopf (1982), using a similar technique in the Californian desert tortoise (Gopherus agassizii, also reported lysis of red cells and increased levels of lactic dehydrogenase and serum aspartate aminotransferase as a result of milking to promote blood flow. In the present study, only free-flowing samples were accepted. Haemolysis was not seen in samples centrifuged for r-ev measurement. Friar (1977) reviewed the published literature on the RBC and PCV values in T graeca and T hermanni. Of the 14 RBC counts quoted, nine have unreported sample sizes or the figures are based on single samples from fewer than five individuals. Despite this the range of RBC counts of O' 54 to O' 73 X 1012 litre "! compares well with the counts recorded in the present study. The reported figures for the r-ev are limited to only seven sources, of which three derive their figures from five or less individuals. These figures range from 0'18 to O' 32 Iitres litre" J which are comparable to the June and October means, but the revs in late hibernation are significantly higher (0' 33 to O' 38 Iitres litre-I). Gilles-Baillien (1973) reported monthly variation in PCV, RBC count and MCV in T hermanni. The figures

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Tortoise haematology for January, March and June mirror the present study but those for October are consistently lower. This may represent the difference between the long-term captives of this study and the tortoises used in GillesBaillien's study, which were being hibernated for the first time since collection from the wild. The recently collected tortoises lost significantly more weight during hibernation (mean decrease of 20 per cent) whereas the weight loss recorded in the study group over a 156 day hibernation was: Tgraeea 5'22±4'91 per cent (range 0'0 to 20·0 per cent); T hermanni 4'55±2'05 per cent (range 2·04 to 8·0 per cent). The marked fall in RBC count and r-ev with the rise in MCV in December was also reported by GillesBaillien (1973) but it was demonstrated in the November sample. The pattern of MCV changes are similar in both studies, although the seasonal range of 350 to 720 fI is wider than the 350 to 550 fI reported by GiIles-Baillien (1973). It has been generally assumed that 'hibernation' in terrestrial reptiles is accompanied by dehydration. It is often proposed that seasonal variations in haematological data are associated with haemoconcentration caused by this dehydration (Gregory 1982). GillesBaillien and Schoffeniels (1965), however, showed that the water content of the small intestine, colon and muscle in T hermanni is unchanged in hibernating individuals. This suggests that the changes in rev, RBC count and haemoglobin concentration are caused not by dehydration but by a relative decrease in the volume of the extravascular space (Gilles-Baillien 1973). The changes in these parameters do not occur until after their precipitate drop and recovery during November or December. These sudden changes may represent marked shifts in water distribution within the body; indeed, the increase in MCV is probably caused by a decreased plasma osmolarity associated with an influx of water into the circulatory system. A further consideration is that these changes, previously believed to be caused by dehydration, still occur in tortoises in the absence of any apparent weight loss and cannot be directly related to weight loss in individuals. Since the reduction in extracellular water volume occurs in the absence of weight loss, it must be redistributed within the body. It was noted by GillesBaillien (1914) that the volume of the bladder in a hibernating tortoise slowly increases during hibernation until it takes up a large portion of the abdominal cavity. Water is therefore lost into the bladder which may act as a water store. However, Gilles-Baillien (1969) reported that the water net flux, measured in vitro across the bladder mucosa, is significantly decreased in torpid tortoises. Thus the water stored in the bladder becomes less available to the animal as hibernation progresses and reduction in extracellular volume may be unassociated with a weight loss.

229

The possibility that the rapid PCV, RBC count and haemoglobin concentration changes during hibernation could be caused by red cell destruction followed by an increase in erythropoiesis was considered. No direct evidence for this proposition can be presented and in view of the long half-life, some II months, of erythrocytes in the box turtle (Terrepena species) (Brace and Atland 1955) and the unlikelihood of a burst of active erythropoiesis occurring during dormancy, this proposition must be considered doubtful. The findings cannot be attributed to a technical artefact because haemoglobin, RBC and rev levels were measured by entirely independent techniques. Haemodilution was described by Musacchia and Sievers (1956) in turtles (Chrysemys picta; exposed to cold. These workers proposed a loss of red cells from the circulation into the liver and spleen as the cause, rather than an influx of water. Further study will be necessary to elucidate the exact mechanisms. The differential WBC count showed seasonal variation similar to that described by Duguy (I 963a, 1963b, 1967) in hibernating species of snakes, lizards and chelonia in France. He described an annual cycle with a marked reduction in lymphocytes during the winter and a winter eosinophilia with a summer eosinopenia. The published observations suggest the annual blood cycle of reptiles is of intrinsic origin and changes may be initiated by complex physiological modifiers which occur during the late summer or autumn, the postulated' Zeitgeber' being a combination of falling temperatures and shorter days.

References BRACE, K. C. & ATLAND, P. D. (1955) American Journal of Physiology 183. 91-94 COLLINS, P. (1980) Journal of the British Chelonia Group I, 27-40 DUGUY, R. (1963a) Bulletin de la Societe Zoologique de France 88, 99-108 DUGUY, R. (l963b) Vie Milieu 14, 311-443 DUGUY, R. (1967) Bulletin de la Societe Zoologique de France 92, 23-37 FRIAR, W. (1977) Herpetologia 33, 167-190 FRYE, F. L. (1981) Biomedical and Surgical Aspects of Captive Reptile Husbandry. Kansas, V.M. Publishing. p 65 GILLES-BAILLlEN, M. (1969) Biochimica Biophysica Acta 193, 129-136 GILLES-BAILLlEN, M. (1973) Archives lnternationalesde Physiologie et de Biochimie 81, 723-732 GILLES-BAILLlEN, M. (1974) Chemical Zoology. Vol IX. New York and London, Academic Press. pp 353-376 GILLES-BAILLlEN, M. & SCHOFFENIELS, E. (1965) Annates de la Societe Royal Zoologique de Belgique 95, 75-79 GREGORY, P. T. (1982) Biology of the Reptilia. Vol 13. London, Academic Press. pp 53- 154 LAMBERT, M. R. K. (1980) Proceedings of the European Herpetological Symposium, Cotswold Wildlife Park, Burford, Oxford. pp 17-23

230

K. Lawrence, C. Hawkey

MUSACCHIA, X. J. & SIEVERS, M. L. (1956) American Journal of Pathology 187, 99-102 NATT, M. P. & HERRICK, C. A. (1952) Poultry Science 31, 735-738 ROSSKOPF, W. J. (1982) Veterinary Medicine/Small Animal Clinician 77, 85-87

SAMOUR, H. J., RISLEY, D., MARCH, T., SAVAGE, B., NIEVA, O. & JONES, D. M. (1984) Veterinary Record 114, 472-476

Accepted October 25, 1985