HEMATOLOGY I

Name : Aficko Razaky Pratama

Student ID : B1B017040
Group : VIII
Subgroup :3
Assistant : Wakhyuningsih

PRACTICAL REPORT OF ANIMAL PHYSIOLOGY I

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
BIOLOGY FACULTY
PURWOKERTO

2018
 I. INTRODUCTION
A. Background
Blood is a liquid matrix and is a specialized binding network formed from
free cells (Bryon & Doroth, 1973). Blood is a tissue that fills almost half of the
body. Blood works as a circulatory system that delivers all the chemicals,
oxygen and nutrients needed by the body. Vertebrate animals have almost the
same blood composition. Blood cells can be divided into erythrocytes (red blood
cells) that bind oxygen, leukocytes (white blood cells) that play a role in the
body's immunity and defense, and platelets that play a role in homeostasis
(Beverlander & Judith, 1979).
Blood is very important for organisms, if deficiencies or excess blood cells
result in abnormal physiological processes of an organism causing an illness.
Oxygen transport in the blood depends on the iron component in the respiration
pigment, usually hemoglobin. Hemoglobin is a part of red blood cells that binds
oxygen. Blood consists of cells and cell fragments that are freely present in a
liquid medium called blood plasma (Dukes, 1995).
Blood plasma is a liquid part of the blood consisting of 99% water and 8
-9% protein. Blood can be a medium to show and identify the effects of stress,
the environment and health of fish, blood in fish is very important to evaluate the
health of species. In recent years blood physiology has been used to clinically
diagnose fish because of the close relationship between the circulatory system
and the external environment (Kimball, 1988).
B. Purpose

The objective of this lab activity are to provide atudent skills how to take
animal blood, and to calculate the red bood cell, white blood cell, hematocrit
value and hemoglobin levels in animal.
 II. MATERIALS AND METHODS
A. Materials

The tools that used in this practical class are haemocytometer kit (pipette
thoma for erythrocyte and leukocyte & hemocytometer), drop pipette, capillary
pipette, haemometer kit (pipette sahli, sahli tube, stirrer bar), beaker glass, petri
dish, micro-centrifuge, capillary tube, haematocrit reader, hand counter, light
microscope, and 1 ml syringe.
The materials that used in this practical class are hayem solution, turk
solution, HCl 0,1 N, EDTA, aquades, clay, blood of Nilem (Osteochillus
vittatus) fish, mice (Mus musculus), and chicken (Gollus galus), tissue, cotton,
alcohol 70%.

B. Method
The work procedures that used in this practical class are:
1. Blood sampling were taken from fish through the cor, mice through the tail,
and chicken through the wing, and collected in petri dish consist of EDTA.
2. Erythrocyte and leukocyte counting.
 The blood was taken and collected in a petri dish consist of EDTA.
 The blood was sucked by using pipette thoma for erythrocyte/ leukocyte
until scale 1.
 Hayem solution was sucked until scale until scale 101 (for erithrocyte)/
turk solution was sucked until scale 11 (for leukocyte), and homogenized.
 The first or up to second drop was removed.
 Haemocytometer was set up on the microscope.
 The blood with the solution was dropped in the gap of haemocytometer
and counted using formula:
Total Erytrocyte ¿ 5000× E
Total Leukocyte ¿ 25 × L
3. Hemoglobin measurement.
 HCl 0,1 N was dropped into sahli tube until scale 2
 The blood from the speciment was added by using sahli pipette, stired by
using stirrer bar, wait until 3 minutes to see the color changing as the
formation of hematin acid.
 Aquadest was added until the color of the tube becomes similar with
comparaive tube.
 The color inside the tube was compared to the comparative wube and the
hemoglobin level was noted.
4. Hematocrit measurment.
 The blood was taken by capillary tube.
 The tip of the capillary tube was closed by the finger
 The tube was centrifuge on micro-centrifuge hematocrit at 3500 rpm for
15 minutes
 Hematocrit value was read by using hematocrit reader (in percent unit).
 III. RESULT AND DISCUSSION
A. Result
Table 3.1 Result of Observation of Total Blood Cell, Hb level, and
Hematocrit Value
The Amaount of Blood Cell
Sub Tested Hb Level Hematocyte
(cell/mm3)
Group Animal (%) Value (%)
Leukocyte Erytrocyte
1. Mice 5425 10,27 x 106 14 5
2. Nilem fish 171,275 2,2 x 106 7,6 15
3. Mice 16425 4,19 x 106 5 18
4. Chicken 6150 2,05 x 106 9 25
5 Nilem fish 162500 3,4 x 106 5 8

Calculation Data of Group 3

Total of leukocyte per mm3 = L x 25
= 25 x 657
= 16425 cell/mm3

Total of erytrocyte per mm3 = 5000 x E

= 5000 x 838
= 4,19 x 106 cell/mm3
B. Discussion

Hematology is the study of the aspects of blood anatomy, physiology and

pathology. The blood component consists of plasma and blood-forming elements
namely erythrocytes, leukocytes and platelets (Nurcholis, et al., 2013).
Hoffbrand and Pettit (1987) argue that, hematology can be known through
measurements which include measurement of hemoglobin levels, calculation of
the total number of erythrocytes and leukocytes and hematocrit. Leukocytes
have a role in the body's defense against foreign substances and are in charge of
fighting antigens. It is widely known that white blood cells are the most sensitive
to radiation or the highest radiosensitivity (Aryawijayanti & Susilo, 2015).
Leukocyte are involved in the defends of organism under several conditions,
such as stress, inflammation, and parasitism, the number of which could be used
as an indicator for infectious diseases. Hematology and serum biochemistry are
important tools for assessing health status in human and animals. It is well know
that many hematology and serum parameters vary with sex, age, season, and
physiological state (Peng et al., 2016).
Fish blood is taken approximately 0.5 ml through the caudal artery. Blood
collection was carried out using a 1 ml syringe which had been given Ethylene
Diamine Tetraacetic Acid (EDTA) then the needle was inserted deep enough
through the lateral line, the blood vessels which were just below the vertebrae
(Salasia et al., 2001). Taking blood samples in chickens according to Syukron
(2013) blood is taken from the wing vein (brachial vein). The hair around the
brachial vein is removed and disinfected with alcohol. Blood collection in the
brachial vein with syringe. The area around the blood vessels is first cleaned
using a cotton swab soaked with alcohol to find the blood vessels more clearly.
The syringe is then inserted between the blood capillary branches. After
entering, slowly pull the syringe so that the blood is sucked. The method of
taking blood in mice can be done through the plexus reorbitalis in the eye, the
lateral lateral vein, the saphena vein found in the legs and the direct retrieval
through the heart. The trick is, the tail of the mice is stretched and first
moistened with alcohol, then the cut is 0.2-2 cm from the base of the tail with a
razor blade or sterile scissors. Blood collection in humans can be done using
lancets inserted in the fingers (Gay, 1987).
 Syringe is used to take blood which will then be tested. Petri dishes serve to
accommodate the blood of test animals. Hemocytometer functions to calculate
the number of erythrocytes and leukocytes. The microscope functions to observe
and calculate the erythrocytes and leukocytes of test animals. Sahli Tube plays a
role in measuring blood hemoglobin levels of test animals. The syringe serves to
take the blood of test animals. Comparator is a tool used in comparing the color
of the solution contained in the Sahli tube with the color of the indicator solution
to determine the amount of hemoglobin in the blood. The hematocrit reader
plays a role in determining hematocrit values in the blood of test animals
(Eliawardani, 2015).
The materials used include Hayem solution, Turk solution, 0.1N HCl
solution, plasticine, EDTA, and distilled water. According to Dukes (1995),
Hayem's solution was a physiological solution consisting of 1 g NaCl, 5 g
Na2SO4, 0.5 g HgCl2 and 200 mL distilled water. This physiological solution is
used to thin the blood so that blood can be calculated because it must be isotonic
and fixative on erythrocytes. Turk solutions have the ability to demolish red
blood cells and contain aniline dyes (to color the cell nucleus). 0.1 N HCl
solution functions to form hernatin acid which is dark brown so that the color of
the solution will be the same as the indicator color after going through dilution
using distilled water. Plasticine acts as a clog at the end of the capillary pipette
so that the solution does not come out. EDTA solution (Ethylen Guaranteed
Tetraacetic Acid) functions as an anticoagulant or substance that causes blood to
not freeze.
According to Paulsen (2000), hematocyte is the percentage ratio between
the number of erythrocytes and blood plasma per unit of blood volume. The
hematocyte value can be used as a clue about the low content of dietary protein,
vitamin deficiency or infection. Increased hematocrit values indicate animals in
a state of stress. The standard hematocyte value is around 45%, but this value
can vary depending on the species. The hematocrit value can be used as an
indication of the low protein content of feed, vitamin deficiency or disease
infection (Frandson, 1992).
Normal blood sugar levels according to WHO when people are in normal
conditions, precisely 90 minutes after eating is 10 mmol / L or about 180 mg /
dL. Normal human blood sugar levels at night are 8 mmol / L or 144 mg / dL.
Normal blood sugar levels when fasting is 4-7 mmol / L or about 72-126 mg /
dL. The yield of sugar obtained from probandus group 3 is 204 mg / dL. These
results are probandus likely to have diabetes or consume excess sugar levels
before the blood sugar level test is carried out (Martin, 2012).
According to Soetrisno (1987), physiologically the difference in the number
of erythrocytes is affected by Sex, in male animals the number of erythrocytes is
more than females, age, the older you age, the less the number of erythrocytes,
body condition, in healthy conditions the number of erythrocytes will be more,
daily activity, high number of erythrocytes at active time, stress, if stress the
number of erythrocytes will decrease.
The amount of leukocytes is always influenced by the number of
erythrocytes, where the number of leukocytes is always lower than the number
of erythrocytes (Bevelander & Judith, 1979). Fluctuations in the number of
leukocytes in each individual are quite large in certain conditions such as stress,
age, physiological activities and others. Leukocyte counts are more produced if
the body's condition is sick if the blood circulation is less than the number of
leukocytes compared to the erythrocytes (Dukes, 1995).
Plasma glucose levels are determined at a time by the balance between the
amount of glucose entering the bloodstream and the amount that leaves it. The
release of antagonistic hormones to insulin increases blood sugar levels by
stimulating the release of glucose from the liver and inhibiting the body from
using glucose. Excessive use of alcohol can cause hypoglycemia because alcohol
decreases glucose release by the liver (Ganong, 2001).
Hemoglobin levels in the blood are influenced by several factors such as
iron adequacy and metabolism in the body. According to Schechter (2008)
Acidity increases & levels of H+ ions increase will weaken the bond between O2
and Hb so that the affinity of Hb for O 2 decreases so that Hb releases more O 2 to
the tissue.
Based on the practicum carried out by group 3 with mice animal test, the
results of the number of erythrocytes were 4,19 x 106 cells / mm3, the number of
leukocytes was 16425 cells / mm3, the hematocyte value was 18% and the
hemoglobin level was 5%. According to Branco et al. (2011), leukocyte values
in the blood of male mice (6.9±0,6x10³/mm³) and values for females
(5.8±0.5x10³/mm³) at different ages. Ponte (2008) presented 8.725±0.6/mm3 and
7.640±0.5/mm3, for males and females, respectively. In research with animals
Verçosa et al. (2004) presented mean values, in females of 25 to 30g, (2.96±1.22
x103/ mm3); and values for males of 2.81±0.39x10³/ mm³, lower than the total
leukocytes values obtained in the present work. When comparing the absolute
values of 5.04±1.84/μL to 3.07±1.20/μL for females and 4.92±2.29/μL to
3.97±2.64/μL for males, erythrocyte values 9.51±0.63x106/μL as the maximum
value and 7.94±0.42x106/μL as the minimum value. The number of red blood
cells was lower in the males than in the females, with observed maximum values
of 9.80±0.46x106/μL in the animals at 120 days and minimum values of
8.24±0.97x106/μL at 30 days. According to Restel (2014), hematocyte value was
difference in the comparison between age and gender. In the present study the
females presented higher values than the males in relation to the hemoglobin
parameter
 IV. CONCLUSION
Based on the result and discussion, it can be conclude that To take the
mice blood sample, blood samples were collected from a tail nick. To take fish
blood sample, blood analysis were obtained through heart puncture by sterilized
syringe in fish. The last is Chicken blood were collected at the end of treatment
was taken from the wing vein. We obtain leukocyte 10.925 cell/mm 3, erythrocyte
14,46 × 106 cell/mm3, Hb (Hemogoblin) level 5% and Hematocrite value 16%.
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