A contribution to clinical investigate methods for birds, with special reference to the racing pigeon, COLUMA LIVIA DOMESTICA

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A Contribution to

Clinical Investigative Methods for Birds,

with Special Reference to the Racing Pigeon,

Columba livia domestica

I T. Lumeij

rat' Prrit

P

A Contribution to Clinical Investigative Methods for Birds, with Special Reference to the Racing Pigeon,

A Contribution to Clinical Investigative Methods for Birds,

with Special Reference to the Racing Pigeon%

Columba livia domestica

Een bijdrage aan klinische onderzoeksmethoden bij vogels,,

met speciale aandacht voor de postduif,.

Columba livia domestica

/met een samenvatting in het Nederlands)

PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Rijksuniversiteit ite Utrecht

op gezag van de Rector Magnificus Prof. Dr. J.A. van Ginkel

volgens het besluit van het College van Dekanen

in het openbaar te verdedigen

op ,donderdag 10 december 1987 des namiddags, te, 2.30 utcr

door

Johannes Thomas Lumeij

Promotor: Prof. Dr. H.W. de Vries

Faculty of Veterinary Medicine, State University Utrecht

Referent: Dr. B.M. Freeman

Agricultural and Food Research Council

Institute for Animal Disease Research, Houghton Laboratory

(formerly Houghton Poultry Research Station)

Houghton, Huntingdon, Cambridgeshire, U.K.

CIP-GEGEVENS KONINKLLIKE BIBLIOTHEEK, DEN HAAG Lumeij. Johannes Thomas

A contribution to clinical investigative methods for birds with special reference to the racing pigeon, Columba uric domes/lea / Johannes Thomas Lumeit, [ill.: Bert Janssen). - [S 1 s.n.].

- Ill.

Proefschrift Utrecht. - Met lit. opg. - Met samenvatting in her Nederlands. ISBN 90-9001845-X geb.

SISO 634.61 UDC 619:636.686(043.3) Trefw.: postduiven / diergeneeskunde.

Vakgroep Geneeskunde San he Kleine Huisdier Facukeit der Diergeneeskunde

Rijksuniversheil Utrecht, 1987 ISBN 90-9001845-X

Aan mijn vrouw A nk, die ondanks haar vogelangst, een onmisbare steun is geweest bij de totstandkoming an dit proefschrift.

This thesis was written and revised on a Olivetti NI 19 Personal Computer using Display Write 3 as word processing software. The text was converted to a MCS phototypesetter and set in Times by Drukkerij Nicoa. de Bruijn BY, Deventer. The thesis was printed 'by Drukkerij,Elinkwijk BV, Utrecht.

The lay out was performed and illustrations were made by Bert Jansseif.

Publication of this thesis was financially supported by: Boehringer Mannheim GmbH, Mannheim (BRD)

Chevita GmbH, Pfaffenhofen/Ilm (BRD) Duphar Nederland BV, Amsterdam

Gist Brocades Animal Health BY, De Bilt Janssen Pharmaceutica, Beerse (13)1 Natural Granen NV, Schoten (B) Stapler Instrumenten BV, Utrecht

CONTENTS

GENERAL 'INTRODUCTION

CHAPTER 4

GENERAL CONSIDERATIONS

AVIAN CLINICAL PATHOLOGY. GENERAL

CONSIDERATIONS

(Lumeij, J.T. The Veterinary Quarterly, 1987; 9: 249-254)

THE INFLUENCE OF BLOOD SAMPLE TREATMENT ON

PLASMA POTASSIUM CONCENTRATIONS IN AVIAN BLOOD

(Lumetj, J. T. Avian Pathology, 1985; 14: 257-260)

BLOOD CHEMISTRY REFERENCE VALUES IN RACING

PIGEONS

(COLUMBA LIVIA DOMESTICA)

(Lumed, J.71, de Brutjne, ii.. Avian Pathology, 1985; 14:401-408)

THE INFLUENCE OF BLOOD SAMPLE TREATMENT,

FEEDING AND STARVATION ON PLASMA !GLUCOSE.

CONCENTRATIONS IN RACING PIGEONS

(Lumeij, J.T.)

CIRCADIAN RHYTHM OF PLASMA GLUCOSE IN' FASTED,

RACING PIGEONS (COLUMBA LIVIA DOMESTICA)

(Lutneij, J. T., Kantor, A. and van den Brom, WE.)

CHAPTER ii

'DIAGNOSIS OF HEPATOBILIART DISEASE.

BLOOD CHEMISTRY FOR THE DIAGNOSIS OF

HEPATOBILIARY DISEASE IN BIRDS. A REVIEW

(Lutneij, J. T.. Westerhof, I. The Veterinary Quarterly,, 1987:

9: 255-261) ₃₆

BLOOD CHEMISTRY REFERENCE VALUES FOR USE IN'

COLUMBINE HEPATOLOGY

(Lutneij, J. T. and Wolfswinkel, J.)

ENZYME ACTIVITIES IN PIGEON TISSUES

(Lumetj, LT, de Bruijne, Li)

_46,

7 8 15 19 26 31 35 44

ELIMINATION HALF-LIFES OF HOMOLOGEOUS LIVER AND

MUSCLE ENZYMES IN THE RACING PIGEON (COLUMBA

LIVIA DOMESTICA).

(Lumeij, J. T., de Bruijne. Li and Rothuizem J.I

56

CHANGES IN PLASMA CHEMISTRY AFTER

D-GALACTOSAMINE AND ETHYLENE GLYCOL INDUCED

LIVER DISEASE IN RACING PIGEONS, COMPARED TO

CHANGES DUE TO MUSCLE NECROSIS.

(Linnet',

f. T., Meidam, M., Wolfswinkel, J., van 'der Sage, Mil.

and Dorrestein, G.M.)

62:

TISSUE ENZYME PROFILES OF THE BUDGERIGAR,'

MELOPSITTACUS UNDULATUS

(Lumeij, f. T. and Wolfswinkel, J.)

CHAPTER 11111

PROTEIN AND NON-PROTEIN NITROGEN

791

THE DIAGNOSTIC VALUE OF PLASMA PROTEINS AND

NON-PROTEIN NITROGEN SUBSTANCES IN BIRDS

(Lumeit J.T. The Veterinary Quarterly, 1987; 9: 262-268)

80'

EVALUATION OF THE REFRACTOMETRIC METHOD FOR'

THE DETERMINATION OF TOTAL PROTEIN IN AVIAN

PLASMA OR SERUM

(Lumeij, f. T., de Bruijne, II Avian Pathology, 1985; 14: 441-444Y

88

PLASMA UREA, CREATININE AND URIC ACID

CONCENTRATIONS IN RESPONSE TO DEHYDRATION IN

RACING PIGEONS

(Lumeit J.T. Avian Pathology, 1987; 16: 377-382)

92

!COLLECTION AND ANALYSIS OF URINE IN RACING

PIGEONS, COLUMBA LIVIA DOMESTICA)

W.

B., Alberts. H., de Bruijne, J.J. and Lamed f. T.,

Avian Pathology; accepted) ₉₈

CHAPTER IV

ENDOCRINOLOGY

103,

CLINICAL ENDOCRINOLOGY IN BIRDS, WITH SPECIAL

'REFERENCE TO SOME FUNCTION TESTS IN RACING

PIGEONS

(Lumeti, f. T. Proceedings European Symposium on Bird Diseases of the Netherlands Association of Avian Veterinarians, Beerse,

Belgium, 5 and 6 March, 1987; pp. 14-20

qO4

ACTION OF ACTH'' UPON PLASMA CORTICOSTERONE

CONCENTRATIONS IN RACING PIGEONS (COLUMBA LIVIA

DOMESTICA)

(Lumeij, IT., Boschma, Y., Mol, J., de Kloet. ER.,, wan den

Brom, W. Avian Pathology, 1987,:, 16: 199-2(14)

71

(Halsema,

CLINICAL EVALUATION OF THYROID FUNCTION IN

RACING, PIGEONS

(COLUMBA LIVIA DOMESTICA)

(Lumeij, J.T. and Westerhof, L Avian] Pathology; accepted) 316

A WATER DEPRIVATION TEST FOR THE DIFFERENTIATION

OF POLYURIC DISORDERS IN BIRDS

(Alberts, H., Halsema, W. B., de Bruijne, ii., Lumelj, J. T. Avian Pathology; accepted)

CHAPTER V

ELECTROCARDIOGRAPHY

127

CLINICAL ELECTROCARDIOGRAPHY IN RACING PIGEONS

(Lutneij, J. T. Proceedings V. Tagung Tiber Vogelkrankheiten der Deutsche veteriniirmedizinische Gesellschaft e. v. Miinchen, 6. und

7. Marz 1986, pp. 19-33) ₁₂₈

ELECTROCARDIOGRAM OF THE RACING PIGEON

(COLUMBA LIVIA DOMESTICA)

(Limed, J.T., Stokhof, A.A. Research in Veterinary Science, J985;

38:275-278) 440

ANAESTHETIC FATALITIES IN GOSHAWKS

(Lumeij, J. T. Proceedings V. Tagung (Ther Vogelkrankheiten der Deutsche Veteriniirmedizinische Gesellschaft e, v. Munchen, 6. mid

7. Mar:, 1986, pp. 201-207) 1146

CHAPTER Ve

ENDOSCOPY

3511

ENDOSCOPIC APPROACHES FOR THE, RACING PIGEON

(COLUMBA LIVIA DOMESTICA)

(Linnet], J. T. and Hasselaar, LC.), 4152

CLINICAL ENDOSCOPY IN BIRDS

(Lume), Westerhof, I. Paper presented at the 11th World Congress of the World Small Animal Veterinary Association, Paris-4-7 December 1986. Translated in French and accepted for

publication in: Pratique medicale el chirurgicale de /'animal compagniel

SUMMARY AND CONCLUSIONS,

057

SAMENVATTING' EN' CONLUSIES

I73

ACKNOWLEDGEMENTS

183

CURRICULUM VITAE

_18-6

J. T.,

de

GENERAL INTRODUCTION

Avian medicine is a speciality of veterinary science which since the Second World War can be characterized by diagnosis, treatment and prevention of diseases at the level of the flock. Because of the economic importance of poultry farming this was an obvious development. Important progress within this speciality has been made in the fields of husbandry, nutrition, microbiology (including serology), parasitology, preventive medicine and post mortem examination. Because individual birds had no economical' or emotional value, it was no problem to cull a few birds in order to establish a diagnosis (Zander, 1984). This flock approach proved also functional' for the veterinary approach of aviary birds which are kept in large numbers.

Apart from technical difficulties, the relative ease with which it was decided to perform a post mortem examination in a few birds from the rock, was perhaps a reason that the developments which were made with regard to clinical diagnostic techniques in birds lagged behind the developments which were made in medicine of other companion animals, like dogs.

With the increasing number of pet birds the veterinarian was confronted with a rising demand for veterinarycare of birds which had a high emotional and/or economical' value for the owner. However, these birds often had to be denied the achievements of clinical veterinary medicine. Examination of the patient was often restricted to anamnesis and physical examination, whereby the latter, for anatomical reasons, could be less comprehensive then the physical examination of other companion animals. Supplementary investigations were often microbiologically or parasitologically oriented. The necessity for a different approach to the individual' avian patient was realized: the flock approach was unsuitable for individual patients. At the same time the possibilities with regard to advanced diagnostic techniques increased because of technical progress. A good example is the application of human arthroscopic equipment to endoscopic examination of birds (Lumeij et al., 1982). Other supplementary diagnostic techniques like haematology, blood chemistry, analysis of urine, radiography and electrocardiography are now also applied (Lunieljt

and Stain, 11985), From the literature and from experience it became clear that large differences exist between avian and mammalian blood. Blood chemistry in birds has

its own peculiarities and the metabolically active nucleated avian erythrocytes cause problems when performing total white cell counts. Furthermore the erythrocytes cause chemical changes in the plasma after a blood sample is collected. Methods which were developed for the diagnosis of adrenocortical insufficiency in dogs, are unsatisfactory

for birds. The collection of a urine sample from a bird presents practical problems.

Even the avian electrocardiogram appears to be inverted when compared with the

more familiar mammalian electrocardiograms.

The aim of the present study was to investigate to what extent a number of clinical

investigative methods which are routinely used in (veterinary) medicine can be applied in avian medicine. Rather than collecting at random clinicopathological data from a

variety of avian species with different diseases, it soon became clear that it was

necessary to collect data under controlled experimental conditions. This was important

to be able to draw statistically sound conclusions. Besides characterization of the

normal bird, clinicopathological changes during disease had to be studied. Because

research with regard to avian haematology (C. Hawkey, Zoological Society of

London) and radiography (A.

Rubel,

University of Zurich) was in progress these

subjects were not systematically investigated.

The racing pigeon, Columba livia domestica, was chosen as an experimental animal in this study. Racing pigeons are easily obtained and are well suited for the purpose. They are very docile animals which are easy to handle and relatively large amounts of

blood can be collected from individual birds. Furthermore, pigeons can sometimes

have a high economical value (sometimes up to Dfl 50,000,-) which can justify an

extensive

clinical examination during disease. Pigeons are used extensively

in

behavioural and

physiological research (Abs, 1983) and also in

studies on

atherosclerosis (Santerre, 1972). In addition this animal is used as an indicator species for the lead contamination of the urban environment (Hutton and Goodman, 1980). All these uses make a clinical characterization of this species desirable.

In CHAPTER I general aspects with regard to clinical pathology are discussed. How

much blood can be collected from birds and how should the blood sampling be

performed? Which anticoagulants can be used, and how should blood samples be

treated once they are collected? In which way should reference values be established

and what are the reference values for pigeon blood? Finally, which intrinsic or

extrinsic factors can influence blood chemical variables?

In CHAPTER 11

a review is given of current knowledge with regard to blood

chemistry for the diagnosis of hepatobiliary disease in birds. Reference values of

plasma chemical variables considered to be of use for the diagnosis of hepatobiliary disease in the pigeon are reported. Tissue enzyme profiles of the major organs of the

pigeon and the budgerigar

(Melopsittacus

undula(us)are presented and changes in

plasma chemistry after experimentally induced liver and muscle injury are shown. In CHAPTER III the diagnostic value of plasma proteins and non-protein nitrogen substances in birds is discussed. First a review is presented, which includes reference values for various avian species and examples of changes in plasma proteins induced by some diseases. Experimental results are given with regard to the reliability of the

refractometric method for the determination of total protein in avian plasma.

Furthermore the effects of experimental water deprivation on non-protein nitrogen

substances in pigeon blood are reported. A method is described for collection of urine

under clinical circumstances and reference values for variables in pigeon urine are

In CHAPTER IV a review is given of reported or supposed endocrinopathies in birds. Results of studies which were performed to develop an adrenocortical function test, a thyroid function test and awater deprivation test are presented.

In CHAPTER V

_{a review of avian electrocardiography is given. an addition}

reference values for the normal pigeon electrocardiogram are shown, and examples of abnormal pigeon electrocardiograms. A paper is included wherein the application of electrocardiography in research relating to avian anaesthesia is illustrated.

In CHAPTER VI results are presented of a study which was performed to determine the best endoscopic approaches to the various organs in the pigeon. Furthermore the basic equipment and technique for avian endoscopy are described and the indications for its use in avian medicine are given.

An interpretive summary is given at the end ofthis thesis.

In present day avian medicine the sound advice which was given 1200 yearsago in the first Arabic book on falconry still holds true (Cooper, 1979):

the recognition of diseases is refined by the observations of symptoms.. Do not change the disease through medicine before its recognition and diagnosis... But ascertain and investigate until you understand the disease. As soon as you are firmly convinced of your diagnosis quickly start the treatment and do not hesitate...'

The experimental work described

in this

thesis was performed to supply the practitioner with additional clinical investigative methods to further rationalize diagnosis (and by that treatment) in avian medicine.,

REFERENCES

Abs, Al. (1983). Physiology and behaviour of the pigeon. London: Academic Press, Cooper, J.E. (1979). The history of hawk medicine. Veterinary History, 1: 11-18.

Hutton, Al. and Goodman, 0. T. (1980). Metal contamination of feral pigeons, Columba /Ana,. from the London Area: Part - Tissue accumulation of lead, cadmium and zinc. Environ.Pollui. Ser.A 22: 207-217.

ukutneij, J.T, Hasselaar, J.C, Zwart, P., Stan, I. WE, and Frankenhuis, M.T. (1982).. Endoscopy as a diagnostic loot in avian medicine. Proceedings Voorjaandagen 1981. Netherlands Small Animal Veterinary Association, pp. 1)4-124.

tueneij, J. T. and Stain, I. WE. (1985). Mogelijkheden voor nadere diagnostiek bij postduiven., Proceedings studiedag duiveziek ten en therapie. Ainerongen: Netherlands Association of Avian Veterinarians, pp. 2-7.

Santerre, R.F., Wight, TN., Smith, S C., Brannigan, D. (1972). Spontaneous atherosclerosis in pigeons. The American Journal of Pathology, 67: 1-22.

Zander, D. V. (1984). Principles of disease prevention: diagnosis and control. in: Diseases- of poultry. Edited by Hofstad et al. Ames, Iowa State University Press.

CHAPTER I

The Veterinary Quarterly, 9: 249-254, 1987

AVIAN CLINICAL PATHOLOGY - GENERAL CONSIDERATIONS

J.T. LUMEIJ

Department of Avian and Exotic Animal Medicine, Small Animal Clinic, State University Utrecht, Yule/oar; 8, 3584 CM Utrecht. The Netherlands

SUMMARY

General aspects of avian clinical pathology are reviewed. It is concluded that in a clinical setting a volume of blood equivalent to 1 per cent of body weight can be collected safely from avian species for laboratory examinations. The anticoagulant of choice for most

labora-tory investigations is lithium heparin. In most bird species theright jugular vein is the preferred site for routine blood sampling. The use of a vacuum system greatly facilitates the procedure. The importance of immediately processing blood samples is explained. The many variables that may influence haematological or biochemical parameters are discussed and the reasons for determining blood chemical reference values by non-parametric methods are emphasised.

INTRODUCTION

Clinical signs in birds are often non-specific and the information gained by physical

examination is limited. Hence it is essential for the veterinarian to rely on additional

clinical investigative methods in order to rationalise diagnosis and treatment in

avian medicine (5. II. 19). In this paper general aspects in relation to clinical

pathology will be discussed.

SIZE OF BLOOD SAMPLES

Important considerations when taking blood samples in birds, especially the

smaller species, are total blood volume, response to blood loss and the amount of

blood needed to perform the diagnostic test(s).

Bond and Gilbert (3) showed that blood volume in aquatic species is significantly

higher than in non-aquatic species. Total blood volume in the mallard,

Anas platyr-hynchos,

was 11.3 ± 0.4 m1/100 g bw (mean ± s.e.m.). The lowest value observed by

these authors in non-aquatic birds (4.8 m1/100 g) was in the ring-necked pheasant,

Phasianus

colchicus. Of the non-aquatic birds only 'pigeons' were reported to have a

blood volume (9.2 ± 0.3 m1/100 g) which approached that of aquatic species. In

100 0.---, o' ,,'

,r.

,, o Rat/

x/V.

./

i

, 1117-,/ _{n Cat} ;'' ' 000g Pheasant *Crow

/

Hen

_..,4!64

,/._V

... -, Neon A Duck 2 3 4 5 6 7 8 9 10

Bleeding volume (per cent body weight)

Fig.I. Mortality after identical blood losses in various avian and mammalian species (ahcissa). Every

hourI%_{body weight of blood was withdrawn from every animal (ordinate). The percentage of animals}

lost during the hour following bleeding has been recorded and plotted. (After Kovachet al., 1969).

found to be 16.3- 20.3 m1/100 g, compared to 4.9- 14.9 m1/100 g in chickens, Gallus

domesticus. For comparison, total blood volume in the dog is 8.3 - 10.1 m1/100 g

(35). Kovach et al. (17) studied the mortality of various avian and mammalian

species following blood loss and showed that birds are better able to tolerate

severe

blood loss than are mammals, although variation existed among the avian species

tested (Fig. I). Birds are less easily brought to the level of irreversible shock than

most mammals, due to their much greater capacity for extravascular fluid

mobilisa-tion (6, 38). The study of Kovach et al. (17) revealed that in healthy individuals, the

amount of blood that can be removed without deleterious effects is 3 per cent of

body weight in ducks and pigeons, 2 per cent in chickens and 1 per cent in crows and

pheasants.

In avian patients,

1

per cent of body weight would appear a safe limit for the

amount of blood that can be collected for diagnostic purposes. Certain disease

conditions may affect the patient so much that, especially in small birds, it would be

safer to start with supportive therapy and collect blood samples later. The amount

needed, and whether blood, serum or plasma is required for the various diagnostic

tests, is dependent on the laboratory. Consulting the laboratory before blood

samples are taken is a wise precaution. When dealing with small birds the

use of

micromethods is a necessity.

ANTICOAGULANTS AND TREATMENT OF BLOOD SAMPLES

Although it has been stated that 'serum enzymes and protein electrophoresis

cannot be run on plasma' (19), in general, plasma is the material of choice for blood

chemistry in birds, for reasons mentioned below.

Significant decreases in plasma potassium concentrations occur if whole pigeon or

chicken blood is stored at room temperature (21). The phenomenon observed in

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40 0

Z

20 0 1

chemical parameters. Therefore blood for chemical determinations should be

centrifuged within a few minutes of collection, to separate plasma from cells. When

serum is prepared for blood chemistry it is inevitable that the blood has to stand for

a certain period to allow coagulation.

Although total protein is traditionally determined in serum, for clinical purposes it

is possible to use plasma for both total protein determination and protein

electro-phoresis. An advantage of using plasma instead of serum is that a relatively greater

amount of fluid can be collected for blood chemistry from the same amount of

blood. Total protein in plasma is slightly higher because it includes plasma

fibrino-gen (2 1 b, 23).

When measuring blood gases and acid-base status in avian species it is of

para-mount importance to realise that the nucleated avian erythrocytes,

which possess

virtually all enzymes typical of metabolically active cells, consume oxygen seven to

ten times faster than do mammalian red blood cells (2,12). The delay between blood

collection and analysis should be kept as short as possible. Collection in precooled

syringes and storage on ice will virtually immediately stop metabolism.

For differential leucocyte counts, blood smears should be made at the time of

collection, without using an anticoagulant. Even when an anticoagulant is used it is

best to make smears immediately after collection to prevent changes in the cell

morphology.

EDTA is regarded as the best anticoagulant for preservation of cellular

morphol-ogy and for good staining characteristics of human blood

(31), but only limited

information is available on the choice of anticoagulant for avian haematology.

Fourie (8) studied the effects of different anticoagulants in various concentrations

on some haematological parameters in the pigeon. Columba livia, and heparin was

found to be the most suitable anticoagulant.

Both EDTA and lithium heparin have been used in avian clinical haematology.

Hawkey et al. (II) reported that EDTA produced progressive haemolysis in blood

samples from crowned cranes (Balearica sp). Dein (5) also reported an unusual

reaction of the blood of some birds ('crows, jays, brush turkey, hornbills') to

EDTA. After about 15 minutes the blood became dark and viscous. Although the

blood did not clot, the cells were disrupted enough to make the sample useless. We

have observed similar reactions in blood from crows (Corvus corone) and magpies

(Pica pica).

The procedure adopted for small bird species in our clinic is as follows.

Immedi-ately after collection of a heparinised blood sample a blood smear is made, a

microcapillary tube for measuring Ht is filled and the amount of blood needed fora

total white cell count is collected in a diluting pipette. The remaining sample

is

centrifuged to separate plasma for chemical determinations.

For the determination of lead, which is the most widely reported and clinically

described poison in caged and wild birds, whole unclotted blood should be sent to

the laboratory, since in birds (as in mammals) more than ninety per cent of the lead

in whole blood is associated with the red blood cells (20). Lithium heparin is a

suitable anticoagulant. EDTA should not be used, because this may interfere

with

the measurement of lead.

SAMPLING SITES AND SAMPLING PROCEDURE

The preferred vein for blood collection in birds varies with the species.

In most species the right jugular vein is better developed than the left and it appears

to receive blood from the left jugular vein via an oblique anastomosis at the cranial

end of the neck. Often a featherless tract of skin overlies this vein.

If this vein can be located it is preferred for routine blood collection above all other

sites (18,25,34). Even in one- to two-day-old sparrows, Passer domesticus, this vein

is sufficiently large for blood collection.

In small birds it is possible for one person working alone to collect a blood sample

from this site. The bird is held on its back in the left hand with the first and second

fingers braced against the skull and extending the neck. The neck is swabbed with

alcohol, which separates the feathers and exposes the featherless tract of skin

overlying the jugular vein. Slight pressure at the base of the neck with the thumb

causes distention of the vein with blood, and the syringe can be operated with the

right hand (25). After collecting the blood sample, some pressure is exerted on the

puncture site with the thumb to prevent haematoma formation.

In larger psittaciformes (e.g. African grey parrot, Psittacus erithacus) sampling

from this site is best accomplished with two persons, one holding the bird and

stretching the neck and the other obtaining a blood sample.

In larger falconiformes (e.g., goshawk, Accipiter gent//is) sampling is easiest with

three persons. It is important that the strong talons arc controlled and do not harm

the persons involved.

When dealing with dangerous species such as the African ostrich (Struthio came/us)

or the cassowary (Casuarius casuarius), sedation is necessary. as these animals can

disembowel and kill a man with a quick forward thrust of their clawed feet (9).

The vein running along the medial side of the tarsometatarsus, the medial

metatar-sal or caudal tibial vein, (Vena metatarmetatar-salis plantaris superficialis medialis), is

especially useful for multiple sampling of small blood volumes in larger birds such

as pigeons. There is almost no haematoma formation. In birds with pigmented or

wrinkled skin and/or short bones it can be difficult to visualise this vein. Blood can

be collected using a needle and syringe, or a blood lancet.

In the racing pigeon, Columbia livia domestica, the jugular vein is not clinically

accessible and thus the medial metatarsal vein is used routinely for blood collection

in this species at our clinic.

One person (preferably the owner) holds the pigeon in an upright position with one

hand, facing the pigeon towards him, and uses the other hand to stretch the

unhanded leg downward. To expose the vein, the overlying skin is thoroughly

rubbed with a piece of cotton moistened with diethyl ether. Then the skin and vein

distal to the intertarsal joint are pierced with a blood lancet which is twisted ninety

degrees after penetrating the vein. The blood is allowed to run freely into a

collecting tube. After the sample has been obtained, the collection site is covered

with adhesive tape wrapped around the tarsometatarsus. The leg distal to the tape

should be checked for swelling in an hour or two. The tape can be removed after

three days.

The 'wing vein' (Vena cutanea ulnaris) is the vein which is traditionally used in

poultry medicine. After being swabbed with alcohol the vein is punctured with a

blood collecting tube (10). In pet birds the use of a blood lancet at this site cannot be

needle is used. The advantage of the cutaneous ulnar vein is that it can be located in all avian species. To obtain a sample with this method the bird has to be firmly restrained on its back with one wing extended. The vein is punctured proximal to the humeroradial joint, with the needle inserted in a proximal direction.

Three different methods of heart puncture have been described for collection of blood samples, namely, the lateral approach (I), the furcular approach, i.e., along

the ventral margin of the thoracic inlet (24), and the sternal approach (33, 36).

Because the procedure is potentially dangerous in unskilled hands, heart puncture

is not recommended for use in avian clinical practice, although it is useful for

obtaining large blood samples before euthanising a bird. .

Recently a blood collection technique has been described in ducks and geese (Anse-riformes) using the venous occipital sinus, located at the junction of the base of the skull and the first cervical vertebra (37). The head is held with the beak perpendicu-lar to the neck and a needle connected to a vacuum tube assembly is inserted at an angle of 30-40 degrees to the skull. When the skin is punctured the vacuum tube is

perforated and the needle is pushed a few millimeters deeper, until blood is

obtained. With this method large volumes of blood can be obtained.

When it is essential to obtain a blood sample and all other acceptable methods have failed, the sample can be obtained by clipping a toenail. The disadvantages of this method are that it is painful for the bird, the sample may become contaminated with tissue fluids, it may cause damage to the nail bed, and the amount of blood that can be obtained in this way is often limited. After obtaining a sample, bleeding can be stopped by means of a silver nitrate stick or a cottonwool swab soaked with ferric subsulphate or ferric chloride. Before obtaining a sample the nail should be thor-oughly cleaned to avoid erroneously high uric acid values due to urate

contamina-tion from the urine (29). In our clinic a vacuum system is routinely used for

collecting blood samples from the jugular and cutaneous ulnar veins, which greatly facilitates the procedure (Venoject, Omnilabo. Breda, The Netherlands).

REFERENCE VALUES

The results obtained from the laboratory have to be interpreted by the clinician. This requires reference values from a clearly defined reference

population of the

species concerned, measured by the same methods. Often the reference range is defined as the mean plus and minus two standard deviations. This method, how-ever, can only be used if the valuesobtained are Gaussian in distribution. The wide

belief that biological data have a Gaussian distribution is

inappropriate (13). Therefore, in most cases, non-parametric statistics should be used to establish reference values (4,14,30).

An example is the reference value for plasma uric acid in theracing pigeon. Lumeij

and de Bruijne (22), using non-parametric statistics,

determined the reference

values to be 150-765 mo1/1. The reference range calculated (inappropriately) from the mean plus and minus two standard deviations would have been -49 to 799 limo1/1, whieh is meaningless, since the lowest observed uric acid concentration in the reference population was 95 gmo1/1.

A number of haematological and biochemical measurements may follow a circa-dian or a circannual rhythm. Age, sex differences, altitude, nutritional status and egg laying are other variables that may cause variation (7.16,27,32,26). Different bleeding sites (e.g., venous blood versus blood collected by cardiac puncture) may also cause variation (15).

It is important for avian clinicians to be aware of all these possible variables and,

especially when publishing haematological or biochemical data, materials and

methods and animals should be specified, so that the validity of comparison with

results from other laboratories can be evaluated.

REFERENCES

I. _{Andrews FX. Simplified heart puncture in poultry diagnosis. J Am Vet Med Assoc 1950; 16: 38-9.} Besch EL. Respiratory activity of avian blood cells. J Cell Physiol 1966; 67: 301-6.

Bond CF. Gilbert PW. Comparative study of blood volume in representative aquatic and non-aqua-tic birds. Am .1 Physiol 1958; 194: 519-21.

Conover) WI Practical Non-parametric Statistics. New York: John Wiley & Sons, 1971. Dein FJ. Hematology. In: Harrison GJ and Harrison LR, eds. Clinical Avian Medicine and Surgery. Philadelphia: WB Saunders Co, 1986: 174-91.

Djojosugito AM, Folkow B, Kovach AGB. The mechanism behind the rapid blood volume restoration after hemorrhage in birds. Ada Physiol Scand 1968; 74: 114-22.

Driver EA. Hematological and blood chemical values of Mallard, Arras p. plat.vrhynchos, drakes before, during and after remigc moult. J Wildlife Dis 1981; 17: 413.

Fourie F le R. Effects of anticoagulants on the hematocrit, osmolarity and pH of avian blood. Poultry Sci 1977; 56: 1842-6.

Fowler ME. Restraint and handling of wild and domestic animals, Ames: Iowa State University Press. 1978: 264-8.

Gratz! E, Koehler H, eds. Spezielle Pathologic end Therapie der Gefluegelk rank hei ten'. Stuttgart: Ferdinand Enke Verlag 1968; 14.

II. Hawkey C. Samour .IH, Ashton DG et al. Normal and clinical hematology of captive _cranes (Gruiformes). Avian Pathol 1983; 12: 73-84.

Hunter AS, Hunter FR. A comparative study of erythrocyte metabolism. J Cellular Comp Physiol 1957; 49: 479-562.

International Federation of Clinical Chemistry. The concept of reference values. Clinica Chimica Acta 1978; 87: 459F-65F.

International Federation of Clinical Chemistry (1984). Statistical treatment of collected reference values. Determination of reference limits. Clinica Chimica Acta 1984; 137: 97F-1 I4F.

Kern MD, de Graw WA. Differences in the composition of venous and cardiac blood from white-crowned sparrows. Condor 1978; 80: 230.

Kocan RM, Pits SM. Blood values of the canvasback duck by age, sex and season. J Wildlife Dis 1976; 12: 341.

Kovach AG B, Szasz E, Pilmayer N. Mortality of various avian and mammalian specks following blood loss. Acta Physiologica Academiae Seientarium Hungaricae 1969; 35: 109-16.

Law GRJ. Blood samples from jugular vein of turkeys. Poultry Sci 1960; 39: 1450-2.

Lewandowski AH, Campbell TW, Harrison GJ. Clinical chemistries, In: Harrison GJ and Harri-son LR, eds. Clinical Avian Medicine and Surgery. Philadelphia: WB Saunders Co, 1986; 192-200. Lumeij IT. Clinicopathologic aspects of lead poisoning in birds: a review. Vet Quart 1985; 7: 133-8. Lumeij JT. The influence of blood sample treatment on plasma potassium concentrations in avian blood. Avian Pathol 1985; 14: 257-60.

2 in. Lumeij IT. The diagnostic value of plasma proteins and non-protein nitrogen substances in birds. Vet Quart 1987; 9: 262-8.

Lumeij JT, de Bruijne JJ. Blood chemistry reference values in racing pigeons, Columba livia domestica. Avian Pathol 1985; 14: 401-8.

Lumeij JT, de Bruijne.1.1. Evaluation of the refractometric method for the determination of total protein in avian plasma or serum. Avian Pathol 1985; 14: 441-4.

MacArthur EN. An improved method of obtaining blood from the chicken heart. Poultry Set 1944; 23: 542-44.

McClure HE, Cedeno R. Techniques for taking blood samples from living birds. J Wildl Managem 1955; 19: 477-8.

McGrath JJ. Acclimation response of pigeons to simulated high altitude. J Appl Physiol 1971; 31: 274-6. O. 18. .19. 20. :22. 26.,

Mon JG, George JC. Seasonal changes in serum levels of certain metabolites, uric acid and calcium in the migratory Canada goose(Brun ta canadensis interior).Comp Biochem Physiol B 1978; 59: 263.

Palomeque J, Planas J. Blood volume in domestic pigeons. Comp Biochem PhysiolA 1978; 59: 413-7.

Ross kopfWJ, Woerpel RW. Rosskopf G, van de Water D. Hematologic and bloodchemistry

values for common pet avian species. Vet Med/Small Anim Din1982; 77: 1233-9.

Riimke CL, Bezemer PD. Methods for the determination of normal values II. New methods. Ned Tijdschr Geneeskd 1972; 116: 1559-68.

Schmidt CH,Hane ME, Gomez DC. A new anticoagulant for routine laboratory procedures. A

comparative study. U.S. Armed Forces Med J 1963; 4: 1556.

Simkiss K. Calcium metabolism in the laying bird. In: Calcium in reproductive physiology. A

comparative study of vertebrates, London: Chapman & Hall Ltd, 1967: 155-96.

Sooter SA. A technique for bleeding nestling birds by cardiac puncture for viral studies. J Wildl Managem 1954; 18: 409-10.

Stevens RWC, Ridgway GJ. A technique for bleeding chickens from the jugular vein. Poultry Sci

1966; 45: 204-05.

Swenson Mi. Physiologic properties, cellular and chemical constituens of blood (blood volume).

In: Swenson MJ, ed. Dukes' Physiology of domestic animals 8th ed. CornellUniversity Press, 1970:

23-9.

Utter JM, Le Febyre EA,Green law IS. A technique for sampling blood from small passerines. Auk

1970; 88: 169-71.

Vuillaume A. A new technique for taking blood samples from ducks and geese. Avian Pathol 1983;

12: 389-91.

WyseDO,Nickerson M. Studies on hemorrhagic hypotension in domestic fowl. Canad J Physiol and Pharmacol 1971; 49: 919-26.

27:

34,

Avian Pathology, 14: 257-260, 1985

THE INFLUENCE OF BLOOD SAMPLE TREATMENT ON PLASMA POTASSIUM CONCENTRATIONS IN AVIAN BLOOD

J .T. LUMEIJ

Small Animal Clinic, State University Utrecht, Yalelaan 8, 3584 CM Utrecht, The Netherlands

SUMMARY

Plasma potassium concentrations were determined in racing pigeons

(Columba lima domestica), chickens (Gallus domesticus) and man

(Homo sapiens) after different storage times of uncentrifuged blood

samples at room temperature.

In the

pigeon plasma potassium concentrations declined

rapidly.

After 10 min the reduction was 9.5% and declined by 65% after 120

min. No further reduction was noted up to 24 hours.

In chickens decreases were also noted but were smaller overall with

a 29% decrease being noted after 2 hours.

No significant changes of plasma potassium concentrations in human

blood were found during the first 2 hours of storage.

The need to describe the treatment of blood after sampling is

indi-cated. It

is concluded that for most blood chemical measurements

plasma is preferred to serum and blood samples should be centrifuged

within minutes of sampling.

INTRODUCTION

The use of clinical biochemistry in avian medicine is of growing importance. Plasma

electrolyte concentrations are used to aid diagnosis of dehydration, renal disease

and adrenocortical insufficiency. The use of clinical biochemistry however, is

dependent upon knowledge of appropriate reference values and upon correct

sample treatment.

A time lag of up to 60 min between collection of a human blood sample and

separation of plasma and cells is common (Laessig et al., 1976). This study was

performed to investigate the influence of storage of uncentrifugecl blood samples

at room temperature on plasma potassium concentrations in avian and human

blood.

MATERIALS AND METHODS

Table 1. The effect of storage of uncentrifuged blood samples at roomy

Temperature for 10, 30 and 120 min and 24 hours on the plasma

potassium concentration in blood samples from racing pigeons, Columba livia domestica, chickens, Gallus domesticus and man, Homo sapiens.

2 mean decrease as percentage of initial value b Standard deviation (On

-I)

rc Standard error of mean in

n

d-slumber of subjects

J

volunteers (Homo sapiens) were used as experimental subjects. From each subject 3 nil of venous blood was collected using heparinised vacuum tubes (Venoject heparin

tubes. Terumo Corporation, Brussels, Belgium). Immediately after collection, the

blood samples were divided into portions,, which were centrifuged after standing at

room temperature for up to 24 hours.

Plasma potassium concentrations were determined immediately following

centri-fuging of the samples (Flame photometry with lithium as an internal standard,

Beckman Klinafname, Beckman Instruments, Fullerton, CA, USA). The

photo-meter precision was monitored by a commercial control serum which was analysed

concurrently with each group of samples (Beckman). The statistical evaluations

were based on Student's t-test for paired observations.

RESULTS

Initial plasma potassium' concentrations of pigeons ranged between 3.9 and 6.1

mmo1/1 (median 4.6 mmo1/1). There were significant decreases at storage times

up to 120 min but not thereafter (PG0.005). Initial plasma potassium concentra-,

lions of chickens ranged between 4.2 and 5.7 mmo1/1 (median 5.3 mmo1/11).,

Again storage up to 120 mm led to significant decreases (P<0.005).

Initial' plasma potassium concentrations, in human blood ranged between 3.8 and

0 mm 110 min 30 mm 120 mm 24 hours pigeon E%a 0

9.5

30.3

64.6

66.3

sdb

_3.9

_6.6

_10.1

_3.7

sem c 1.11

2.0

2.5

1.9'

nd

_{16 12} 11 116

4

chicken

E%

0

10.5 29.2

sd'

o 3.01 3.8 i sem 0 0.91 1.1 n 12 112 12 man E70 0 0.5 01.7 sd 10 1.8 2.1 .sem 0 1.4 01.9 n. 6 6 6 -0 0

4.9 rnmo1/1 (median 4.1 mmo1/1). No significant changes were seen during 2

hours of storage of whole blood samples at room temperature (Table 1).

DISCUSSION'

The results of this study dearly demonstrate that significant decreases, in plasma

potassium concentrations occur if whole pigeon or chicken blood is stored at room

temperature. The observed time dependent changes are related to contact between

plasma and cells. The findings are in contrast to the findings in human blood,

where no significant changes in plasma potassium concentrations were seen as a

result of prolonged contact with the blood cells. The observed phenomenon

_may

lead to a false impression of normokalaemia in birds with hyperkalaemia due to

adrenocortical insufficiency. Furthermore reference values for plasma potassium

concentrations in pigeons and chickens reported in the literature may be too

low due to incorrect sample treatment, e.g. Erchis and Fontaine (1978), for the

pigeon.

The phenomenon observed in pigeons and chickens may also occur in other avian

species and involve other blood chemical parameters. When serum is prepared for

blood chemistry in birds it is inevitable that the blood has to stand for a certain

period to allow coagulation. Therefore analyses on serum should be avoided

when-ever possible; plasma is the material of choice. Plasma and cells should be separated

within minutes of collection.

REFERENCES

ErclOs, A. and Fontaine, R. 119781 Ermittlung von Normalwerten des flutes bei drei

Tauben-rassen in Abha'ngigkeit von Alter und Geschlecht.Arehiv far Geflugelkunde 42: 34 44.

Laessig, Pit, Indriksons, AA., Hassemer, DJ, Paskey, TA. and Schwartz, TN. (1976).

Changes in serum chemical values as a result of prolonged contact with the clot.

Amen--can Journal of Clinical Pathology 66: 598-604.

RESUME

Influence des modalites de traitement des echantillons de sang

d 'oiseaux sur les concentrations plasmatiques en potassium

Les concentrations en potassium du plasma ont Ote determinees chez

des pigeons'

voyageurs (Columba livta domestica), des poulets (Gallus do rnesticus) et rhomme

llorrio sapiens) apres differents temps de stockage des ,echantillons sanguins

non

centrifuges a la temperature ambiante.

Chez le pigeon, les concentrations plasmatiques en potassium declinent

_rapidement.

Apres 10 minutes la reduction etait de 9.5% et atteignait 65% apres 120

mn.

Aucune diminution supplementaire n'a etd notee jusqu'a 24 H. Chez les poulets,

les memes diminutions etaient observees mais moindres car la reduction n'etait

que de 29% en 2 heures. En cc qui concerne Ehomme, aucune modification

signifi-cative du taux de potassium plasmatique n'a ete enregistree pendant

les deux

premieres heures de stockage. Le besoin de preciser les conditions de traitement

des echantillons est mentionne et

il

est conclu que pour la plupart des analyses

chimiques du sang, le plasma doit etre prefere

au serum et que les &chant illons de sang doivent etre centrifuges dans les minutes qui suivent le prelevement.

ZUSAMMENFASSUNG

Der Einflufl der Blutprobenbehandlung auf die Konzentration des Plasmakaliums im aviiren Slut

Die Konzentration des Plasmakaliums wurde inch unterschiedlicher

Aufbewah-rungszeit bei Zimmertemperatur der nicht-zentrifugierten Blutproben von

Reise-tauben (Columbia livia domestiea), Fliihnern (Gallus ,domesticus)und von Menschen (Homo sapiens) bestimmt.

Im Taubenplasma sinkt die Kaliumkonzentration schnell. Nach 10 Minuten betrug

die Reduktion 9.5% und nach

120 Minuten 65%. Innerhalb von24

Stunden wurde

kein weiterer Abfall beobachtet. Auch im Eliihnerplasma nahmen die Werte ab,

abet wesentlich weniger, im allgemeinen nach2Stunden urn 29%.

Vrahrend der ersten beiden Lagerungsstunden wurden im menschlichen Plasma.

keine signifikanten Veranderungen der Plasmakaliumkonzentration beobachtet. Es wird auf die Notwendigkeit, die Behandlung der Blutproben nach der Entnahme.

ft beschreiben, hingewiesen. Far die Messung der meisten chemischen

Blutpara-meter 1st die Verwendung von Plasma statt Serum vorzuziehen. Die Blutproben

sollteni innerhalb von Minuten nach der Entnahme zentrifugiert werden.,

RESUMEN

La influencia deL tratamiento de muestras de sangre en las concentraciones de potasio plasmatic° en la sangre de las ayes

Se determinaron las concentraciones de potasio en el plasma de palomas de carrera

(Columba Wee domestica) y del hombre (Homo sapiens) despues de diferentes

tiempos de almanenamiento en muestras de sangre no centrifugadas a la

tempr-ratura ambiente.

En la paloma las concentraciones de potasio plasmatic° declinaron rkpidamente.

Despues de 10 minutos, la reduccion fue de 9.5% declinando un 65% despues

de 120 minutos. No se observaron mayores reducciones hasta las 24horas.

Avian Pathology, 14: 401-408, 1985

BLOOD CHEMISTRY REFERENCE VALUES IN RACING PIGEONS (COLUMBA LIVIA DOMESTICA)

J.T. LUMEIJ and J.J. de BRUUNE

Small Animal Clinic, State University of Utrecht, Yalelaan 8, 3584 CM Utrecht, The Netherlands

SUMMARY

Reference values for 23 blood chemical parameters in racing pigeons

(Columba livia domestica) were established for use in clinical

patho-logy. The inner limits are given for the percentiles P2

.5 and P9 7.5

with a probability of 90%. A minimum of 50 blood samples collected

between September and January from different animals aged between

6 months and

12 years (median 1.5 years) was used for each

para-meter. Reference values obtained in the present study are compared

with values published previously.

INTRODUCTION

The diagnostic application of clinical pathology in human and veterinary medicine

is a well-established additional procedure for the establishment of a diagnosis in

certain disease conditions. Earlier demands for large blood sample volumes and

limited veterinary involvement in pet bird disease were major obstacles to a parallel development in avian medicine.

The introduction of micro methods in clinical laboratories has removed a major

obstacle to the application of clinical pathology in pet bird medicine. The use of

clinical pathology in any species, however, is dependent upon an extensive

know-ledge of reference values obtained from clearly defined normal populations.

The present study was undertaken to establish reference values for some blood

chemical parameters in racing pigeons (Columba livia domestica), and to compare

these with values published previously (Erdos and Fontaine, 1977, 1978; Parker

and George, 1976; Ramis and Planas, 1978; Wagner, 1978).

MATERIALS AND METHODS Experimental animals

Racing pigeons from a wide variety of breeds were used. About half the number

of birds had been used for racing and had been housed in our pigeon loft (Small

Animal Clinic, State University of Utrecht, The Netherlands) for at least 2 months

prior to the investigation. The animals had not been exposed to stressful situations

during this period (e.g. racing). They were given about 30 g of a commercial pigeon

ration '(Wine Molen, Meeuwen, The Netherlands) each day at 13.30 hour (MET.)

and water ad libitum. The remainder of the birds belonged to the breeding stock of

a commercial racing pigeon breeding farm (Eijerkarnp B.V. Zutphen, The

Nether-rands).

On clinical examination all pigeons appeared to be 'healthy. Throat swabs were

negative for trichomonads and faecal examinations were negative for parasite ova,

oocysts and salmonellae. All pigeons were vaccinated against paramyxovirus disease with an inactivated Newcastle disease vaccine (Newcavac NobiIts, intervet, Boxmeer, The Netherlands) at least

I month prior to the experiment.

The age of the birds ranged between 6 months and 12 years (median 1.5 years)

and their weights between 456 and 610 g (median 518 g). Blood sampling was done

between September and January to exclude changes in blood chemical parameters,

in relation to the breeding cycle and at evenly spaced times between 08.00 and

18.00 (M.E.T.) to exclude confounding data due to possible variations arising from

a circadian rhythm in the metabolic status of the birds or due to a postprandial

effect. All birds were exposed to the local day-night rhythm.

A minimum of 50 blood samples from different animals was used to establish

[reference values for each parameter.

Male and female pigeons were approximately equally represented. Sampling

Samples were collected from the vena metatarsalis plan tans superficialis tnedialis,

To expose the vein the' overlying skin was rubbed with a piece of cotton, wool

moistened with diethyl ether. The vein was punctured with a blood lancet and the

blood was allowed to flow freely into blood collecting tubes.

For serum total protein estimations and serum protein electrophoresis the blood

samples were allowed to coagulate for 1 hour in plain blood collecting tubes before

centrifuging, to prevent latent fibrin formation in the serum.

For glucose estimations blood was collected in tubes containing 4 mg fluoride

and 2 mg K2 EDTA for 2 ml of blood.

All other parameters were determined in plasma. Blood was collected in tubes

containing 45 US? units of lithium heparin per 3 ml of blood. Immediately after

drawing of blood plasma and cells were separated by centrifuging.

Chemistry

All analysis were done on the same day the samples were taken, except uric acid,

iron and iron-binding capacity, for which samples were stored at -20°C.

All enzyme determinations were carried out according to the Recommendations

of the German Society of Clinical Chemistry (1972). The analytical methods and

the coefficients of variation (V.C.) showing the day to day variation of these

methods. are tabulated (Table I).

Statistics

Reference values were established using a distribution free method (Conoven

1971; Iblimke and Bezemer, 1972). The inner limits are given for the percentiles

P2.5 and P97.5 with a probability of 90%. To make comparison with data from

Table I. Plasma or serum chemistry reference Values for racing pigeons. -P2.5 and P52.5 are given.

Materials and methods

flame photometry flame photometry atomic absorption spectrophotometry atomic absorption spectrophotometry colorimetry, TC 124 974 BMc coulometty, amperometry colorimetry, Guanidine-ferrozine method, Baker Chem. colorimetry, Guanidine-ferroaine method, Baker Chem. vapour pressure osmometry MCAd, Hexokinase method, TC 124 338 BM MCA, Jaffd kinetic reaction at 30° C. photometry, uricase method TC 124 737 BM, MCA, 30° C, TC 181 188 BM MCA, 30° C, TC 415 278 BM MCA, 30° C, TC 158 186 BN1 MCA, 30° C, TC 158 178 BM MCA, 30° C, TC 244 236 BM MCA, biuret method with blank correction

}Serum

protein electrophoresis was performed

using cellulose acetate membranes. Barbital buffet pH 8.6, 20 minutes, 200 Volts, Staining with Ponceau S

b

Day to

day variation coefficient of analytical method.

d Multistat FLS Centrifugal Analyser.

Allied Instrumentation Laboratory, London, UK.

-Ti P2.5 -P9'7.5 Median Mean ± s.e.mean V.C.%b 68 141449 mmo1/1 145 145 ±0.3 0.9 52 3.94.7 mmo1/1 4A 4.4 ±0.06 1.4 52 1.9-2.6 mmo1/1 2.2 2.3 ± 0.03 50 1.1-1.8 mmo1/1 1.3 1.3 ±0.03 53 0.57-1.33 mmo1/1 0.81 0.83 ± 0.04 3.0 SS 101-113 mmo1/1 107 107 ±0.5 50 11-33 prno1/1 22 23 ± 2.1 4.7 binding capacity, 50 3045 i.ano1/1 37' 37.5 ± 0.8 5.2' 55 297-317 mOsm/kg 305 306 = 0.9 47 96 12.9-20.5 mmo1/1 16.4 16.6 = 0.2 3.2 52 23-36 mmol/1 27 28 ± 0.6 2.4 50 150-765 firno1/1 330 375 ±30 4.0 SO 130-380 U/I 170 203 = 13.5 4.6 65 160-78013/I 320 367 = 25 4.3 50 32-107 U/I 50 57 ± 3.2 6.0 50 40-85 U/I 55 58.6 ± 2.5 5.1 50 18-31 U/I 23 25 ±1.6 3.8 93 21-33 g/I 28 27 = 0.4 2.9 58 14 g/1 2 2.8 ± 0.13 58 13-22 g/1 17 17.5 ± 0.36 58 2-3 g/I 2 2.29 ± 0.08 58 3-6 g/1 4 4.3 ±0.14 58 1-3 g/I ,2 1.9 ± 0.1 a 2.1 2.1 3.2

RESULTS

Reference values for 23 plasma or serum chemical parameters of racing pigeons,

as established in this study, are shown in Table I.

Fig. 1 shows a densitometer scan and the electrophoretic pattern from a

represen-tation pigeon serum.

Plasma glucose concentrations in morning samples were significantly lower than

plasma glucose concentrations in samples taken between 14.00 and 18.00 hr

(P<0 .01 ).

DISCUSSION

A subdivision in age-groups, sex and time of sampling would have been impractical for clinical use, although variation due to these factors cannot be excluded.

The wide belief that biological data assume a Gaussian distribution is

inappro-priate (International Federation of Clinical Chemistry, 1978). Also in this study

most of the data could not be described by a Gaussian distribution. Therefore

non-parametric statistics had to be used to establish reference values (International

Federation of Clinical Chemistry, 1984). However, a less appropriate parametric

estimation technique was also applied (mean ± s.e.mean) to enable comparison with

reference values from other authors who did not give information about

distribu-tion of data and may have misused this method in ignorance of its basic

assump-tions.

The plasma uric acid concentration as determined in this study was 375 ± 30

prno1/1 (mean ± s.e.mean). Wagner (1978) found plasma uric acid concentrations

of 219 ± 13 pmo1/1 (mean ± s.e.mean) in white Carneaux pigeons. These lower

values might be attributed to the fact that this parameter was measured in fasting

subjects. ErdOs and Fontaine (1977) determined reference values for blood

chemi-cal parameters in healthy racing pigeons but did not include uric acid in their

study. In analogy with mammals they determined serum urea concentrations as a

parameter for renal function. Because pigeons are uricotelic it seems more

appro-priate to measure plasma or serum uric acid concentrations to assess renal function

in this species.

The plasma potassium concentrations in the present study was 4.4 ± 0.06 mmo1/1

(mean ± s.e.mean). Erdos and Fontaine (1977) have found serum potassium values

of 3.8 ± 0.1 mmo1/1 (mean ± s.e.mean, n = 20) in 4-year-old female racing pigeons.

However. in their study the serum was separated from the cells after the blood

samples had been standing for

1

hour at room temperature. In a recent study

(Lumeij, 1985) it has been shown that plasma potassium values in pigeons start to

decrease immediately after collection of the blood sample. It was demonstrated

that reliable results could only be acquired if plasma and cells were separated

immediately after collection.

ErdOs and Fontaine (1977), using the biuret reaction for the determination of

serum total protein, give reference values for racing pigeons within the range of

the reference values presented in this article. The electrophoretic results, however,

Cannot be compared because different materials and methods were used.

Pre-alb. Alb.

CI

Fig; Densitometer scan and electrophoretic pattern from a representative pigeon serum.

Pre-albumin, albumin, a-,0-and ry globulin.

IL

A

was found. Morning values were lower than afternoon values, suggesting a circadian

rhythm in plasma concentrations or a postprandial hyperglycaemia in this species.

The reason that other authors report lower reference values for plasma glucose

values in racing pigeons might be associated with different times of sampling.

Erdos and Fontaine (1978), for example, report values of 10.8 ± 0.4 mmo1/1

(mean ± s.e.mean, n = 20) for plasma glucose in 4-year-old female racing pigeons.

Sampling was done between 8 am. and 9 a.m. (Koln/Germany, February).

Accord-ing to our findAccord-ings, values in the lower range are to be expected at that time of

the day. Parker and George (1976) reported a plasma glucose concentration of

110'.1

± 0.15 (mean ± s.e mean, n = 26) in randomly bred white Carneaux pigeons

(Columba livia). The lower values reported by the latter authors might be

associate-with breed differences. The question whether a circadian rhythm can be found in

plasma glucose concentrations and the existence of a postprandial hyperglycaemia

in pigeons should be investigated.

Reference values for plasma iron were in agreement with values published

previous-ly by Erclos and Fontaine (1978) and Ramis and Planas (1978). Reference values

for total iron-binding capacity however, were markedly lower than reported by the

above mentioned authors. No explanation was found.

Reference values for ALAT, ASAT and AP cannot be compared with values

report-ed previously (Erdris and Fontaine, 1978) because different methods were usreport-ed

(Bessey, Karmen and Wroblewski respectively). These methods can be, considered

less accurate and inappropriate to modern instrumentation..

REFERENCES

Erdos, A. rind R. Fontaine (1977). Ermittlung von Normalwerten des Mines bei drei Tauben-rassen in Abhangigkeit von Alter und GeschIccht. I. Mitteilung. Archly flit Geflugel-kunde 41. 238-245.

Erd6s, A. und K Fontaine (1978). Ermittlung von Normalwerten des Mutes bei drei Tauben-rassen in Abhangigkeit von Alter und Geschlecht. 2. Mitieitung. Archly far Geflugel-kunde 42, 3444.

Conoven, W.I. (1971). Practical Nonparametric Statistics. John Wiley & Sons, New York.,

International Federation of Clinical Chemistry (1978). The concept of reference values. Clinica Chimica Acta 87, 459F465F.

International Federation of Clinical Chemistry (1984). Statistical treatment of collected

refer-ence values. Determination of referrefer-ence limits. Clinica C7zimica Acta 137, 97F-114F.

Gummi, IT (1985). The influence of blood sample treatment on plasma potassium

concen-trations in avian blood. Avian Pathology 14, 257-260.

Parker, GA. and J.C. George (19761. Changes in blood glucose, blood and muscle lactate, and plasma lactic dehydrogenase levels in the pigeon on acute exposure to cold. Archives

Intemationales de Physiologie et de Biochlmie 84, 517-526.

Ramis. J. and I Planas (1978). Iron metabolism in pigeons. Quarterly Journal of Experimental

Physiology 63, 383-393.

Recommendations of the German Society for Clinical Chemistry (1972)k Journal, of Clinical

Chemistry and Clinical Biochemistry 10, 281-291.

Rumke. CL. and P.D. Bezemer (1972). Methods for the determination of normal values II .

New methods. Nederlands Tildschrift voor Geneeskunde 116, 1559-1568.

Wagner, W.D. (1978). Risk factors in pigeons genetically selected for increased(atherosclerosis,

susceptibility. Atherosclerosis 31, 453-463.

-RESUME

Valeurs de reference des constantes biochintiques dui sang chez le pigeon voyageur (Columba livia domestica)

Des valeurs de reference concernant 23 parametres biochimiques du sang chez

les pigeons. voyageurs (Columba livia dottiest/ca) oat ete etablies pour etre utilisees

en pathologie clinique. Les lirnites interieures sont donnees pour les percentiles

P2.5 et P97.5 avec une probabilite de 90%. Un minimum de 50 echantillons

pre-leves entre septembre et janvier a partir des differents animaux ages de 6 mois

a 12 ans (medianne 1.5) ont ete utilises pour chaque parametre. Les valeurs de

reference obtenues dans is presente etude soot comparaes avec les valeurs publiees

precedemment.

ZUSAMMENFASSUNG

Referenzwerte fiir die Blutchemie bei Reisetauben (Columba livia domestica)

Fiir den Gebrauch in der Iclinischen Pathologic wurden Referenzwerte von 23

Blutparametern bei Reisetauben (Columba livia domestica) aufgestellt. Die

Giiltig-keit wurde beschrankt auf die Prozentwerte zwischen P2,5 und P97.5 mit ether

Wahrscheinlichkeit von 90%. EM Minimum von 50 Blutproben wurde zwischen

September und Januar von verschiedenen Tieren im Alter zwischen 6 Monaten

und 12 Jahren (Mittelwert 1.5 Jahre) filr die Bestimmung jeden Parameters

aus-gewertet. Die in dieser Untersuchung gewonnenen Referenzwerte wurden mit

anderweitig veroffentlichen verglichen..

RESUMEN

Valoxes de referencia de la quirnicasanguinenn

palomas de Carreras

Los valores de referencia pars 23 parametros de quimica sangulnea en palomas

de carers (Columba livia domestica) fueron establecidos pare su uso en patologl a

clinica. Los 'finites internos son expresados por los porcentajes P2.5 y P27.5

!con una probabilidad del 90%. Un minim° de 50 muestras de sangre colectadas

'entre septiembre y enero a partir de anitnales diferentes, con edades de 6 meses

a 12 Mos (promedio de 1.5 altos) fueron empleados para cada parametro. Los

valores de referencia obtenidos en el presente estudio son comparados con los

THE INFLUENCE OF BLOOD SAMPLE TREATMENT, FEEDING AND

STARVATION ON PLASMA GLUCOSE CONCENTRATIONS IN

RACING PIGEONS (COLUMBA LIVIA DOMESTICA)

J.T. LUMEIJ

Department of Avian and Exotic Animal Medicine, Small Animal Clinic, State University Utrecht, Yalelaan 8, 3584 CM Utrecht, The Netherlands

SUMMARY

In avian blood samples it is not necessary to prevent glycolysis for reliable glucose determinations, as long as the blood is not stored for more than 2

hours. This is contrary to the situation in mammals where sodium fluoride is

often used for this purpose.

No hypoglycaemia was observed in pigeons up to 4 days of starvation, but rather a starvation hyperglycaemia after 3 days.

The high blood glucose concentrations which were observed in the present and a previous experiment in afternoon blood samples, 4 hours after feeding, were

attributed to a postprandial effect.

In fasted pigeons plasma glucose concentrations in afternoon blood samples

were significantly lower than in morning samples, suggesting a circadian rhythm of plasma glucose in fasted subjects.

INTRODUCTION

D-glucose is

the major circulating carbohydrate in birds, and plasma glucose

concentrations in birds are significantly higher than plasma glucose concentrations in mammalian species (Hazelwood, 1986).

To get reliable results of glucose determinations in mammalian blood samples it is

important to prevent glycolysis by the erythrocytes, e.g. by adding sodium fluoride to

the anticoagulant. It has been stated that also in avian blood contact of serum with

the clot will result in 5 per cent per hour loss of serum glucose due to red blood cell metabolism (Lewandoski et al., 1986). Avian erythrocytes, however, consume very little, if any, glucose (Hazelwood 1986) and preventing glycolysis seems therefore not necessary in avian blood samples.

It has been stated that presurgical fasting will predispose 'birds' to hypoglycaemia

(Harrison,

1986). However, no controlled experiments seem to substantiate this

statement.

In a previous experiment plasma glucose concentrations in blood samples taken in the afternoon were higher than in morning blood samples (Lumeij and de Bruijne, 1985).

In this study it was suggested that this could be due to a circadian rhythm or a

postprandial hyperglycaemia. In chickens

(Gallus gees domesticus)

a circadian

rhythm in plasma glucose concentrations has been demonstrated, daytime glucose

levels being higher than nighttime glucose levels (Twiest and Smith, 1970).

The aim of the present study was threefold. First the hypothesis was tested that it is not necessary to prevent glycolysis by avian erythrocytes with fluoride for accurate

glucose determinations. Blood from racing pigeons,

Columba liviu domestica, was

used for this

purpose. Second the

effect

of starvation on plasma glucose

concentrations in

racing pigeons was determined. Third the occurence of a

postprandial hyperglycaemia in this species was investigated.

MATERIALS AND METHODS

Experiment I.

Heparinized blood samples from 10 healthy adult racing pigeons (5

male and 5 female) were divided in three portions directly after sampling. The blood

sampling procedure has been described previously (Lumeij, 1987). In one portion

plasma and cells were separated immediately by centrifuging

= 0 h). The other

portions were first stored at room temperature (20°C) for 2 h and 24 11, respectively.

Plasma glucose concentrations were determined in all samples with a hexokinase

method on a Multistat FLS centrifugal analyser (Allied Instrumentation Laboratory, London, UK) with a commercially available test combination (TC 124338, Boehringer

Mannheim, West Germany). Differences between t =0 h, t = 2 h and t = 24 h were

tested for significance using Student's t-test for paired observations. The difference

between values found at t = 0 h and reference values established previously, with a

method whereby fluoride (4 mg for 2 ml of blood) was added to the anticoagulant

(Lumeij and de Bruijne, 1985), was tested for significance using Student's t-test for independent observations.

Experiment 2. Six healthy adult racing pigeons (3 male, 3 female), with body weights

varying from 413 to 507 (median 432) g, were starved up to 96 h. Plasma glucose

concentrations after 24 h, 48 h, 72 h and 96 h of starvation, were compared with

plasma glucose concentrations directly after feeding (t = 0 h). Blood was collected, plasma obtained and glucose determined as in experiment 1. Differences were tested for significance using Student's t-test.

Experiment 3. Plasma glucose concentrations were established at 07.30 (clock hours, GMT) in 48 healthy adult racing pigeons (20 male, 28 female), which were used to get

fed at 11.30. Body weights varied from 397 to 573 (median 464) g. Blood was obtained,

plasma prepared and glucose determined as in experiment I. Half of these animals (10

male, 14 female) were fed at 11.30 with 30 g of a commercial pigeon diet (Witte Molest,

Meeuwen, The Netherlands), while the rest of these animals was starved. At 15.30 a second blood sample was collected from all the animals. Differences in plasma glucose concentrations between morning and afternoon samples in the two groups were tested for significance using the signed rank test. Differences between the two groups in the morning and in the afternoon were tested for significance using the

Wilcoxon-Mann-Whitney test.

All experiments were performed in October 1986 in the Netherlands. Plasma's were stored at 4°C pending analysis, which was always performed within 24 h of collection

of a blood sample.

RESULTS

Experiment I. Mean ( ± sd) reference values in fluoridized samples reported previously (Lumeij and de Bruijne, 1985) were 16.6 ± 1.95 mmol/litre, while in this experiment values of 16.4 ± 1.13 mmol/litre were found in heparinized blood samples which were not fluoridized. This difference was not significant. At t = 2 h values of 16.3 ± 1.25 mmol/litre were found. These values were not significantly different from those at it = 0 h. At t = 24 h values of 15.3 ± 1.36 mmol/litre were measured, which represented a significant 6.6 per cent reduction when compared to values at t = 0 h (P<0.05). Experiment 2. No decrease in plasma glucose concentrations was observed in response to starvation

for 96 h (Table

1). Contrary to expectations plasma glucose concentrations were significantly higher after 3 days of starvation in pigeons, when compared to plasma glucose concentrations directly after feeding (P<0.05).

Table I. Effects of fasting up to 96 h on plasma glucose concentrations (mina/litre) in adult racing pigeons (n=6). Male and female pigeons were equally represented.

Experiment 3. Plasma glucose concentrations were significantly lower in afternoon samples than in morning samples in pigeons that were starved at 11.30 (P<0.001), while pigeons that were fed had significantly higher afternoon plasma glucose concentrations (P<0.001). The initial values were not significantly different between

the two groups, while the difference at 15.30 was significant (P<0.01) (Table 2).

Table 2. Effect

of feeding

or starvation on plasma glucose concentrations (mmol/litre) in adult racing pigeons (times are clock time, GMT).

0 h 24 h 48 h 72 h 96 h 07.30 h 15.30 h 07.30 h 15.30 h mean 18.9 17.6 18.5 20.8 min 12.6 16.2 17.2 17.4 max 26.4 19.0 20.1 32.8 sd 2.3 0.8 0.2 3.4 mean 14.8 15.0 15.6 18.1 16.5 sd 1.8 0.6 0.8 0.8 0.6

pigeons starved at 11.30 h pigeons fed at 11.30

(n = 24) (n = 24)