Textbook Physiology

Methods & Protocols
for Practical Training

in Physiology
- Part 1
Eva Matalová
Jaroslav Doubek
Department of Physiology
University of Veterinary and Pharmaceutical Sciences
Brno, Czech Republic

Matalová-Doubek-2021-Department of Physiology-UVPS Brno-Czech Republic
Author for correspondence:
Prof. Dr. Eva Matalová, Ph.D.

Department of Physiology and Pathophysiology
University of Veterinary and Pharmaceutical Sciences
Palackého 1-3
612 42 Brno
Czech Republic
Copyright ©Eva Matalová et al., 2021
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Table of Contents
Laboratory Animal Science 5

CELLULAR PHYSIOLOGY 7
Cell Cycle – Cell Proliferation 7
Organ Explants Cultures and their targeted Modulations 10
Cell Homeostasis 14
INTERNAL ENVIROMENT 17
Internal environment – AB status 17
Internal environment - Electrolytes 20
PHYSIOLOGY OF BLOOD 22
Suspension Stability of the Blood 22
Functions of Plasma Proteins 25
Haematocrit 28
Erythrocyte Count 30
Osmotic Resistance of Erythrocytes 33
Blood Indices 36
Agglutination of Erythrocytes 38
Count of Leukocytes 42
Permanent Blood Smear 45
Leukocyte Differential Count 48
Functions of Leukocytes in the Immune System 52
Haemostasis, Coagulation 55
Automated Haematology 59
METABOLISM AND THERMOREGULATION 64
Basal Metabolism 64
Physiological Heat Production 66
Physiological Heat Losses and their Metabolic Compensations 68
Thermoregulation in Homoiothermic and Poikilothermic Animals 71
Adipose Tissue and its Function 73
SKIN PERCEPTION 76
PHYSIOLOGICAL REGULATIONS 79
REFERENCES 82
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We hope this textbook will help students prepare for practical courses, perform experiments,
and elaborate protocols. The topics of the protocols are listed to correspond to the practical
courses in the Department of Physiology, University of Veterinary and Pharmaceutical
Sciences Brno, Czech Republic, in the first term (Physiology I) with English as the
communication language. The protocol style follows the simple description of aims,
experimental design, results, and discussion with space for conclusions, self-evaluation, notes
and comments.
Thank to Dr. L. Zdražilová Dubská and other co-workers who have helped with preparation
of this textbook.
Authors
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LABORATORY ANIMAL SCIENCE
Laboratory animal science can be defined as a multidisciplinary branch of science,

contributing to the human use of animals in biomedical research and to the collection of
informative, unbiased and reproducible data. Laboratory animal science encompasses the
study of the biology of laboratory animals, their husbandry, genetic and microbiological
standardization procedures, the prevention and treatment of diseases, the optimization of
experimental techniques, and the improvement of anaesthesia, analgesia and euthanasia.
Ethical aspects of animal experimentation, together with the search for alternatives, also fall
within the domain of laboratory animal science.
Basic methodical approaches in Physiology:
– Experience/observation
– Dissection/autopsy
– Clinical study (adspection, palpation, auscultation, percussion)
– Experiment (in vivo, ex vivo, in vitro, in ovo, in silico)
– Modelling (observation, verification, computers, mathematics, living models/model
organisms)
An animal experiment means any scientific procedure involving animals. Most animal
experiments are performed in the fields of medical, biological, veterinary and agricultural
science. Animals are used for a wide range of purposes. The major areas are drug research,
vaccine testing, and toxicity testing and cancer research. Other purposes involve basic
biomedical research, genetic studies, diagnosis, experimental surgery, education etc. The
number of animals used for the various purposes may vary among countries. Animal
experiments are subjected to international and national legislative regulations. Performance of
an animal experiment is not permissible if the result can be reasonably and practically
obtained without the use of the animal.
Alternatives to animal experiments involve particularly:
– In vitro and ex vivo techniques

– The use of lower organisms
– Immunological techniques
– Quantitative structure-activated relationship analysis
– Mathematical modelling of physiological processes
– Human models
– Other alternative methods (e.g. video and computer based demonstrations for
education)
The 3 R Principle (Replacement, Reduction, Refinement) is the main guideline for the
responsible use of animal in experiments (introduced by Russell and Burch in 1958).
5
Replacement refers to the substitution of living animals by alternative techniques,

computerized models, videos, films, etc. which yield the same results without the use of live
animals.
Reduction refers to a decrease in the number of animals required for a given experiment. This
can be achieved by choosing suitable experimental procedures, by controlling environmental
factors and by standardizing the animal population.
Refinement refers to any decrease in the incidence or severity of painful or distressing
procedures applied to animals. This can be achieved by the adjustment of the environment to
suit the behavioural (e.g. environmental enrichment) and physiological needs of the animal,
improvement of experimental procedures and methods of anaesthesia etc.
FELASA (Federation of European Laboratory Animal Science Associations) was established
in 1978 to advocate responsible scientific conduct with animals in the life sciences with
particular emphasis on ensuring animal welfare including the 3Rs of Laboratory Animal
Science. FELASA (www.felasa.eu) maintains relations with national, international and
governmental bodies concerned with laboratory animal science particularly in Europe, notably
the Council of Europe, the European Commission and European Parliament.
Good laboratory practice (GLP) principles refer to the methods of conduct, management of
the research and the circumstances under which it is carried out. They stimulate accuracy in
the conducting and reporting on animal experiments.
Legislation in laboratory animal science is based on the premise that, under certain conditions,
it is morally acceptable to use animals for experimental and other scientific purposes. There is
an obligation to notify the authorities (governmental authority, local ethics committee) in
advance about the proposed experiments and who will be conducting them. The person must
have certified competence in planning and performing animal experiments.
Two important documents issued in Europe create the basis for controlling the use of animals
in experiments - Convention for the Protection of Vertebrate Animals used for Experiments
and other Scientific Purposes (ETS 123) and Directives for the Protection of Vertebrate
Animals used for Experiments and other Scientific Purposes 86/609/EEC). All European
Union members are compelled to implement the provisions of the EC Directives via their
national laws (e.g. 246/92 Sb. in the Czech Republic, modified by the law 359/2012 Sb.).
Task 1: Search for laboratory animal science policy related to your home country and provide
comparison with legislation in the Czech Republic.
Task 2: Learn about animal welfare, list major approaches and historical milestones.
NOTES & COMMENTS
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CELLULAR PHYSIOLOGY
CELL CYCLE – CELL PROLIFERATION
INTRODUCTION
Cells as the basic functional units of the organisms undergo multiple division (proliferation).
During cell division, DNA replicates (chromosomes are doubled) and distributed into
daughter cells. Timing and control of proliferation is based on specific mechanisms of the cell
cycle. In the first phase (G1), the cell increases its volume, further DNA synthesis occurs (S
phase), the cell becomes ready for division (G2) and enters mitosis (M).
The most common proliferation markers are PCNA (proliferating cell nuclear antigen) and
Ki-67 (product of the MKI67 gene). One of possible detection methods is the
immunohistochemistry using histological sections.
AIMS
 Evaluation of the cell cycle in the small intestine

 Immunohistochemistry of proliferating cell nuclear antigen
 Evaluation of proliferation rate of the tissue and within specific cell populations
EXPERIMENTAL DESIGN
By visualization of PCNA protein in cell nuclei can be detected cells undergoing proliferation
within a tissue. Such detection is based on specific antigen-antibody binding and antibody
visualization. This straight forward procedure applies particularly for fluorescent labelling, for
conventional light microscopy (and long-term preservation of the sample), a multiple-step
system is preferred: 1) antigen-antibody (primary antibody) reaction, 2) primary antibody –
secondary (e. g. biotinylated) antibody reaction, 3) biotin - enzyme (e.g. POD – horse radish
peroxidase) binding, 4) enzyme-substrate (chromogenic) reaction. To distinguish the positive
cells more clearly, so called counterstaining is performed before mounting.
Histological section - small intestine:

(for preparation method see histology)
– Deparaffinization: xylen, and rehydration: ethanol series (100%-96%-70%-50%), distilled
water
– Pretreatment - antigen revitalization (if necessary) using citrate buffer
– Wash in PBS (Phosphate-Buffered-Saline)
– Inhibition of endogenous peroxidase: H2O2 treatment
– Wash in PBS
– Primary antibody (30 - 60 min/RT/humidified chamber)
– Wash in PBS
Note: if biotinylated primary antibody is used, skip the following (secondary antibody) step and go
directly to Biotin-POD
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– Secondary antibody – against IgG of the species of origin (30 min/RT/humidified chamber)
– Wash in PBS
– Biotin-POD complex (30 min/RT/humidified chamber)
– Wash in PBS
– Chromogenic substrate (e.g. DAB – diaminobenzidine)
– Wash in PBS
– Counterstaining – haematoxylin (5 min + 10 min tap water)
– Dehydration (ethanol series), mounting
RESULTS
 Labelled tissue section with PCNA positive (proliferating cells) with brown nuclei and
PCNA negative (non-proliferating cells) with blue nuclei
Histological section of mouse duodenum (after PCNA immunohistochemistry, positive cells

appear brown, haematoxylin background is blue).
 Evaluation of labelled section with respect to distribution of positive cells (crypts, villi
etc), identification of particular cell types – repetition from histology:
 Expression of proliferation rate of Paneth cells:
 Expression of proliferation rate in the entire crypt:
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DISCUSSION
Immunohistochemical reactions are based on compatibility of an antigen and corresponding

antibody. Antigen (antibody generating) is any substance able to evoke an immune response
(antibody production). This fact is widely used also in research and practice since against any
antigen, the antibody can be produced. There are many ways for antibody preparation, such as
injection of the antigen into an animal (mostly rabbit or mouse) followed antibody separation
from the serum, or preparation in hybridomas (immortal antibody producing cells) cultures
and antibody collection from the culture medium.
Proliferating Cell Nuclear Antigen (PCNA) takes part in topological interaction between DNA and
DNA-polymerase during replication (DNA clump). Therefore, the antigen is located in the nucleus
with the highest level in the S-phase of cell cycle. Apart of DNA replication, PCNA is important for
DNA repair.
Cell cycle markers can be used also for evaluation of the explant growth in the culture.
Proliferation activity (rate) refers to the percentage of proliferating cells form the entire
number of cells within the cell population of interest. Proliferation rate evaluation is an
important part of tissue investigation related to renewal (cell cycle) and belongs also to the
tumour tissue examination. High cellularity, high proliferation rate, infiltrative growth and
necrosis are generally considered as markers of tumour aggressiveness.
CONCLUSIONS
NOTES & COMMENTS
9
ORGAN EXPLANT CULTURES

AND THEIR TARGETED MODULATIONS
INTRODUCTION
Animal experiments are still an important background for the rapid progress in biomedicine.
Alternative methods however, prefer to follow the 3R principle (reduction, refinement,
replacement) as much as possible. Organ explant cultures eliminate animal suffering during
experiments but simultaneously allow investigation of intact organ, tissue and cell systems
with preserved interactions corresponding to the situation in vivo. Explant cultures refer to
ex vivo systems and a mouse model is usually used due to acceptable size of the organs/tissues
for cultivation. Explants are grown on a Millipore paper placed on a supportive grid in the
middle of a plastic dish. A humid environment is kept by using round, distilled water soaked
filter paper. Explants are cultured in a thermostat incubator at 37 °C with a 5 % CO2
atmosphere.
The explants can be manipulated in several ways such as the implantation of beads soaked in
purified signalling proteins or the separation of epithelium and mesenchyme which allows
heterotypic/chronic recombination. Moreover, cells can be transplanted into specific regions
of the explants and their fates followed. DNA (gene constructs) can be electroporated into
specific areas of the tissue to misexpress genes, inhibit protein function using dominant
negatives or inhibit translation using morpholino antisense oligonucleotides. A transfer into
renal capsules can finalize further development of embryonal organs.
Explant cultures are frequently used in studies to reveal the molecular basis of development,
physiological principles of tissue homeostasis and to investigate medical disorders at the cell
and molecular levels.
AIMS
 Preparation of culture dish

 Preparation of culture medium
 Preparation of explants culture
 Cultivation of explants culture
 Discussion
EXPERIMENTAL DESIGN
Culture dish preparation

– Under sterile conditions open the well organ culture dish (Falcon).
– Position a sterile supportive metal grid in the centre in exactly horizontal position.
– Insert a moist filter paper into the outer ring of the dish.
– Label the dish with experiment indices and your name.
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Culture medium preparation

– Under sterile conditions prepare the culture medium consisted of DMEM (Dulbecco´s
Modified Eagle´s Medium) supplemented with glutamine, pen/strep and 10 % fetal
calf serum.
– Fill in the inner ring of the dish with the medium to cover the grid.
Explant preparation
– Under stereoscopic microscope dissect the organ/tissue of interest (keep as much
sterility as possible).
– Place the explants on a culture filter paper and position on the supportive grid (the
paper must be cover by medium to guarantee nutrition but there must be kept an
interface of the explants with the atmosphere).
– Deliver fresh medium everyday as long as you keep the culture.
– Keep cultures at 37 C, 5 % CO2 and modified according to your experimental needs.
RESULTS
Species and organ used for preparation:
Explants in a culture dish, ready for cultivation.
DISCUSSION
Explant culture represent ex vivo approaches in physiological research. Explant

micromanipulation techniques have got a great future not only thanks to easy culturing,
investigation and reliable results but also due to the support of the 3 R Principles in laboratory
animal science and good laboratory practice approaches. Moreover, they can be used in stem-
cell-based tissue-engineering approaches to create organs and tissues for transplantation.
The organ/tissue of interest supplemented with culture medium and ready for cultivation and
further experimental modifications. One example is modulation of cell cycle demonstrated e.
g. by application of doxorubicin to duodenal cultures. Doxorubicin is an anthracycline
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antibiotics used in anti-cancer therapy. Along with cancer cells it targets all tissues with a
rapid renewal such as in the duodenum. The functional enterocytes in mammals undergo
renewal in a few day intervals by differentiation of the stem cells. Doxorubicin blocks the cell
cycle due to DNA damage. Evaluation of morphology and proliferation can be done e.g. by
immunohistochemistry using PCNA or Ki67 proliferation markers (see the previous course).
Mouse duodenal explants with clearly seen villi, ready for cultivation and treatment.
Ki67 staining in the control (left) and doxorubicin (right) treated explants clearly shows the
destructive effect of doxorubicin on rapidly cycling cells.
Chemotherapies in general target cancer cells with high proliferation activity. Cytostatic
effect can be simultaneously seen also in other cells and tissue with a rapid renewal. Thus, the
cytostatic effect causes inhibition of enterocyte renewal from the stem cells in the duodenum
and consequently digestion complications in the patient.
Discoveries related to „Cell cycle and its regulations“ were awarded by the Nobel Prize in
Physiology or Medicine in 2001 (Leland Hartwell – US, Tim Hunt – UK, Paul Nurse – UK).
Another novel ex vivo approach in cellular and molecular physiology, so called organ slices,
allows even the testing of hypotheses concerning specific clusters of cells within an organ.
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This methodical procedure enables easy observation of the cell population within an organ.
The sliced organs can not only be clearly seen in the culture but also modulated according to
experimental needs.
CONCLUSIONS
NOTES & COMMENTS
13
CELL HOMEOSTASIS
INTRODUCTION
Homeostasis as a dynamic constancy of internal environment refers to cells, tissues, organ and
the entire body. Selected simulations of external environment alterations for free cells (blood
elements) enable to evaluate the ability of cells to balance such alterations and demonstrate
the impact of such changes on plasma membrane, cell survival and other physiological
aspects.
AIMS
 To follow and microscopically evaluate the effect of different environment on animal

cells:
o Detergent
o Ethanol
o Direct osmotic changes
EXPERIMENTAL DESIGN
Control sample:
– Heparin treated blood, PBS will be use as alternative of tested solutions
Experimental samples:
– The effect of detergent on animal cells: cover one drop of blood on a slide with the
cover slip, add one drop of Septonex to the cover slip edge (the Septonex will travel
underneath of the cover slip) – observe reaction on the blood/Septonex interface
– The effect of ethanol on animal cells: cover one drop of blood on a slide with the
cover slip, add one drop of 100 % ethanol to the cover slip edge (ethanol will travel
underneath of the cover slip) – observe reaction on the blood/ethanol interface
– Osmotic alterations in the environment: a) cover one drop of blood on a slide with the
cover slip, add one drop 1.5 % NaCl to the cover slip edge, b) cover one drop of blood
on a slide with the cover slip, add one drop 0.3 % NaCl to the cover slip edge, NaCl
solution will travel underneath of the cover slip – observe reaction on the blood/NaCl
interface
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RESULTS
 Description of microscopically observed events at the cellular level after treatment

with altered environment.
 Comparison with control samples and discussion of findings.
DISCUSSION
Homeostasis plays a central role in maintenance of physiological functions, however, cell-

friendly environment is necessary also in handling tissue samples. Particularly, cell lysis of
erythrocytes (haemolyses) – so called haemolytic samples – makes many diagnostics analysis
very difficult or almost impossible. Therefore, this aspect must be considered in the case of
blood sample collection and sample treatment.
Cell lysis can be of different kinds (based on the causing agent):
• Osmotic: hypotonic, hypertonic environment (solutions)
• Physical: ultrasound, flicking, mixing, temperature
• Chemical: reactions with membrane lipids
• Toxic: bacterial, snake or protozoa toxins
• Immunological: complement – membrane defects (pathological)
The mammalian bodies have four major buffer systems:

 Bicarbonate buffer system (carbonic acid) – H2CO3/HCO3-: primary ECF
(extracellular fluid) buffer against non-carbonic-acid changes.
 The protein buffer system: primary ICF (intracellular fluid) buffer, but also buffers
ECF.
 The haemoglobin buffer system: primary buffers against carbonic acid changes.
 The phosphate buffer system – H3PO4/H2PO4-/HPO42-: important urinary buffer, but
also buffer ICF.
Note: Phosphate Buffered Saline (PBS) is used as an isotonic, non-toxic solution for ex vivo
works with mammalian cells/tissues/organs, substance dilutions etc. The salty solution
contains sodium chloride, sodium phosphate, and potassium phosphate in order to maintain a
constant pH
CONCLUSIONS
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NOTES & COMMENTS
16
INTERNAL ENVIRONMENT – ACID BASE STATUS
INTRODUCTION
Balance between acidic and basis substances in the inner environment is crucial for normal
function maintenance and morphological stability of the organism. The balance is established
if the production and uptake of protons (H+) on one side corresponds to its consumption and
elimination on the other side. Examination of the acid-base status is an important segment
in the laboratory diagnostics of the internal environment disorders and several organ or
systemic failures.
AIMS
 Determination of major AB parameters – pH, HCO3-, base excess, pCO2 in blood in

selected species
 Comparison among species
 Discussion
EXPERIMENTAL DESIGN
Sample collection
– Fix the animal, collect venous blood
– Respect rules of preanalytical handling of the sample (anaerobic collection with
minimal compression, storage at 1-4 °C – on ice, analysis no later than 2 h after
sample collection etc.)
Sample analysis:
– Perform the analysis using an automatic analyser in the clinical laboratory
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RESULTS
Records of analysed parameters
Sample/Species pH HCO3-, BE pCO2

mmol/l mmol/l kPa
Reference values
Species pH HCO3-, BE pCO2
mmol/l mmol/l kPa
Cat 7.28 – 7.40 17.5 – 20.7 -0.5 - +4 4.8 – 6.4
Cow 7.38 – 7.43 23.5 – 27.0 -0.5 - +4 5.3 – 6.4
Dog 7.30 – 7.42 19.1 – 24.1 -0.5 - +4 5.2 – 6.7
Horse 7.32 – 7.44 24.6 -3,0 - +3,0 5.0 – 6.1
Pig 7.33 – 7.41 23.0 – 26.5 -2 - +3.6 5.6 – 7.7
According to Clinical Laboratory for Small Animals (UVPS Brno)
DISCUSSION
Acid-base maintenance requires effective regulations because the hydrogen ion is very
reactive. Proton production and consumption occur in different metabolic pathways. Proton
producing reactions include e. g. non-oxidative glycolysis, ketogenesis, urea synthesis,
lipolysis, whereas, proton consuming reactions include e. g. gluconeogenesis (from lactate),
lactate oxidation. The sources of protons are particularly tissue respiration and acid
production (H3PO4, H2SO4, uric acid) during metabolizing of proteins, phospholipids etc.
Proton homeostasis is granted by buffer systems and also by regulatory functions of organs,
particularly of lungs, kidneys, bones and liver.
Imbalance in AB status accompanies acidosis or alkalosis which may be either metabolic or
respiratory (based on the cause). The case, when pH of the blood becomes stabilized but some
other marker/parameter is deviated, refers to compensated AB disorder.
CONCLUSIONS
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NOTES & COMMENTS
19
INTERNAL ENVIRONMENT - ELECTROLYTES
INTRODUCTION
Electrolytes belong to basic components of the inner environment which contribute

particularly to acid-base balance and body fluid volume maintenance, muscle activity and
transduction signal in the nervous system. Ions of sodium and potassium associated with
chloride anions have the greatest importance in these functions. Concentration of these ions in
plasma is regulated via kidneys, skin, GIT and system of inner secretion.
Analysis of electrolytes is one of screening methods related to homeostasis and its
regulations.
AIMS
 Determination of the Na/K/Cl electrolyte level in blood serum/blood plasma in

selected animal species
 Discussion
EXPERIMENTAL DESIGN
Sample collection (e.g. from the rabbit's central ear artery):

Sample preparation:
– Separate plasma by centrifugation of the collected blood, 10 min/15 000 rpm
– Collect the supernatant (plasma) into a 0.5 ml tube and close the lid
Sample analysis:
– Insert the sample into the tray (carousel) of the analyser
– Once all samples are positioned, lock the carousel lid
– Start analysis
RESULTS
Records of the Na/K/Cl levels
Evaluation of data
Sample/Species Na K Cl
mmol/l mmol/l mmol/l
20
Electrolytes reference values

Species Na K Cl
mmol/l mmol/l mmol/l
Cat 150 – 160 3.5 – 5.0 117 – 123
Cow 132 – 152 3.9 – 5.8 97 – 111
Dog 145 – 155 4.7 – 5.5 105 – 115
Horse 132 – 146 2.4 – 4.7 99 – 109
Pig 135 – 150 4.4 – 6.7 94 – 106
Rabbit 131 – 155 3.6 – 6.9 92 – 112
Reference values according to Doubek et al. 2010
DISCUSSION
Natremia refers to the plasma level of sodium. Its regulation includes particularly the
reabsorption effect of aldosterone on the distal tubulus and collecting duct of the kidney.
There is also a secondary effect of the antidiuretic hormone (ADH) as Na+ transport is linked
with water reabsorption in the distal tubulus as supported by this hormone. An opposite,
natriuretic effect, has atrial natriuretic peptide (ANP) which works against the renin-
angiotensin-aldosterone system (RAAS) and plays an important role in heart tissue
maintenance.
Kalemia refers to plasma level of potassium. Also kalemia is regulated by aldosterone from
the adrenal cortex but as aldosterone aims to increase natremia, it decreases kalemia.
Aldosteron supports secretion of potassium in the distal tubulus and collecting duct of the
kidney.
Chloridemia (chloremia) refers to plasma level of chlorides and is linked with regulations of
sodium and potassium ions. The major regulatory organs are therefore kidneys, then also
intestine and skin (due to the effect of aldosterone).
CONCLUSIONS
NOTES & COMMENTS
21
PHYSIOLOGY OF BLOOD
SUSPENSION STABILITY OF THE BLOOD
INTRODUCTION
Erythrocyte sedimentation rate (ESR, sed rate) is a method for evaluation of suspension
stability of the blood. ESR measures the speed in which red cells settle to the bottom of the
test tube and thus represents a simple, rapid and very common method of blood
investigations. This method is unspecific and cannot ultimately constitute a diagnosis. The
sedimentation rate value, however may serve to alert that there is an inflammation in process,
an allergic reactions and/or an infectious disease. ESR also increases in cases of anaemia,
malignancy (blood or solid), higher fibrinogen or γ – globulin levels, and under physiological
conditions such as menstruation or the end of gravidity.
AIMS
 Sedimentation rate evaluation in different species

 Comparison with the reference values and among species
 Discussion
EXPERIMENTAL DESIGN
Sedimentation measurement is performed in a sedimentation capillary filled with

anticoagulant-treated (citrate) blood:
– Fill the blood reservoir in the basis up to the strip (approx ¾ of total volume).
– Screw the reservoir into the lid until the blood is pushed up in the capillary to the zero
level.
– Fix the capillary in the sedimentation holder in the upright position.
– Read the sedimentation rate given by the plasma level in the capillary, which clears
after separation of blood elements.
– Repeat reading the sedimentation values after 15, 30 and 60 minutes.
– Express the results in mm per hour [mm/h].
RESULTS
Sedimentation rate in species under study:
Sample/Species 15 min 30 min 60 min

mm mm mm/h
22
The horse is an animal with the highest physiological sedimentation rate (70 mm/h), whereas
the cow is the opposite extreme with 0-1 mm/h.
Schematic principle and result of blood sedimentation.
DISCUSSION
Sedimentation principle
Erythrocyte density is higher than plasma density and therefore in the anticoagulated blood
the force of gravity causes the sinking of blood elements to the bottom.
Sedimentation speed indicates the suspension stability of the blood. Suspension stability
of the blood is balanced by the electric charge on the plasma membrane of erythrocytes
(negative electric bilayer) and by plasmatic proteins.
Thus, sedimentation depends on erythrocyte aggregation (vertical columns – rouleaux) and
the content of the plasmatic protein above. Other factors such as the number, size and shape
of erythrocytes play a role.
Increase in globulin and/or fibrinogen fractions increases the speed of sedimentation (by
increased aggregation of erythrocytes), indicating immune defence processes (production of
antibodies) and increased protein degradation in the organism.
CONCLUSIONS
23
NOTES & COMMENTS
24
FUNCTIONS OF PLASMA PROTEINS
INTRODUCTION
Plasma proteins are organic components of blood plasma. Plasma proteins include
albumines, globulines and fibrinogen. The functions of plasma proteins cover transport and
storage, they work as coagulation factors and antibodies. Plasma proteins contribute to
oncotic pressure and suspension stability of the blood. Blood serum refers to plasma without
fibrin/fibrinogen.
Proportion of individual protein depends on species and physiological/pathological status of
the organisms. One of methods for detection and separation of plasma protein is
electrophoresis of blood serum or plasma. The method represents a semiquantitative
evaluation and is based on protein separation in a gel by electric current according to the size
and charge of individual proteins.
AIMS
 Separation of plasma proteins from serum

 Results evaluation
 Discussion
EXPERIMENTAL DESIGN
Sample preparation
– Blood plasma or serum can be used
Sample spotting
– Take out IFC gel and dry it by using filtration paper
– Fix the template in the centre of the IFC gel into the position A (Albumin)
– Pipette 5 μl of the sample and let it diffuse for 1 min
– Keep ladder position in the middle
Electrophoresis
– Insert the gel into the glass bath filed with the B2 barbital buffer; in the proper
orientation (direct current)
– Close the electrophoretic bath
– Set the electric field: 90V/20 min
Staining of separated samples
– Dry the fixed gel in the desiccator, 15 min/80 °C
– Continue with staining using Amidoblack, 4 min
– Wash 3x in dH2O and dry the gel
25
RESULTS
The result of electrophoresis is electrophorogram, which shows individual fractions of

proteins in the sample. This method serves as screening method, which finds out the deviation
of normal proteins spectrum in the serum but also in urine or liquor.
Agarose gel electrophoresis pattern of plasma proteins.
 Identification of plasma proteins and discussion of their functions

Protein Specification (e. Functions
g. α,β,γ)
26
DISCUSSION
Electrophoresis is based on ability of charged particles to migrate in the electric field. The
speed of migration depends on their size, surface charge, molecular configuration and also
concentration. Electrophoretic gel is a 3D net, through which the particles travel to the
electrode with opposite polarity. Mole molecules move quicker than the big ones and are
therefore identified closer to the opposite electrode. Position of individual fractions can be
précised using specific markers (protein of known size). Further specification requires 2D
electrophoresis, capillary electrophoresis and other modifications. Simple protein separation is
a screening method to evaluate organic components of blood plasma.
CONCLUSIONS
NOTES & COMMENTS
27
HAEMATOCRIT
INTRODUCTION
Blood is the suspension of blood elements in liquid blood plasma. Red blood cells are the
most numerous cellular elements in the blood and occupy approx 45 % of the total volume of
blood in adult humans. The volume of erythrocytes, expressed as a percentage of the total
blood volume or L/L, is called the haematocrit (HCT, Ht) or packed cell volume (PCV).
AIMS
 The determination of the microhaematocrit in selected animal species

 Discussion
EXPERIMENTAL DESIGN
Microhaematocrit. The plasma and erythrocyte ratio is measured by centrifugation in

microcapillaries.
– Soak anticoagulated blood (e.g. heparinized) in a capillary, the capillary ends are
sealed by plastic lids.
– Perform centrifugation 10 min/3000 rpm.
– Plasma and blood elements become clearly separated.
– Evaluate the haematocrit value. A thin leukocyte layer is ignored by the haematocrit
evaluation. In case of microcentrifugation, the haematocrit value is read using a
special reading frame.
1.0 (100 %)
Plasma
Buffy coat
Erythrocytes
Schematic principle and result of hematocrit. 0.0 (0 %)
28
RESULTS
Microhaematocrit values
Cow:
Dog:
Horse:
Other species:
Comparison with the reference values, among species and discussion.
Haematocrit references values in different species

Cat Cow Dog Goat Horse Pig Rabbit Sheep
0.25 - 0.24 – 0.37 – 0.22 – 0.32 – 0.32 – 0.33 – 0.27 –
0.45 0.46 0.55 0.38 0.53 0.50 0.50 0.45
According to Doubek et al. 2010
DISCUSSION
The haematocrit value depends on:

 The individual number of erythrocytes.
 The sex of the animal. Testosteron stimulates erythropoiesis via erythropoietin
released from the kidney and liver.
 The size of erythrocytes (macrocytes, normocytes, microcytes).
 The hydration of the organism (dehydration: lower plasma level – increased
proportion of erythrocytes – higher HCT).
Within the automatic haematological analyzer, haematocrit is not directly measured but
calculated by multiplying the red cell count (RBC) by the mean cell volume (MCV) as
average volume RBC (see the next chapters).
CONCLUSIONS
NOTES & COMMENTS
29
ERYTHROCYTE COUNT
INTRODUCTION
The count of erythrocytes represents one of the most common measurements in laboratory
blood investigations. The number of blood elements is counted in a special Bürker chamber
or automatically (analyser). The counting chamber contains a polished thick slide, which is
modified to create a space of a high space of 0.1 mm after being covered by a cover slip. The
surface of the chamber is divided into individual fields differing in shape and size – bigger
squares of 1/25 mm2, smaller squares of 1/400 mm2 and rectangles of 1/100 mm2.
Erythrocytes are counted in smaller squares or rectangles. All of the elements inside the area
and those touching just two flanks in a right angle (e.g. upper + right, or lower + left) are
counted. This is known as the Bürker rule.
AIMS
 The determination of the count of erythrocytes in selected animal species

 The comparison with physiological reference values
 Discussion
EXPERIMENTAL DESIGN
– Pipette 4 975 µl of Hayem solution (Na2SO4, NaCl, HgCl2, H2O) in a flask.

– Add 25µl of the anticoagulant-treated blood sample (dilution 200x) - make sure, the
exact blood volume is in the flask. Wash the pipette tip properly!
– Seal the flask, mix gently and keep for 3 min at the room temperature.
– Mix gently again and then either drop the sample directly on the Bürker chamber
surface or place it on the cover glass first and let the blood sample draw in by capillary
force.
– A microscopic magnification of 400x (ocular 10x, objective 40x) is generally used to
count the red blood cells in 20 rectangles or 80 small squares of the chamber by a
meandering movement of the sample.
30
RESULTS
Counting of cells in the Bürker chamber.
Number of erythrocytes in the investigated undiluted blood sample and comparison with
reference numbers.
Result calculation – example:

Calculations (an example): 20 rectangles counted (rectangle surface 1/100 mm2, depth 1/10
mm), dilution 200 times, 560 erythrocytes found
Volume of 1 rectangle: 1/1 000 mm3
Cells per one rectangle: 560/20 – therefore, 560/20 cells in volume of 1/1 000 mm3 (200 times
diluted blood)
Cells in 1 mm3of the evaluated sample (200 times diluted blood) = 560/20 x 1 000 = 28 000
Cells in 1 mm3of the original (undiluted) blood sample = 560/20 x 1 000 x 200 = 5 600 000
Cells in 1 l of the original blood sample = 560/20 x 1 000 x 200 x 106 =5.6 x 1012
Final result: the erythrocyte count in the blood sample is 5.6 x 1012 per litre (=5.6 T/l)
Number of erythrocytes in the evaluated sample:
Reference values
Species Erythrocytes (x1012/l)
Dog 5.5 – 8.5
Cat 5 – 10
Horse 6.5 – 12.5
Cow 5–7
Pig 5–8
Sheep 8 – 16
Rabbit 4-6
According to Doubek et al. 20003, 2010
31
DISCUSSION
Automated erythrocyte count

Within the automatic haematological analyzer, the red blood cell count (RBC) is measured as
sphering of RBC and detection of laser light scatter interfering with erythrocytes (provides
information about count, size, and the haemoglobin level in RBC).
Erythrocytes – advantages of biconcave disc shape (doughnuts): 30 % larger surface for

oxygen diffusion, travel easily in thin capillaries
Anisocytosis means striking differences in erythrocyte size. Poikilocytosis refers to
occurrence of erythrocytes of abnormal shape. Erythrocytosis refers to an increased number
of erythrocytes (dehydration, stress, neoplasm). Erythrocytopenia means a decreased
number of erythrocytes (anaemia).
The number of erythrocytes does not change with temperature, climate or nutrition. Nor does
it oscillate during the day. However, the number of erythrocytes is sex and species dependent.
CONCLUSIONS
NOTES & COMMENTS
32
OSMOTIC RESISTANCE OF ERYTHROCYTES
INTRODUCTION
Erythrocytes keep the membrane intact in an isotonic environment, such as the blood. An
isotonic solution has the same osmotic pressure as the blood plasma. However, erythrocytes
are sensitive to different chemical and physical impacts. Those stimuli may dismantle the
erythrocyte membrane in a reversible or an irreversible ways. The intracellular mass is
released into the cell environment by a process called haemolysis. The osmotic resistance
(or fragility) test is one of the procedures for investigation of the cause of haemolysis,
together with the Ham (Acid serum lysis) test, the sucrose lysis test, or the mechanical
resistance test. Osmotic resistance of erythrocytes refers to a concentration interval
(resistance interval) without any haemolytic effect.
AIMS
 The estimation of the concentration of NaCl which causes partial and total haemolysis
of erythrocytes
 The demonstration of the maximal and minimal osmotic resistance of erythrocytes
 Discussion
EXPERIMENTAL DESIGN
– Pipette NaCl and distilled water into six tubes as given in the table.
– Add 2 drops of blood to each tube, stir gently and wait for 30 minutes.
– Perform the “reading test” to evaluate the results:
o Take a printed page and place it behind the tubes.
o The tube content will be transparent (letters can be easily read) because the
maximal resistance of erythrocytes was trespassed and hemolysis was
completed.
Tube Nr. 1 2 3 4 5 6
1% NaCl 8 ml 7 ml 6 ml 5 ml 4 ml 3 ml
dH2O 2 ml 3 ml 4 ml 5 ml 6 ml 7 ml
% NaCl 0.8 0.7 0.6 0.5 0.4 0.3
RESULTS
Animal species under study:
Concentration of NaCl causing partial haemolysis:
33
Concentration of NaCl causing total haemolysis:
Maximal osmotic resistance of erythrocytes:
Minimal osmotic resistance of erythrocytes:
no partial total
hemolysis hemolysis hemolysis
„Reading test“ to evaluate results:

take a printed page and place it behind the test tubes,
if the test tube content is transparent and letters can
be easily seen, the maximal osmotic resistance
of erythrocytes was trespasses and hemolysis was completed.
DISCUSSION
In laboratory-clinical practice, more exact osmotic resistance tests are applied. The
concentration range is between 0.24 and 0.7 % NaCl and samples are centrifuged to clearly
see the results. Optical evaluation or ELISA readers can be applied.
Decreased osmotic resistance is associated with erythrocyte defects such as sickle cell anemia,
thalassemia or conditions connected with target cells. Increased osmotic resistance may be
observed in cases of hereditary spherocytosis (a very rare disease).
Osmotic pressure works on a semi-permeable membrane interface between a hypotonic and
a hypertonic solution and is caused by molecules of the diluent (usually water) passing
through the membrane to equilibrate pressure on both sides of the membrane and may cause
so called osmotic haemolysis.
HYPOTONIC HYPERTONIC
water
water molecules
molecules ERYTHROCYTE
34
In a hypotonic environment, erythrocytes absorb water which enters through the membrane
to equilibrate the pressures. Erythrocyte volume increases and the cell cracks. Hemoglobin
gets released into the solution which becomes red and the cell debris sinks to the bottom.
In a hypertonic environment, the intracellular water passes through the erythrocyte
membrane into the solution. Erythrocyte volume decreases and the cell shrinks and breaks
down.
Anaemia is the most common disorder of the blood. The three main classes of anaemia
include excessive blood loss (acutely such as a haemorrhage or chronically through low-
volume loss), excessive blood cell destruction (haemolysis) or deficient red blood cell
production (ineffective haematopoiesis). The cause of anaemia is determined by evaluation of
patient history, clinical and physical examination findings, and hematologic laboratory results
(concentration of haemoglobin, haematocrit, RBC and their morphology, reticulocyte count,
MCV, MHC, MCHC).
CONCLUSIONS
NOTES & COMMENTS
35
BLOOD INDICES: MCV, MCH, MCHC
INTRODUCTION
The RBC parameters are determined along with the haematocrit, haemoglobin and
erythrocyte numbers to evaluate erythropoiesis. These RBC indices involve average
erythrocyte size (MCV), average haemoglobin amount per erythrocyte (MCH) and
haemoglobin concentration per red blood cell (MCHC). RBC indices are used for
differential diagnosis of the causes of anaemia.
AIMS
 Calculations of Mean Corpuscular Volume, Mean Corpuscular Haemoglobin, Mean

Corpuscular Haemoglobin Concentration in red blood cells of selected animal species
 Comparison of the results with reference values
 Discussion
EXPERIMENTAL DESIGN
– MCV (Mean Corpuscular/Cell Volume) - [fl]
haematocrit (l/l) x 1000

number of erythrocytes (1012/l)
– MCH (Mean Corpuscular/Cell Hemoglobin) -

[pg]
haemoglobin (g/l)
number of erythrocytes (1012/l)
– MCHC (Mean Corpuscular/Cell Haemoglobin Concentration) - [g/l]
haemoglobin (g/l)
haematocrit (l/l)
Example for calculation:

Haematocrit: 0.4 l/l
Number of erythrocytes: 6x1012/l
Haemoglobin concentration: 144 g/l
MCV:
MCH:
MCHC:
36
Index of RBC count in different animal species

MCV [fl] MCH [pg] MCHC [g/l]
Cat 35-50 12-17 290-340
Cow 40-60 11-17 300-360
Dog 65-75 22-25 300-340
Goat 16-25 5-8 300-360
Horse 37-58 12-20 310-380
Pig 50-68 12-30 300-340
Rabbit 50-75 17-23 270-340
Sheep 28-40 8-12 310-340
According to Doubek et al 2003, 2010
DISCUSSION
MCV differences include:

Higher value: macrocytosis (pathological: in anemia caused by vitamin B12/folic acid
deficiency)
Lower value: microcytosis (anemia caused by iron-deficiency – most common blood disease)
MCH differences include:

Higher levels: hyperchromia (physiological in newborns)
Lower levels: hypochromia
Anaemias are defined based on cell size (MCV) and the amount of Hgb (MCH): microcytic
anaemia, normocytic anaemia, macrocytic anaemia, hypochromic anaemia, normochromic
anaemia, hyperchromic anaemia; e.g. microcytic/hypochromic anaemia from iron-deficiency
vs. microcytic/normochromic anaemia from kidney failure (lack or erythropoietin).
NOTES & COMMENTS
37
AGGLUTINATION OF ERYTHROCYTES
INTRODUCTION
Identification of the blood groups (types) allows successful transfusion and transplantation.
The discovery of blood types in humans has been connected with the names of Karl
Landsteiner (Austrian – Nobel Prize Winner) and Jan Janský (Czech) who classified human
red blood cells into four groups in the AB0 system according to antigen on the cell surface
(agglutinogens) and antibodies in the blood plasma (agglutinins). Many other blood systems
may be recognized in humans, and of a particular importance is the Rh factor. Erythrocytes of
common domestic animals possess several species specific antigens belonging to many blood
group systems. Erythrocyte clumping as a consequence of agglutinogen-agglutinin reaction
refers to agglutination.
AIMS
 Determination of blood groups (blood typing) in the human AB0 system using
purified antibodies
 Discussion
EXPERIMENTAL DESIGN
– Use a commercially available kit with purified antibodies.

– Drop the antibodies on the corresponding area of the testing sheet (blue circle – anti-
A, yellow circle – anti-B).
– Drop the blood sample treated with anticoagulant into the red circles.
– Mix the blood and antibodies using a wooden or plastic stick.
– Observe and evaluate the results.
RESULTS
Agglutination reaction with anti-A antibody: positive – negative
Agglutination reaction with anti-B antibody: positive – negative
Blood sample type:
38
Agglutination test – blood types AB0 - design.
Agglutination test – blood types AB0 – results: (left), negative reaction (right).
Red blood cell
Agglutinogen A Agglutinin anti-A

Agglutinogen B Agglutinin anti-B
Agglutination test – blood types AB0 - principle.
39
DISCUSSION
Blood groups in animals

 Antigens in different species are arbitrarily designated using the same letter system.
This does not imply that the antigens are related (antigen A of the dog is not the same
as antigen A of humans).
 Most blood groups in animals are inherited as simple Mendelian dominant characters.
 Many blood groups systems in various animals are comprised of several blood group
factors, e.g. the B system of cattle has got more than 300 alleles.
 Blood group antigens vary in antigenicity.
Blood transfusion
 Transfusion therapy is an attempt to replace blood or its component when life is
threatened without such a restoration.
 Blood-typing (pre-selection of appropriate donor for the recipient according to their
blood groups) followed by cross-matching (test for agglutination of the candidate
donor red cells by recipient plasma) to select a proper donor-recipient pair is a step to
safeguard against a severe transfusion reactions.
 Blood-typing may not be feasible in general veterinary practice because of a lack of
suitable reagents. Therefore, cross-matching is the most practical approach.
 It is a generally accepted principle in veterinary medicine that the first transfusion can
be given safely without regard to blood-typing. However, subsequent transfusions
require proper cross-matching. Remember the secondary immune response!
Alternative methods for blood typing

In the clinical practice, gel-bound antibodies immobilized directly in the test tube are widely
used. Blood sample is dropped into the tubes, results are evaluated after centrifugation.
Principle of blood typing using immobilized antibodies and centrifugation. Schematic position
of the antibodies (colourless in the reality): anti-A (blue), anti-B (yellow), anti-D (green).
40
CONCLUSIONS
NOTES & COMMENTS
41
COUNT OF LEUKOCYTES
INTRODUCTION
Leukocytes (white blood cells, WBC) are the mobile units of the body´s immune defence
system. Leukocytes and their tissue derivatives defend against invasion of pathogens by
phagocytizing the foreigners or causing their destruction by more subtle means. They identify
and destroy cancer cells that arise within the body and function as a “cleanup crew” that
removes debris resulting from dead or injured cells e.g. wound healing and tissue maintenance
and repair. The count of leukocytes can be determined in the Bürker chamber (for details see
the chapter “Erythrocyte count”) using a Türk solution which stains the cell nuclei and
dissolves the blood sample.
AIMS
 Determination of the count of leukocytes in selected animal species

 Comparison with physiological reference values
 Discussion
EXPERIMENTAL DESIGN
– Insert 475 μl of Türk solution (water solution of gentian violet and acetic acid) into a
test tube.
– Add 25 μl of the blood sample (treated by anticoagulant).
– Flick the tube gently.
– Drop the sample on the chamber grid.
– Place the sample under the microscope, use meander evaluation of the slide and count
the cells in 50 large squares (each 1/25 mm2). Do not forget the Bürker rule: count all
the cells inside the square plus the cells touching two sides in a right angle from inside
or outside of the square area.
– Calculate the number of leukocytes in one litre of the blood.
Leukocytes in the Bürker chamber, meander evaluation of the slide, and the Bürker rule.
42
RESULTS
The number of leukocytes in the original blood sample, comparison with reference numbers.
Result calculation – example:

Calculation (an example): 50 large squares (square surface 1/25 mm2, depth 1/10 mm), 20
times diluted, 80 leukocytes found
Volume of 1 square: 1/250 mm3

Cells per one square: 80/50 – therefore, 80/50 cells in 1/250 mm3 (of 20 times diluted blood)
Cells in 1 mm3of the evaluated sample (20 times diluted blood) = 80/50 x 250 = 400
Cells in 1 mm3of the original blood sample = 80/50 x 250 x 20 = 8 000
Cells in 1 l of the original blood sample = 80/50 x 250 x 20 x 106 = 8 x 109
Final result: the leukocyte count is 8 x 109 per litre
Number of leukocytes in one litre of the examined blood sample:
Species Leukocytes (x109/l)

Dog 17
Cat 7 – 17
Horse 5.4 – 14.3
Cow 4 – 12
Pig 11 – 22
Sheep 4 – 12
Rabbit 6 - 12
DISCUSSION
Within the automatic hematological analyzer, the white blood cell count (WBC) is measured
by identification according to optical characteristics of the cells (reflect their morphology).
The leukocyte count is not sex dependent. It depends on the physiological/pathological
conditions of the animal, gravidity, circadian alterations, nutrition, temperature, hypoxia,
stress etc.
Leukocytosis (neutrophilia, lymphocytosis, monocytosis) refers to an increased number of
leukocytes in circulation (infection diseases, inflammation, lymphomas, leukemia).
Leukopenia (neutropenia, lymphopenia, monocytopenia) refers to a decreased number of
leukocytes (after drug treatment, irradiation, higher sensitivity to infection attack).
Leukemia is malign proliferation of blood cells (usually white blood cells) and thus an
increased number of WBC.
Myeloid leukemia are characterized by the purposeless proliferation of one or more of the
nonlymphoid marrow cell lines (granulocytic, monocytic, erythroid, or megakaryocytic).
Myeloid leukemia occurs as acute or chronic.
43
Lymphoid leukemia indicates a neoplastic condition of lymphocytes present in bone marrow

and/or blood that is not associated with a solid tumour(s). Lymphoid leukemias are further
classified as acute or chronic depending on the maturity of the cells involved.
Leukocytes and their derivates (such as cytokines) fulfil the following major missions in the
body:
 They defend against invasion of pathogens
 They identify and destroy cancer or potentially dangerous cells that arise within the
body.
 Granulocytes and macrophages function as a “cleanup crew” by phagocytosis of
debris resulting from dead or injured cells. This is essential for wound healing and
tissue repair.
CONCLUSIONS
NOTES & COMMENTS
44
PERMANENT BLOOD SMEAR
INTRODUCTION
A blood smear is a microscopic film obtained by a smooth rubbing of a blood drop on a

microscopic slide and by the staining of it. Blood smears are used for the visualization of
leukocytes. They are necessary for leukocyte differential and enable the evaluation of the size
and shape of blood elements. Permanent blood smears can be long-term stored for repeated
evaluations.
AIMS
 Preparation of permanent blood smears from the peripheral blood of selected animal
species
 Staining of permanent blood smears using the Pappenheim protocol
EXPERIMENTAL DESIGN
– Place a blood drop on a slide.

– Use another slide to pull the drop, rubbing it on the slide in a thin film (never push the
drop!).
– Allow the sample to dry and stain it using the Pappenheim protocol.
– Pappenheim protocol:
o Apply 20 drops of May-Grünwald staining solution for 3 min (staining of the
cell cytoplasm).
o Add 4 ml of distilled water for 3 min.
o Add diluted Giemsa-Romanowski solution (15 drops in 10 ml of water) for 15
minutes, resulting in the staining of the cell nuclei.
o Wash the sample in distilled water, dry and investigate under the microscope.
45°
Permanent blood smear preparation.
45
RESULTS
A stained blood smear for further evaluation, such as the leukocyte differential count.
DISCUSSION
Blood
7 –10 % of the entire body weight
55 – 90 ml/kg live weight - amount of the blood in mammals
Volume of blood per weight in different species

Species Cat Cow Dog Horse Goat Pig Sheep
ml blood/
60-70 70-90 75-90 65-75 65-70 55-65 55-65
kg live weight
According to Doubek et al. 2003
Blood elements
Erythrocytes
Number: x1012/l of blood
Longevity: 120 days
Leukocytes
Number: x109/l of blood
Longevity: depends on the cell type
 granulocytes: neutrophil, eosinophil, basophil
 monocytes (macrophages): normal, inflammatory
 lymphocytes: B-lymphocytes, T-lymphocytes, NK-cells
Thrombocytes (platelets)
Number: .x109/l of blood
Longevity: 9 – 12 days
46
Plasma
Anorganic substances: water (91–92 %), Na+, Cl-, HCO3-, K+, Ca2+, Mg2+, phosphates and
sulphates. Functions: maintenance of osmotic pressure and acido-basic balance (hydrogen-
carbonate and phosphate buffer systems
Plasma proteins: albumins, globulins and fibrinogen. Functions: maintenance of plasma

volume, transport of various ions and molecules, contribution to the proteolytic systems such
as hemocoagulative, kallikrein-kinin, fibrinolytic and complement, buffer systems, nutrition,
and the maintenance of the suspension stability of the blood.
Other organic components: glucose, triacylglycerols, phospholipids, cholesterol, urea,

aminoacids, lactate, nitrogenous non-protein substances, free fatty acids, hormones, vitamins,
enzymes, and eventually drugs.
CONCLUSIONS
NOTES & COMMENTS
47
LEUKOCYTE DIFFERENTIAL COUNT
INTRODUCTION
There are five types of leukocytes in the peripheral blood which fall into two main categories,
depending on the appearance of their nuclei and the presence or absence of granules in their
cytoplasm when viewed microscopically. Neutrophils (bands + segments), eosinophils, and
basophils are categorized as polymorphonuclear granulocytes. Monocytes and lymphocytes
are known as mononuclear agranulocytes. These populations of leukocytes are produced at
varying rates depending on the changing needs of the body. Percentage distribution of each
population within the total leukocyte count, so called the WBC differential count, represent
a basic physiological parameter of WBC.
AIMS
 Identification (morphological evaluation) of peripheral leukocytes in selected animal

species
 Determination of the differential WBC count
 Comparison among species and comparison with reference numbers
 Discussion
EXPERIMENTAL DESIGN
– Prepare a permanent blood smear of the blood sample.

– Place permanent blood smear under immersion and focus under and focus with
magnification of 100x (objective).
– Use the meander movement of the slide to evaluate 100 leukocytes.
– Sort these leukocytes into populations (neutrophils bands and segments, eosinophils,
basophils, lymphocytes, monocytes) based on their morphological appearance.
– Express the final differential WBC count in the sample.
Differential count evaluation.
48
RESULTS
Differential WBC count:
Leukocyte Count % x109/l

Neutrophils - bands
Neutrophils - segments
Eosinophils
Basophils
Lymphocytes
Monocytes
Schematic morphological appearance of neutrophil granulocytes (band, segment).
Schematic morphological appearance of eosinophil (red-pink granules) and basophil (blue

granules) granulocytes.
Schematic morphological appearance of agranulocytes (lymphocyte, monocyte).
49
Differential WBC count - reference value

Leukocyte Cat Cow Dog Horse Pig Rabbit
population
Neutrophils 0.90–0.80 0.25–0.30 0.60–0.82 0.55–0.60 0.30–0.45 0.45–0.55
Eosinophils 0.02–0.08 0.05–0.06 0.02–0.04 0.01–0.05 0.02–0.05 0.01–0.03
Basophils 0.00–0.05 0.00–0.04 0.00–0.02 0.00–0.02 0.00–0.01 0.01–0.05
Monocytes 0.01–0.04 0.05–0.08 0.04–0.05 0.01–0.04 0.01–0.03 0.04–0.06
Lymphocytes 0.20–0.50 0.55–0.65 0.13–0.32 0.30–0.40 0.50–0.60 0.35–0.55
DISCUSSION
WBC is measured manually from the permanent blood smear or by the automatic
haematological analyzer. Within the automatic haematological analyzer, WBC are
differentiated according to their size, granularity, cellular peroxidase and resistance to acid
lyses in six populations: lymphocytes (small, no peroxidase, no granules), monocytes (large,
intermediate level of peroxidase, few granules), neutrophilic granulocytes (large, high
granularity and level of peroxidase), eosinophils (very high granularity and level of
peroxidase), basophils (granules and resistance to acid lyses), atypical leukocytes.
Morphology of leukocytes
Polymorphonuclear granulocytes have a segmented nucleus with several lobes of varying
shapes, and their cytoplasm contains an abundance of membrane-enclosed granules. The three
types of granulocytes can be distinguished on the basis of the varying affinity of their granules
for dyes. Therefore eosinophil granules appear red, basophil blue and neutrophil pink.
Mononuclear agranulocytes have a single, large, non-segmented nucleus and a few
granules. Monocytes are the larger with an oval or kidney shaped nucleus. Lymphocytes
differ in size and have a large spherical nucleus that occupies most of the cell.
The morphological appearance of leukocytes differs slightly among species.
Functions and life span of different leukocytes

Among the granulocytes, the neutrophils are phagocytic specialists, and are called
microphages. They invariably are the first defenders during bacterial invasion and are very
important in inflammatory responses and work as scavengers. An increase in circulating
neutrophils (neutrophilia) typically accompanies acute bacterial infection. Eosinophilia is
associated with hypersensitivity conditions or with internal parasitic infections. Basophils are
involved in allergic reaction and inflammation. Their tissue derivatives are called mast cells
and are found working within tissues. Both cell types synthetize and store histamine and
heparin. The life of granulocytes once released from the bone marrow is 4 to 8 hours
circulating in the blood and 4 to 5 days in the tissues.
Among the agranulocytes, monocytes belong to professional phagocytes, which settle down
in various tissues as macrophages. The monocytes have a short transit time, 10 to 20 hours.
However, they may survive in the tissues for years. Lymphocytes belong to specific immune
50
defenders. The B-cells belong to humoral and the T-cells to cell-mediated immune machinery.
T-cells can be distinguished based on their surface markers (receptors) into 3 main
subpopulations: T-helper, T-cytotoxic and T-suppressor cells that have further specific roles.
T-helpers act as initiators of the immune response particularly by communication with antigen
presenting cells and production of the B-cell growth factor. T-cytotoxic lymphocytes are
effectors destroying the targeted infected and transformed cells. T-suppressors balance these
two by suppressing the immune response.
CONCLUSIONS
NOTES & COMMENTS
51
FUNCTIONS OF LEUKOCYTES
IN THE IMMUNE SYSTEM
INTRODUCTION
The most important function of the neutrophils and macrophages is phagocytosis (cellular
ingestion of the offending or foreign agents). When the WBC encounters a large
multimolecular particle (bacterium or tissue debris), it extends pseudopods that engulf the
particle into a vesicle (phagosome). After fusion with intracellular lysosomes, a
phagolysosome forms. Lysosomal enzymes break down the engulfed material into smaller
ingredients (amino and fatty acids or sugars), which can be reused or in case of antigens
presented on the cellular surface. Phagocytic activity is the actual set-up of the non-specific
immune system. The phagocytic index characterized each individual phagocyte. For these
purposes, an experimental approach using artificial particles can be applied. Phagocytic
activity and index may be evaluated for each cell population (neutrophils, monocytes) or all
the phagocytizing cells together.
AIMS
 The determination of the phagocytic index and phagocytic activity of selected cells in
peripheral blood (neutrophil granulocytes, monocytes)
 Discussion
EXPERIMENTAL DESIGN
– Prepare a kit for phagocytic activity and index measurement (commercially available)
and fresh heparinized blood sample.
– Mix the blood sample with microspheric particles from the kit.
– Keep the solution 30 min/37 °C and mix it occasionally.
– Prepare a permanent blood smear from the solution.
– Evaluate the phagocytic parameters microscopically using immersion and
magnification of 100 x (objective).
– Evaluate 100 cells for reliable results.
RESULTS
Cell type Potential Phagocytizing Phagocytic Number of Phagocytic

(population) phagocytes phagocytes activity engulfed index
% particles n/1 cell
All
Neutrophils
Monocytes
52
Phagocytizing cell: Each cells with 3 or more engulfed particles

Phagocytic index: Number of particles engulfed by 1 cell, average of engulfed particles/one
cell
Phagocytic activity: Ratio (percentage) of ultimately phagocytizing cells to all potentially
phagocytizing cells
Phagocytizing neutrophil granulocytes in the permanent blood smear.
DISCUSSION
The phagocyte responsibilities involve not only the destruction of foreign particles or
damaged cells by phagocytosis but also the secretion of chemical mediators that destroy the
targeted material by non-phagocyting means and augment many aspects of inflammation and
other immune processes.
Phagocytes must be selective of the material that is phagocytized in order to prevent
phagocytosis of normal cells and structures in the body. Whether phagocytosis occurs,
depends on three selective procedures in particular.
 Most natural structures in the tissues have smooth surfaces which resist phagocytosis.
Rough surfaces increase the likelihood of phagocytosis.
 Most natural substances of the body have protective protein coat.
 The body has a specific means for recognizing certain foreign material.
The phagocyte responsibilities in homeostasis involve the secretion of chemical

mediators causing:
 Stimulation of kinin activation: action as a chemotaxins (promotion of phagocytic
migration), activation of nociceptors (pain receptors), stimulation of the complement
system, enhancement of vascular response to inflammation
 Stimulation of clotting and anticlotting systems
 Stimulation of histamine release
 Release of lysosomal enzymes, lactoferrin (destruction of target cells - bacteria)
 Stimulation of granulopoiesis
 Release of acute phase proteins (promotion of immune response)
 Stimulation of hypothalamus (development of fever)
 Stimulation of B and T lymphocytes
53
CONCLUSIONS
NOTES & COMMENTS
54
HEMOSTASIS, COAGULATION
INTRODUCTION
Haemostasis represents a physiological mechanism of organism protection against unwanted

bleeding. It covers several steps with participation of blood vessels, thrombocytes and
plasmatic coagulation factors. The vascular spasm in the wounded area eliminates blood
loss, thrombocyte aggregation plugs the vessel along with promotion of the following healing
process and the coagulation cascade contributes by fibrin network formation.
Examinations (tests) in haemostaseology are important components characterizing the blood
status of the organism. Anticoagulation agents are crucial for in vitro work with blood
samples as well as for diagnostics and treatment.
AIMS
 Physiological aspects of haemostasis, focused on coagulation

 Coagulation pathways vs. anticoagulation agents
 Basis of coagulation tests and their importance
 Haemostaseology in veterinary medicine
 Prothrombin time and its determination
 Discussion
EXPERIMENTAL DESIGN
– Sample preparation
– Collect a fresh blood sample and stabilize it by sodium citrate
– Centrifuge the sample to obtain citrate plasma
– Use the set Thromboplastin-S
– Sample analysis
– Use an automatic coagulometer (automated haematology)
– Measure the prothrombin time according to the laboratory protocol
RESULTS
55
Prothrombin time
Analyzed blood sample (species):
Obtained value:
Comparison with reference values:
DISCUSSION
Coagulation pathways
Schematic (classical) model
Damaged vessel (exposed collagen),

in vitro (test tube surface)
Active factor XII

Inactive factor XII
Hageman factor
Inactive factor XI Active factor XI
Ca2+ (factor IV)
Inactive factor IX Active factor IX

Ca2+
Factor V
PF3
Factor X Prothrombin - thrombin

Ca2+ activation
Factor VII
Tissue tromboplastin
(factor III)
Fibrinogen - fibrin
Tissue factor
Ruptured vessel (damaged tissue)
(thromboplastin, factor III) Fibrin stabilization
vs. fibrinolysis
Coagulation tests
There are two main screening tests for the clotting system: the prothrombin time (PT) and the
activated partial thromboplastin time (APTT).
The PT (prothrombin time, Quick’s test) tests the extrinsic coagulation pathway and is
initiated by adding crude thromboplastin (usually from a rabbit brain) containing a high
concentration of tissue factors and phospholipids. CaCl2 (Ca2+ ions) is added to the plasma.
The tissue factor interacts with factor VII leading to direct activation of factor X followed by
thrombin production and fibrin clot formation (see the cascade). PT is mostly used for
monitoring coumarin (antagonists of vitamin K) therapy as vitamin K is required for factor
VII, factor X and prothrombin carboxylation and function. To perform the procedure, the
blood sample must be stabilized by decalcification (citrate). Coagulation factor III (tissue
factor, thromboplastin) is added to separated plasma, the test starts after addition of calcium
ions and the result is time (in seconds) to achieve coagulation of the plasma. Particularly in
56
human medicine, the measured time is often recalculated to INR (international normalized
ration) for better comparability among laboratories. Prolong time means decreased activity of
the prothrombin complex.
APTT involves activation of a contact system which generates the activated factor XI (XIa),
followed by IXa, in turn followed by Xa, thrombin production and fibrin clot formation.
APTT requires an activating surface (kaolin or negative phospholipids) and Ca2+ ions.
Because heparin acts as a potent activator of antithrombin factor Xa- and thrombin-blocking
activity, APTT is used for monitoring heparin therapy.
PT and APTT are clotting test actually measuring the time it takes to form a fibrin clot, but
using different means to initiate the coagulation. Both tests are done in citrated platelet-poor
plasma so the patient’s platelets are therefore irrelevant for the testing. The in vivo platelets
supply of negative phospholipids for coagulation along with other sources of phospholipids
must be supplied for the tests. Ca2+ ions (chelated by citrate to prevent coagulation of the
withdrawn blood) must be added to perform PT and APTT reactions. Clotting tests are done
in coagulation analyzers and are based on the optical or mechanical detection of clot
formation.
Another global clotting test in common use is thrombin time which involves adding
thrombin to plasma and then testing the fibrinogen cleavage and fibrin clot formation.
Further tests for evaluating the coagulation cascade involve fibrinogen concentration,
antitrombin activity, and D-dimer concentration. D-dimers are by-products of fibrin clot
formation in vivo and refer to thrombotic event.
Coagulation in veterinary medicine

 Generally, all mammals share a similar coagulation system. Most of the tests developed
for human investigation are applicable for mammals. Differences in time-to-clot between
species in global tests, such as PT or APTT, do not reflect clotting capacity in vivo but
rather the affinity of plasma coagulation factors to the supplied clot-initiating components
(e.g. thromboplastin). Whole blood clotting tests provide information about the clotting
capacity of blood. Platelets play an important role. Thus plasma-based clotting tests have a
wide-range of usage but the normal reference ranges must be assessed for each species.
 Other mammals may suffer from the same bleeding disorders as humans (hemophilia A,
hemophilia B and von Willebrand factor deficiency).
 Thrombotic episodes and DIC cause more deaths than bleeding disorders in non-human
mammals. DIC (disseminated intravascular coagulopathy) leads to multiple-organ
microthrombosis (multiple organ failure-MOF) and paradoxical bleeding caused by the
inactivation or excessive consumption of platelets and clotting factors secondary to
enhanced fibrinolysis. DIC is not a specific disorder but rather a common pathway in a
variety of disorders. Moreover, DIC constitutes a dynamic phenomenon in which the
patient's status and the results of coagulation tests change markedly, rapidly, and
repeatedly during treatment. This syndrome is relatively common in dogs and cats.
Laboratory findings in DIC show a decrease in platelet time, fibrinogen concentration,
anti-thrombin and an increase: D-dimer, PT, APTT, TT)
57
Anticoagulantion agents
Anticoagulant is a substance that prevents coagulation. It stops blood from clotting,
Pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic
disorders. Anticoagulants are used in medical equipment (ex vivo, in vitro), such as test tubes,
blood transfusion bags, venal ports, and renal dialysis equipment.
 Vitamin K antagonists act by antagonizing the effects of vitamin K, a compound
necessary for the synthesis of several clotting factors. It has a slow effect, in vivo only.
 Heparin and derived substances work by potentiation of antithrombin thrombin-
inhibiting activity and by the inhibition of factor Xa (and other serine proteases)
clotting activity. It has an immediate effect in vitro and in vivo.
 Direct thrombin inhibitors, such as hirudin, that is secreted by the salivary glands of
the medicinal leech, Hirudo medicinalis. It has an immediate effect in vivo and in
vitro.
 Others, such as EDTA, citrate or oxalate are used only in vitro and work by
binding calcium ions, and preventing them from initiation of the clotting cascade.
NOTES & COMMENTS
58
AUTOMATED HEMATOLOGY
INTRODUCTION
Clinical laboratory provides tests for monitoring the physiological function of organisms and
their pathological conditions. The wide scope of testing covers several major areas:
 Chemistry - chemical analysis of body fluids (electrolytes, lipid metabolism, renal
and liver functions)
 Hematology – study and classification of blood cells
 Immunology and serology - qualitative and quantitative testing for antigens, and
antibodies, and the quantitative and functional analysis of immune cells
 Immunohematology or blood banking – blood-typing of recipients and donors,
cross-matching units of blood and identifying atypical antibodies
 Coagulation – testing for defects in the blood clotting cascade
Because clinical lab procedures must be rapid and accurate they have lead to the
implementation of automated analyzers.
AIMS
 A visit to a haematological laboratory.

 Measurement of haematological parameters in human and animal samples.
 Evaluation, discussion.
Clinical Laboratory for Small Animals at the UVPS Brno
59
An example of equipment for routine automated hematological analyses - Advia 2120.
Task: Compare the following automated blood parameters in the available species (dog,
human, pig, cattle).
 CBC parameters
 Myeloperoxidase level in granulocytes
 Leukocyte size
 Reticulocyte count
Example of results from a routine analysis of a real blood sample from a dog
Automated CBC and WBC differential count
A haematology laboratory provides an automated analysis of blood: a complete blood count

(CBC), differential white blood count (WBC) and manual evaluation of blood and bone
marrow smears (red cell morphology, WBC, and morphology).
60
CBC includes Principle of automated test (ADVIA 2120)

White blood count (WBC) Identification according to optical characteristics of the cells
(reflect their morphology)
Hemoglobin (Hb or Hgb) Hemolysis followed by the modified cyanmethemoglobin
method
Red blood cell count (RBC) Sphering of RBC and detection of laser light scatter
Red blood cell indices: interfering with erythrocytes (provides information about
MCV, MCH, MCHC count, size, and the hemoglobin level in RBC)
Hematocrit (Hct) Calculated: multiplying RBC count by MCV (average
volume of RBC)
Platelet count and MPV Detection of laser light scatter interfering with platelets
(mean platelet volume) (provides info about count and size)
WBC are differentiated according to their size, granularity,
cellular peroxidase and resistance to acid lyses in six
Differential white blood populations: lymphocytes (small, no peroxidase, no
cell count granules), monocytes (large, intermediate level of peroxidase,
few granules), neutrophilic granulocytes (large, high
granularity and level of peroxidase), eosinophils (very high
granularity and level of peroxidase), basophils (granules and
resistance to acid lyses), atypic leukocytes
Other parameters derived from automated CBC tests:

RDW (red blood cell volume distribution width) reflects the variance in erythrocyte size and
refers to anisocytosis.
HDW (hemoglobin distributin width) reflects the variance in hemoglobin concentration in
RBC and refers to anisochromia.
Platelet indices: PDW (analogy to RDW), PCT – plateletcrit (analogy to hematocrit), large
platelet, platelet clumps
Flagging of abnormal findings e.g. LEFT SHIFT (increased proportion of bands), IG -
immature granulocytes, BLASTS, MICRO – microcytosis, HYPER - hyperchromia
Other measurements provided by hematological analyzer:

Reticulocyte analysis – The reticulocyte (less mature erythrocyte) is distinguished from RBC
by its content of RNA by the method of RNA staining by florescent dye. The reticulocyte
count involves retic differentiation according to its maturity, retic volume and retic
hemoglobin concentration.
Immunophenotyping
CD (cluster of differentiation) antigens are cell surface molecules present on leukocytes.
The CD system is commonly used as cell markers. This allows cells to be defined based on
what molecules are present on their surface after their staining with fluorescently labelled
antibodies and detected by flow cytometry. For example, within the lymphocyte population
we can recognize T-lymphocytes as CD3+ cells. These can be further differentiated based on
their expression of CD4 or CD8 to T helper (Th) cells or T cytotoxic (Tc) cells, respectively.
B-cells can be recognized as cells with surface expression of CD19+. Natural killers (NK-
cells) can be detected as CD3- CD16+ CD56+ cells.
61
Automated haematology - BLOOD ANALYSIS – results, an example
Reference
Parameter Result Unit Evaluation
level
ACIDOBASIC BALANCE
B_pH 7.375 - 7.360-7.440 *
B_pCO2 6.63 kPa 4.80-5.90 *
B_O2 14.9 kPa 9.9-14.4 *
cal_HCO3 26.8 mmol/l
cal_BE (base excess) 3.20 mmol/l -2.50-2.50 *
B_O2_SAT 97.90 % 94.00-99.000 *
B_sodium 145 mmol/l
B_potassium 3.7 mmol/l
B_chlorides 105 mmol/l
B_glucose 10.10 mmol/l
B_Ca2+ 1.14 mmol/l
BLOOD ELEMENTS
B_erytrocytes 3.490 .1012/l 3.800-4.800 *
B_leukocytes 14.670 .109/l 4.000-10.000 *
B_hemoglobin 111 g/l 120-165 *
B_Htc 0.321 l/l 0.360-0.470 *
B_thrombocytes 98 .109/l 150-350 *
B_MCV 92.0 fl 75.0-90.0 *
B_MCH 31.9 pg 27.0-31.0 *
B_MCHC 347 g/l 330-370 *
B_RDW 16.8 % 11.5-14.5 *
B_HDW 30.1 g/l 22.0-32.0 *
B_MPV 11.4 fl 7.2-11.1 *
DIFFERENTIAL COUNT
B_neutrophiles 0.774 - 0.450-0.700 *
B_eosinophiles 0.089 - 0.000-0.050 *
B_basophiles 0.004 - 0.000-0.020 *
B_monocytes 0.018 - 0.000-0.070 *
B_lymphocytes 0.097 - 0.250-0.400 *
B_atypic leukocytes 0.018 -
B_neutrophile number 11.350 .109/l 2.500-5.600 *
B_eosinophile number 1.300 .109/l 0.030-0.250 *
B_basophile number 0.060 .109/l 0.030-0.160 *
B_monocyte number 0.270 .109/l 0.150-0.580 *
B_lymphocyte number 1.430 .109/l 1.500-3.000 *
B_atypic leukocyte number 0.260 .109/l
B_MPX1 7.5 - -7.0-10.0 *
62
COAGULATION
Pc_thromboplastin test – INR 1.44 INR 0.80-1.20 *
Pc_APTT 38.5 s 20.0-35.0 *
Pc_fibrinogen 4.3 g/l 2.0-4.0 *
Pc_thrombin time 17.8 S 14.0-22.0 *
Pc_ATIII 18.50 % act. 80.00-110.00 *
Pc_D dimers 12.02 μg FEU/ml up to 0.50 *
PLASMA BIOCHEMISTRY
P_sodium 147 mmol/l 130-149 *
P_potassium 4.6 mmol/l 3.6-5.1 *
P_chlorides 107 mmol/l 97-111 *
P_bilirubin 164 μmol/l up to 17 *
P_AST 0.24 μkat/l 0.17-0.58 *
P_ALT 0.19 μkat/l 0.17-0.58 *
P_kreatinin 118 μmol/l 45-84 *
cal_GFR 0.697 ml/s 1.100-2.000 *
P_urea 19.5 mmol/l up to 11.9 *
P_glucose 6.6 mmol/l 4.4-6.4 *
P_alpha-amylase 0.31 μkat/l 0.47-1.70 *
P_LPS 0.15 μkat/l 0.22-1.00 *
NOTES & COMMENTS
63
METABOLISM AND THERMOREGULATION

BASAL METABOLISM
INTRODUCTION
Basal metabolism is expressed as energetic turnover necessary to maintain physiological

functions under conditions of complete rest (physical and emotional), thermoneutrality of the
environment and at postabsorptive stage (14 – 16 h after satiety in monogastric, 32 h and
more in polygastric animals). Energy consumption is expressed in daily doses. Such energy is
used to keep functions of critical organs such as the heart, lungs, liver, kidney, muscles, skin,
nervous, digestive and reproductive systems. One third of the basal energy is necessary for
cell membrane maintenance.
BMR (basal metabolic rate) depends on the weight of the organism, thus also on other aspects
such as pregnancy, breed etc.
AIMS
 Calculation of metabolic active body weight

 Calculation of basal metabolism
 Comparison of basal metabolism in different species
 Discussion
EXPERIMENTAL DESIGN
Determination of the metabolic active weight

– Metabolic active body weight can be approximately expressed as the weight of the
body raised to the power of ¾.
Determination of the basal metabolism (BMR)

– Basal metabolism requires 293 kJ per each kilogram of the metabolic active weight
– Basic formula: BMR = m3/4x293 [kJ]
RESULTS
Determination of the metabolic active weight and BMR in the human

– Metabolic active weight:
– BMR:
64
Determination of the metabolic active weight and BMR in selected species, comparison
Species/animal Metabolic active weight BMR

[kg] [kJ/day]
DISCUSSION
The first estimation of the metabolic active weight dates back to 1932 and is known as the
Kleiber´s law. The low says that BMR depends linearly on ¾ power of the body weight and
is generally accepted in homoiotermic animals.
Among species, the BMR values are indirectly related to longevity predictions, however, the
correlation cannot be taken absolutely. There are several other factors, such as interspecies
and interindividual variations in oxidative phosphorylation, ATP production, amount of
unsaturated fatty acids, production of reactive oxygen species, DNA reparation capacity etc.
BMR also gets increased in the case of stress of diseases.
CONCLUSIONS
NOTES & COMMENTS
65
PHYSIOLOGICAL HEAT PRODUCTION
INTRODUCTION
Organisms obtain energy by oxidation of saccharides, fats and proteins. Energy is released by
the catabolic processes in which the oxygen is consumed and the carbon dioxide is produced.
Both gases can be measured by a gas analyzer and from the data animal heat production can
be determined. Ratio between CO2 release and O2 consumption refers to the respiratory
quotient (RQ). Amount of energy released from the nutrients during oxidation by one litre of
oxygen is the energy equivalent (EE).
AIMS
 Demonstration of the effect of defined physical exercise on the O2 consumption and

CO2 production
 Comparison of the data of the metabolic rate during rest and physical exercise
 Discussion
EXPERIMENTAL DESIGN
Use the gas analyser

Measure heat production under resting and exercise conditions
– A volunteer sits resting on the bicycle ergometer breathing via an air tube.
– The amount of inspired air is measured by the airflow-meter. Respiration
frequency is observed and counted.
– After 5 minutes of adaptation the volunteer is connected through the tube to a
gas analyzer. O2 consumption and CO2 production are determined.
– After 10 minutes of measurement at rest, begin to record the data under physical
exercise (load of 50 or 100 W).
Gas analyser “Spirolyt”.
66
RESULTS
Resting stage Physical exercise

Breathing frequency
Respiratory volume
O2 consumption
CO2 production
RQ value
Heat production
DISCUSSION
Heat production measured in resting human is usually higher than the calculated BMR, and
increases several times during exercise. The ability to enhance the metabolic rate depends
on many factors. These factors include the portion of active body mass, it is efficiency to
utilize O2, and the capacity of the respiratory and cardiovascular system etc.
A bicycle ergometer connected with an analyzer of expired air enables the determination of
the actual metabolic rate of a human submitted to different physical efforts. By means of this
procedure we can also obtain a good survey of other physiological data like heart rate, EKG,
blood pressure, efficiency of respiratory system etc.
CONCLUSIONS
NOTES & COMMENTS
67
PHYSIOLOGICAL HEAT LOSSES

AND THEIR METABOLIC COMPENSATIONS
INTRODUCTION
The heat losses in general can be caused by four means: convection, radiation, evaporation
and conduction. Heat losses thus depend on several parameters including temperature of the
environment, air flow, humidity, and in animals also on body isolation (fur, adipose tissue
etc).
A physical model for simulation of heat loss can be performed using an electric dynamic
katathermometer. This device is based on cooling of subjects by alterations in heat flow
density (HFD).
AIMS
 Determination of the heat production in animals with different types of body

insulation
 Demonstration of the effect of insulation on heat loss
 Evaluation of alteration in the environment using katathermometer
 Discussion
EXPERIMENTAL DESIGN
Use the gas analyser

Measure heat production in animals with and without isolation
– Place a rabbit with intact body insulation (fur) into the metabolic chamber.
– Adjust the inside temperature to 10 °C.
– Adjust the airflow through the chamber in accordance to body weight of the
animal.
– After 15 minutes of acclimation, connect the chamber with analyzer (Spirolyt).
– Determine the O2 consumption and CO2 production from the analysis record
(%).
– Calculate the RQ value and determine the energy equivalent of O2 (EE1l O2).
– Repeat the same measurement for the rabbit without insulation (shorn fur).
– Compare the results from both experiments.
Use the katathermometer

Determine HFD (W/m2)
at the room temperature:
under increased radiation (infrared lamp):
under increased air flow (in front of ventilator):
under increased humidity (wet covered sensor):
68
RESULTS
Animal with intact isolation

O2 consumption (%):
CO2 production (%):
RQ value:
EE (1l O2) [kJ]:
Ventilation of chamber per 1 minute:
O2 consumption per 1 minute:
O2 consumption per day:
Heat production kJ/day = O2 consumption (l/day) x EE1l O2
Animal without isolation

O2 consumption (%):
CO2 production (%):
RQ value:
EE (1l O2) [kJ]:
Ventilation of chamber per 1 minute:
O2 consumption per 1 minute:
O2 consumption per day:
Heat production kJ/day = O2 consumption (l/day) x EE1l O2
Expression of metabolic compensation

Comparison and evaluation of heat loss, animal with isolation contra without
HFD under different conditions
Temperature of the HFD (W/m2)

environment
Room temperature
Increased radiation
Increased air flow
Increased humidity
DISCUSSION
The heat production of an animal increases in cold environment because of higher heat loss
compensation. The dissipation of heat from the body surface is accelerated if the body
insulation is impaired or lost.
A thermal environment has a substantial influence on the energy metabolism especially in
young animals. They have greater body surface and their insulation in not fully developed.
Consequently, the requirement for environmental temperature is higher in comparison to adult
69
animals. Heat loss, which cannot be compensated for by a higher heat production, causes
hypothermia.
Energy equivalent (EE) and respiration quotient (RQ) of different nutrition components
1 g of Lipids Proteins Sugars
RQ 0.7 0.8 1.0
EE of 1l O2 (kJ) 19.6 18.0 21.1
According to Kotrbáček e al. 2010
Note: In the case of oxidation of all components, the average EE is expressed as 20 kJ (= 4.85
kcal).
The HFD from the EDK sensor increases if the temperature of the surrounding air drops lower
than surface temperature of the sensor. In such a situation the HFD is also closely dependent
on air velocity (draught). The HFD underneath an infrared lamp, on the contrary, decreases
rapidly in accordance with the distance of the sensor from the heat source. A black cylinder
absorbs heat radiation in the same manner as the animal skin. Evaporation of water from the
EDK sensor increases the HFD rapidly especially in draught. The EDK measurement is a
suitable method for evaluation of the thermal environment. In comparison with the air
temperature measurement (by means of mercury thermometer), the EDK integrates all the
important parameters of the surroundings and enables the determination of the cooling
properties of the environment. It also enables the prediction of heat loss in the animals.
CONCLUSIONS
NOTES & COMMENTS
70
THERMOREGULATION IN HOMOIOTHERMIC
AND POIKILOTHERMIC ANIMALS
INTRODUCTION
To maintain a constant body temperature and thereby a constant course of biochemical

reactions in an organism, the homoiothermic animal (warm blooded) produces enough heat
to compensate for heat loss. In the poikilothermic animal (cold blooded) body temperature
varies with that of the environment. Consequently their neuroendocrine regulatory
mechanisms under cold conditions are either not operating or are not adequate.
AIMS
 Record of the energy metabolism and of the body temperature of homoiothermic

(mice) and poikilothermic (frogs) animals exposed to changing thermal conditions
 Comparison of the changes
 Discussion
EXPERIMENTAL DESIGN
– Place a group of mice into one metabolic chamber.

– Place a group of frogs (of the same weight) into another metabolic chamber.
– Perform measurement of O2 consumption of both groups in comfort
environmental temperature (30 °C).
– Determine the body temperature at the end of 30 minutes.
– Calculate the heat production.
– Perform the same measurement in cold (10 °C)
– Construct a graph from the results. In the case of body temperature, mark the
average value.
RESULTS
Homoiothermic Poikilothermic
Heat production
at 30 °C [kJ/day]
Body temperature
at 30 °C [°C]
Heat production
at 10 °C [kJ/day]
Body temperature
at 10 °C [°C]
71
DISCUSSION
The metabolic processes of homoiothermic organisms increase in a cold environment to

compensate for higher heat losses. As a result of these changes body temperature remain
nearly constant. A homeothermic organism is, to a large extent, independent of the thermal
environment but needs more energy (food) for its higher metabolism especially in the cold.
Poikilothermic animals on the contrary, reduce the body temperature in cold and their
metabolic rate declines very quickly. Consequently their activity and contact with the
environment decrease up to the point of lethargy.
Poikilothermic animals are more independent on energy supply, but dependent on external
heat sources. For this reason they have not developed any thermal insulation.
CONCLUSIONS
NOTES & COMMENTS
72
ADIPOSE TISSUE AND ITS FUNCTIONS
INTRODUCTION
Adipose tissue consists of adipocytes, vessels, nerve endings, connective tissue and
extracellular fluid. Recently, stem cells were identified in the adipose tissue with a future
therapeutic potential. The major role of the adipose tissue is to create energetic reserves and
secure related metabolic functions. Adipose tissue also belongs to the system of inner
secretion as it produces e. g. leptin, resistin and cytokines (TNFα etc.), moreover, some
hormones such as estrogens increase their activity in the adipose tissue. Adipose tissue
influences functions of other organs and negative contributes to obesity which participates in
the metabolic syndrome.
Screening methods for estimation of adipose tissue weight in the body are based on
adipometers which can evaluate e. g. different conductivity of the adipose tissue.
AIMS
 Determination of percentage proportion of the adipose tissue within human body

 Determination of the weight of the adipose tissue
 Calculate the body mass index
 Evaluation of body score condition in a dog
 Discussion
EXPERIMENTAL DESIGN
Use the Omron adipometer

Set the parameters (body weight, height, age, gender)
Hold the device properly and start the measurement
Calculate the body mass index

– BMI = weight[kg]/height [m2]
73
Assess the body score condition in a dog

Basic evaluation parameters
Rib palpation
Waist palpation
Tail-base area palpation
Visual assessment (side view and top view)
RESULTS
Weight and proportion of the adipose tissue in the human, BMI
Body weight Body hight Adipose Adipose BMI

[kg] [m] tissue [%] tissue [kg] [kg/m2]
Person 1
Person 2
Person 3
Basic categories of MBI for comparison (according to WHO, 1997)

Underweight: less than 18
Normal weight: 18.5 – 24.9
Pre-obesity: 25 – 29.9
Obesity: more than 30
Body score condition in dogs
Ribs Tail-base Side view Top view

Dog 1
Dog 2
5 basic categories from cachexia to obesity exist for the BSC
DISCUSSION
Metabolism of adipose tissue differs in different body parts, e. g. adipocytes located in the
abdominal area are more resistant to insulin. 80 % of adipose tissue is formed by lipids, exact
number differs among species. Lipogenesis is stimulated by insulin and leptin (in monogastric
animals). Lipolysis is supported by hormone-dependent lipases (influenced by adrenalin,
noradrenalin, glucagon, STH, ACTH, thyroxin etc). Leptin is related to satiety/hunger
feelings. Adipometers are widely applied for human, in animals (particularly pets and dairy
cows), the BCS is used instead. BCS is a subjective and semiquantitative method.
74
CONCLUSIONS
NOTES & COMMENTS
75
SKIN PERCEPTION
Receptive Field and Discriminative Ability
INTRODUCTION
The afferent division of the peripheral nervous system sends information about the internal
and external environment to the CNS. The incoming pathway for subconscious information
(blood pressure, CO2 concentration) derived from the internal viscera (organs) is called a
visceral afferent. Input that reaches the level of conscious awareness is known as sensory
afferent. Sensory information is categorized as somatic sensation (somesthetic sensation
from the body surface and proprioception from the muscles, joints, and middle ear) and
special senses (hearing, taste, vision, smell). Receptors of somatosensoric system located in
the skin enable communication with the external environment, particularly receptors with
sensitivity (adequacy) to heat, cold, touch and pain (nociceptors). Integration of stimuli from
different receptors evaluated by the CNS creates the general surface feeling.
AIMS
 Investigation of tactile skin perception

 Comparison of receptive field and receptor discrimination in different areas of the skin
 Discussion
EXPERIMENTAL DESIGN
– Compare the tactile discrimination in different areas of the skin, such as fingertips or
forearms using a hair esthesiometer.
– Evaluate thermoreceptors (heat and cold) in different parts of the skin using a
thermoesthesiometer.
– Score number of correct evaluations out of 10 trials.
RESULTS
Tactile discrimination level and thermoreception of the skin in the tested body parts:
Skin Discrimination level

Tactile reception Thermoreception
Calf
Fingertip
Forearm
Palm
Shoulder
76
DISCUSSION
Each somatosensory neuron responds to stimulus information only within a limited region
called the receptive field. The smaller the receptive field in a region, the greater its
discriminative ability (acuity).The receptor at the site of the most intense stimulation is
activated to the greatest extent. The surrounding receptors are stimulated, however, to a lesser
degree. The most intensively activated receptor pathway halts transmission of impulses in the
less intensively stimulated pathway through lateral inhibition. This process facilitates the
location of the site of stimulation.
Lateral inhibition facilitates location of the stimulus within the CNS. Blockage of further
transmissions of the weaker inputs increases the contrast and the sharp point can be precisely
located. The extent of lateral inhibitory connections within sensory pathways varies for
different modalities. Touch and vision have the most lateral inhibition and thus bring about
the most accurate localizations.
Two-point-threshold
INTRODUCTION
The relative tactile acuity of a given region can be determined by the two-point threshold-
of-determination test. If the two points of a pair of callipers applied to the surface of the skin
stimulate two different receptive fields, two separate points will be felt. If the two points
touch the same receptive field, they will be perceived as only one point. By adjusting the
distance between the calliper points, one can determine the minimal distance at which the two
points can be recognized as two rather than one. This is a reflection of the size of the receptive
fields in the region. With this technique it is possible to plot the discriminative ability of the
body surface.
AIMS
 Determination of the two-point-threshold in different body parts

 Discussion
EXPERIMENTAL DESIGN
– Apply a pair of callipers to the surface of the skin.

– Record if one or two separate points can be felt.
– Adjust the distance between the calliper points and determine the minimal distance at
which the two points can be recognized as two rather than one. This is a reflection of
the size of the receptive field in the region.
RESULTS
The size of the receptive field in the skin regions under study:
77
Receptive field
Skin – region
Calf
Fingertip
Forearm
Palm
Shoulder
DISCUSSION
The two-point threshold ranges from 2 mm in the fingertips (enabling one to read Braille,
where the dots are spaced 2.5 mm apart) to 48 mm in the poorly discriminative field of the
leg.
If the pair of callipers touches two different receptors, two points can be felt (blue). If only
one receptor is touched, just one point can be felt (red).
An estimated 17, 000 tactile mechanoreceptors are present in the fingertips and palms of the
human hand. In contrast the skin over the forearm is served by relatively few sensory endings
within large receptive fields. The distorted cortical representation of various bodies
corresponds with the innervation density - more cortical space is allotted for sensory
reception from areas with smaller receptive fields (which means with greater discriminative
ability). Skin sensitivity is thus given by the density of tactile receptors (average 25 in 1 cm 2,
up to100-130 in the facial area and fingertips).
CONCLUSIONS
NOTES & COMMENTS
78
PHYSIOLOGICAL REGULATIONS
INTRODUCTION
Physiological regulations aim to keep homeostasis, the dynamic constancy of the inner
environment, at different levels. Three major systems operate at the whole organism level:
nervous, inner secretion and immune. The most rapid compensations are provided by the
nervous system and the effects can be easily demonstrated by physical exercise (load)
simulation. The investigated physiological parameters may include body temperature, blood
pressure, pulse, respiratory frequency, tidal volume and peripheral oxygen saturation. The
monitoring applies non-invasive methods based on peripheral evaluation of cardiovascular
and respiratory functions. An example of such monitoring can be evaluation of the
orthostatic vital signs.
AIMS
 Evaluation of investigated physiological parameters under resting conditions

 Simulation of physical load (exercise)
 Evaluation of investigated physiological parameters during and after exercise
 Comparison and evaluation of monitored parameters
 Discussion
EXPERIMENTAL DESIGN
Physiological parameters
Body temperature: digital thermometer
Heart rate: auscultation
Blood pressure: digital sphygmomanometer
Pulse: manual evaluation (palpation of a. radialis) or pulse oxymeter
Respiratory frequency: manual evaluation (stopwatch)
Tidal volume: digital spirometer
Simulation of physical load (exercise)

Orthostatic reflex: change in body position from horizontal (lying) to vertical (sitting)
Exercise bike set on 50 W load, exercise for 10 min
Changes in external environment: temperature and/or humidity
RESULTS
Physiological parameters under resting conditions

Body temperature:
Heart rate:
79
Blood pressure:
Pulse:
Respiratory frequency:
Tidal volume:
Physiological parameters during and after exercise

Type of exercise (load):
Body temperature:
Heart rate:
Blood pressure:
Pulse:
Tidal volume:
Type of exercise (load):

Body temperature:
Blood pressure:
Pulse:
Tidal volume:
DISCUSSION
“Centre” of thermoregulation is located in the hypothalamus, respiratory “centres” in the brain

stem (medulla oblongata, pons of Varoli), cardiovascular “centres” in the medulla oblongata
(nuclei of the reticular formation). These centres dynamically react on changes and needs of
the organism. Communication with the periphery is mediated by the autonomic nervous
system, its sympathetic and parasympathetic parts. Motoric system may also contribute to
compensation including movement related changes of behaviour. Alterations of the
investigated parameters during/after exercise along with compensation speed and return to the
values under resting conditions are indicators of physical status of the organism. Orthostatic
vital signs are commonly used in triangle medicine.
Note: More precisely, the “centre” means neurons, e. g. inspiratory neurons, expiratory
neurons.
CONCLUSIONS
80
NOTES & COMMENTS
81
REFERENCES
BOOKS:
Campbell NA, Reese JB: Biology. 2002, ISBN 0-8053-6624-5

Despopoulus A, Silbergnagl S: Colour Atlas of Physiology. 2003, ISBN 1-5889-0061-4
Duke HH, Reece WO: Physiology of Domestic Animals. 2004, ISBN 0-8014-4238-9
Ganong WF: Review of Medicinal Physiology. 2001, ISBN 0-8385-8282-6
Guzton AC, Hall JE: Textbook of Medical Physiology. 2000, ISBN 0-8089-2187-8
Klein B, Klein B: Cunningham´s Textbook of Veterinary Physiology. 2012, ISBN
9781437723618
McKenzie S: Clinical Laboratory Hematology. 2003, ISBN 0-130-199-966
Sherwood L: Human Physiology from Cells to Systems. 2004, ISBN 0-534-37261-9
Sjaastad OV et al.: Physiology of Domestic Animals. 2010, ISBN 978-82-91743-07-3
Stiene-Martin EA et al.: Clinical Hematology – Principles, Procedures, Correlations. 1997,
ISBN 0-397-553-218
SPECIFIC PAPERS AND RECENT KNOWLEDGE:
https://apps.webofknowledge.com
http://www.pubmed.com
REFERENCE VALUES
Doubek J et al.: Veterinární hematologie. 2003, ISBN 80-86542-02-5
Doubek J et al.: Interpretace základních biochemických a hematologických nálezů u zvířat.
2010, ISBN 978-80-86542-22-5
Kaneko JJ: Clinical Biochemistry of Domestic Animals. 1997, ISBN 978-0-12-370491-7
Quesenberry KE, Carpenter JW: Ferrets, rabbits, and rodents: clinical medicine and surgery.
2004, ISBN 0-7216-9377-6
http://www.cklvfu.cz/pokyny.html#referencni-hodnoty
82
NOTES & COMMENTS
83
NOTES & COMMENTS
84

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Methods & Protocols

for Practical Training

University of Veterinary and Pharmaceutical Sciences

Brno, Czech Republic

Author for correspondence:

Prof. Dr. Eva Matalová, Ph.D.

Copyright ©Eva Matalová et al., 2021

Laboratory Animal Science 5

LABORATORY ANIMAL SCIENCE

Laboratory animal science can be defined as a multidisciplinary branch of science,

Basic methodical approaches in Physiology:

Alternatives to animal experiments involve particularly:

– In vitro and ex vivo techniques

Replacement refers to the substitution of living animals by alternative techniques,

NOTES & COMMENTS

 Evaluation of the cell cycle in the small intestine

Histological section - small intestine:

Histological section of mouse duodenum (after PCNA immunohistochemistry, positive cells

 Expression of proliferation rate of Paneth cells:

 Expression of proliferation rate in the entire crypt:

Immunohistochemical reactions are based on compatibility of an antigen and corresponding

NOTES & COMMENTS

ORGAN EXPLANT CULTURES

 Preparation of culture dish

Culture dish preparation

Culture medium preparation

Species and organ used for preparation:

Explants in a culture dish, ready for cultivation.

Explant culture represent ex vivo approaches in physiological research. Explant

NOTES & COMMENTS

 To follow and microscopically evaluate the effect of different environment on animal

 Description of microscopically observed events at the cellular level after treatment

Homeostasis plays a central role in maintenance of physiological functions, however, cell-

The mammalian bodies have four major buffer systems:

NOTES & COMMENTS

INTERNAL ENVIRONMENT – ACID BASE STATUS

 Determination of major AB parameters – pH, HCO3-, base excess, pCO2 in blood in

Records of analysed parameters

Sample/Species pH HCO3-, BE pCO2

NOTES & COMMENTS

INTERNAL ENVIRONMENT - ELECTROLYTES

Electrolytes belong to basic components of the inner environment which contribute

 Determination of the Na/K/Cl electrolyte level in blood serum/blood plasma in

Sample collection (e.g. from the rabbit's central ear artery):

Records of the Na/K/Cl levels

Electrolytes reference values

NOTES & COMMENTS

 Sedimentation rate evaluation in different species

Sedimentation measurement is performed in a sedimentation capillary filled with

Sedimentation rate in species under study:

Sample/Species 15 min 30 min 60 min

Schematic principle and result of blood sedimentation.

NOTES & COMMENTS

FUNCTIONS OF PLASMA PROTEINS

 Separation of plasma proteins from serum

The result of electrophoresis is electrophorogram, which shows individual fractions of

Agarose gel electrophoresis pattern of plasma proteins.

 Identification of plasma proteins and discussion of their functions

NOTES & COMMENTS

 The determination of the microhaematocrit in selected animal species

Microhaematocrit. The plasma and erythrocyte ratio is measured by centrifugation in

Schematic principle and result of hematocrit. 0.0 (0 %)

Comparison with the reference values, among species and discussion.

Haematocrit references values in different species