Laboratory Manual: Physiology

Practical(ZOO519P)
 Contents

S. No. Practical P. No.

Determination of hemoglobin content.
1 2

2 RBC counting by using Hemocytometer 5

Nerve muscle preparation, Muscle twitch, effect of stimulus strength, effect of

3 10
stimulus frequency (tetany)
Normal cardiac activity in amphibian model, effect of temperature and effect
4 14
of drug
Measurement of blood pressure and effect of exercise on blood pressure.
5 19

Video Study of brains in different animals in relation to complexity of

6 23
functions.
7 Video Study of human brain model and different areas eliciting behaviors. 27

8 Stages in estrous cycle (studies on the effects of hormones in breeding season) 31

Oxygen consumption in fish and effect of temperature (by dissolved oxygen

9 35
meter)and terrestrial animal (mouse)
Study through clinics data on the insulin and glycemia in type1 and type 2
10 39
diabetic subjects.

1
 Practical No: 1
Determination of hemoglobin content.

Learning outcome
 Students will be able to describe the structure and function of hemoglobin.
 To acquire the skill necessary to count the hemoglobin content of a blood sample.
 To interpret hemoglobin count resut from a laborartory test.

General Background
Hemoglobin is a conjugated protein composed of the basic protein globin linked to 4 heme
molecules. Ninety-eight percent of all the iron found in the blood is contained in hemoglobin.
Hemoglobin transports oxygen and carbon dioxide. This important substance reacts with oxygen
to form oxyhemoglobin. In the tissues, oxygen is released and reduced hemoglobin is formed.
Hemoglobin can react with acids, bases, and oxidizing and reducing agents. It also can exist in a
variety of forms. The normal values for hemoglobin determinations are as follows:

Fig 1.1: The volume of hemoglobin in male and female

Compounds of hemoglobin
 Oxyhemoglobin. Oxygen combines loosely with iron (ferrous state) in hemoglobin. The
loosely attached oxygen diffuses into the tissues for oxidative processes. The hemoglobin then
binds carbon dioxide and exists as reduced hemoglobin.
 Carboxyhemoglobin. Hemoglobin combines with carbon monoxide to form
carboxyhemoglobin. Carbon monoxide has an affinity 200 times greater for hemoglobin than
oxygen does. Hemoglobin in this combination is incapable of oxygen transport.
 Methamoglobin. This compound is formed when the ferrous state of the heme is oxidized to

2
 the ferric state. This compound is incapable of oxygen transport.
 Sulfhemoglobin. This compound results from the combination of inorganic sulfides and
hemoglobin. This compound is incapable of oxygen transport. This is an irreversible reaction.
 Cyanmethemoglobin. This compound results when methemoglobin combines with the
cyanide radical. This compound is used in hemoglobinometry.
Hemobloginometry
There are four basic ways to measure the hemoglobin concentration:
1. Measurement of the oxygen-combining capacity of blood (gasometric)
2. Measurement of the iron content (chemical method)
3. Colorimetric measurement of specific gravity (gravimetric method)
4. The cyanmethemoglobin method is the method of choice and is most widely used
Cyanmethemoglobin (hemoglobin-cyanide) method for estimation of hemoglobin
This is the method of choice for estimation of hemoglobin and is recommended by International
Committee for Standardization in hematology. This is because
i. All forms of hemoglobin are converted to cyanmethemoglobin (except sulfhemoglobin)
ii. A stable and reliable standard is available.
Principle
Blood is mixed with a solution of potassium ferricyanide, potassium cyanide and a non-ionic
detergent (Drabkin’s solution). Erythrocytes are lysed producing an evenly distributed hemoglobin
solution. Potassium ferricyanide converts hemoglobin to methemoglobin, and methemoglobin
combines with potassium cyanide to form cyanmethemoglobin (hemiglobincyanide). All forms of
hemoglobin present in blood are completely converted to a single compound, cyanmethemoglobin.
When the reaction is completed, absorbance of the solution is measured in a spectrophotometer at
540 nm. At this wavelength, cyanmethemoglobin has a broad absorbance peak. To obtain the
amount of hemoglobin in the unknown sample, its absorbance is compared with that of the standard
cyanmethemoglo-bin solution (the hemoglobin concentration of which is known) by using a
formula or a previously prepared graph/table.
Equipment
1. Photoelectric colorimeter or spectrophotometer

3
Reagents
Drabkin’s solution (pH.7.07.4)
(Potassium ferricyanide 200 mg Potassium cyanide 50 mg, Potassium dihydrogen phosphate 140
mg’ Non-ionic detergent 1 ml, Distilled water to 1000 ml)
1. Cyanmethemoglobin standard solution with known hemoglobin value.
2. Specimen: EDTA-anticoagulated venous blood or blood obtained from skin puncture.
Method
1. In a test tube, take 5 ml of Drabkin’s solution and to it add 20 μl of blood (1:251 dilution).
Stopper the tube, mix by inverting several times, and allow standing for at least 5 minutes. This
time is adequate for conversion of hemoglobin to cyanmethemoglobin.
2. Transfer the test sample to a cuvette. Read the absorbance in a spectrophotometer at 540 nm
or in a photoelectric colorimeter using a yellow-green filter. Also take the absorbance of the
standard solution. Absorbance should be read against reagent blank (Drabkin’s solution).
3. Hemoglobin value is derived from the formula given below or from the previously prepared
graph or table.

Preparation of graph and table: If a graph or a table is prepared which correlates absorbance with
hemoglobin concentration, result can be obtained quickly. This is particularly suitable when a large
number of samples are regularly processed on the same instrument. Diluted
cyanmethemoglobin standards are available commercially for preparation of a calibration graph.
Alternatively, standard cyanmethemoglobin solution is diluted serially with Drabkin’s solution.
On a linear graph paper, hemoglobin concentration (horizontal axis) in each dilution is plotted
against the absorbance (vertical axis). A straight line joining the points and passing through the
origin is obtained. From this graph, a table can be prepared relating absorbance to hemoglobin
concentration.
Significance
The hemoglobin concentration is directly proportional to the oxygen-combining capacity of blood.
Therefore, the measurement of the hemoglobin concentration in the blood is important as a
screening test for diseases associated with anemia and for following the response of these diseases
to treatment.

4
 Practical No: 2
RBC counting by using hemocytometer

Learning outcome
 Students will be able to comprehend the structure and function of the hemocytometer
 To acquire skills to count RBCs by using a hemocytometer
 To interpret RBCs count resut from a laborartory test

General Background
Although a variety of automated cell counting instruments have been developed, Hemocytometer
remains the most common method used for cell counting around the world. The most frequently
used hemocytometer is the Neubauer’s (or ‘Improved Neubauer’) chamber. Other hemocytometers
include the Burker, Thoma, and Fuchs-Rosenthal. Using these, the particles (e.g., leucocytes,
erythrocytes, thrombocytes, bacteria, fungus spores, pollen) are visually counted under a
microscope. Neubauer’s chamber is a thick glass plate with the size of a glass slide (30x70x4mm).
The counting region consists of two square-shaped ruled areas. There are depressions or trenches
on either side or in between the areas on which the squares are marked thus giving an “H” shape.

The ruled area is 3mm2 divided into 9 large squares each with a 1 mm2 area. The large central
square (which can be seen in its entirely with the 10X objective), is divided into 25 medium squares
with double or triple lines. Each of these 25 squares is again divided into 16 small squares with

single lines so that each of the smallest squares has an area of 1/400 mm2.
The glass cover is a squared glass of width 22 mm. The glass cover is placed on the top of
Neubauer’s chamber, covering the central area. The ruled area is 0.1 mm lower than the rest of the
chamber. So that when a cover slip is kept on the counting region, there is a gap of 0.1 mm
(1/10mm) between the cover slip and the ruled area.

5
Figure 2.1: View of hemocytometer
([Link]

Figure 2.2: Detail of Neubauer’s Chamber or hemocytometer

([Link]

The counting can be done either in the central large square or in the corner squares, depending
on the size of the cells under study.
WBC Counting Area
The four large squares placed at the corners are used for white blood cell count. Since their
concentration is lower than red blood cells a larger area is required to perform the cell count.
RBC Counting Area
The large center square is used for RBC counts. As already stated, this area is subdivided into 25
medium squares, which in turn are each divided into 16 squares. Of the 25 medium squares, only

6
the four corner squares and the center square within the large center square are used to perform
RBC counts.
Materials:
Biologicals: 1. Droplet of blood
Glass/plastic ware:
1. RBC diluting pipette with hose and bulb
2. Trash bin for lancets
3. Plastic droppers
Chemicals
1. RBC diluent 3.2 or 4% w/v Sodium Citrate (Na3C6H5O7) /d.H2O/ Normal Saline
(Saline maintains the normal disk shape of RBC and prevents auto agglutination)
2. 95% Alcohol for rinsing
Instruments
1. Sterile lancet
2. Hemocytometer for RBC and WBC counting
3. Microscope
Others
1. Tissue paper
2. Gloves for use while staining
Method
1. Sample Preparation
1. As before make a pin-prick using a fresh unused lancet on the index- or ring finger. If you have
been pricked before on that finger, use another finger.
2. Bring a cleaned RBC dilution pipette tip close to the droplet of oozing blood. Using the bulb
allow (by capillarity and pressure) blood to enter up to the 0.5 mark.
3. Using a small tube of Sodium-Citrate solution, additionally aspirate this solution to reach the
mark 101 (1:200 dilution)
4. Gently turn the dilution tube in your hand.
2. Preparing Neubauer Chamber
Clean the Neubauer chamber and the cover slip with 70% ethanol. Put the glass cover on the
Neubauer chamber central area. Use a flat surface to place the chamber, like a table or a

7
workbench.
3. Introducing the sample into the Neubauer chamber
With a pipette, carefully draw up around 20 ml of the cell mixture (dilution). Place the pipette tip
against the edge of the cover glass and slowly expel the liquid until the counting chamber is full.
Capillary action will help to ensure that the counting chamber is full, but care should be taken not
to overfill the chamber. A volume of 10 ml is sufficient to fill one counting chamber.

Figure 2.3: Sample introduction to hemocytometer

([Link]

4. Microscope focusing and Cell Counting

1. Place the Neubauer chamber on the microscope stage. Using the 10X objective, focus both onto
the grid pattern and the cell particles.
2. To count the RBCs and Platelets, the microscope must be switched to 40X objective. Count the
cells in the respective areas as stated early.
3. Write down the amount of cells counted
4. If cells are touching the 4 perimeter sides of a corner square, only count cells on 2 sides, either
the 2 outer sides or 2 inner sides.

8
Calculations:

Significance
A complete blood count can help find the cause of symptoms such as weakness, fatigue and fever.
It also can help find the cause of swelling and pain, bruising, or bleeding. To check on a medical
condition. A complete blood count can help keep an eye on conditions that affect blood cell counts.

9
 Practical No: 3 Nerve muscle preparation, Muscle twitch, effect of stimulus strength,
effect of stimulus frequency (tetany)

Learning outcomes
 Students will be able to understand basic knowledge of nerve physiology.
 To prepare nerve-muscle preparation
 To develop the ability to measure muscle twitch in response to a single stimulus

Introduction
The functional unit of skeletal muscle activity is the motor unit. Interposed between the axon
branches and the muscle fibers is a structure called the neuromuscular junction (motor end-plate).
The most convenient way of examining the properties of the motor unit is by using the nerve-
muscle preparation.
Purpose
1. To make a sciatic nerve and gastrocnemius (SNG) preparation
2. To measure the effects of different stimulus intensities on the muscle response
3. To measure a single twitch, incomplete tetanus, and complete tetanus
Chemicals
Ringer’s solution is a solution of recently boiled distilled water containing NaCl 6.5g, KCl 0.14g,
CaCl2 0.12g, NaHCO3 0.20g, NaH2PO4 0.01g per liter
Equipment
 RM6240 biological signal collecting system
 Force transducer

10
 Figure 3.1: RM6240 biological signal collecting system
([Link]
system_fig1_348132519)
Procedure
For intact Nerve-Muscle preparation, pith the toad, resulting in the toad not feeling pain. As soon
as the brain and spinal cord are destroyed, the toad should become flaccid. Then peel the skin of
one leg. Fix the toad on the frog board with the dorsal surface uppermost. Identify the
gastrocnemius muscle. Tie a knot around the Achilles tendon using suture thread. Cut the Achilles
tendon as close to the bottom of the foot as possible. Locate the sciatic nerve in the thigh region.
Use the glass hooks to separate the sciatic nerve. Pass a piece of thread beneath the sciatic nerve.

Figure 3.2: Nerve-Muscle preparation

([Link]

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Hold the knee in place by pushing down two T-pins. Tie the free end of the thread to the hole on
the end of the tongue of the force transducer. Place the stimulating electrode on the sciatic nerve.
Warning:
 Do not touch the nerve with any object that is made of metal; this will render the nerve useless.
 Avoid stretching or otherwise damaging the nerve. Keep the tissue moist with the ringer’s
solution.
Effect of stimulus intensity on twitch amplitude
Set record parameters. The recording trace should be set to zero.
Apply a light load to the muscle by raising the force transducer with the tension adjuster until the
trace moves approximately 2 g above the baseline. Warning: Make sure to lift the muscle vertically
to an angle that is perpendicular to the rest of the frog leg.
Display the stimulation mark. Change the stimulus amplitude to 0.2V. Determine the threshold
voltage by increasing the stimulus voltage in 0.01V increments. Stop and save the recording trace.

Figure 3.3: Threshold stimulus maximal stimulus

Table 3.1: Measure the following values

Intensity stimulus (V) Tension of contraction (g)
Threshold
intensity
Maximal
intensity

Table 3.2: Record the following values in a single twitch induced by maximal stimulus

12
 Duration (ms)
Latent period
Contraction
Relaxation

Effect of stimulus frequency on twitch amplitude

Set record parameters. Click on the record button. Select the frequency ascending mode. Display
the stimulation mark. Begin the recording. Set the stimulus intensity to the lowest value that will
give strong contraction as during the twitch amplitude calculation. Stop and save the recording
trace.

Twitch incomplete tetanus complete tetanus

 Figure 3.4: effect of stimulus frequency on twitch amplitude

Table 3.3: Determine the following values

Frequency of stimulus (Hz) Tension of contraction (g)

Single twitch
Incomplete tetanus
Complete tetanus

Significance
This experiment will help students to understand the basis and application of nerve physiology.

13
 Practical No: 4
Normal cardiac activity in amphibian model, effect of temperature and effect of drug

Learning Outcomes
 Students will be able to analyze and describe normal cardiac activity in an amphibian model.
 To investigate the effects of temperature and different drugs on cardiac activity of amphibian

Introduction
A kymograph is an instrument for recording variations in pressure, as of the blood, or in tension,
as of a muscle, by means of a pen or stylus that marks a rotating drum. A kymograph (which means
'wave writer') is a device that gives a graphical representation of spatial position over time in which
a spatial axis represents time. It basically consists of a revolving drum wrapped with a sheet of
paper on which a stylus moves back and forth recording perceived changes of phenomena such as
motion or pressure.
It was invented by German physiologist Carl Ludwig in the 1840s and found its first use as a means
to intrusively monitor blood pressure, and has found several applications in the field of medicine.
Its primary use was to measure phenomena such as changes in muscular contractions or other
physiological processes, including speech sounds. Kymographs were also used to measure
atmospheric pressure, tuning fork vibrations, and the functioning of steam engines.

Figure 4.1: View of kymograph

14
Cardiac activity
The heart's primary function is simply to act as a pump that provides pressure to move blood to its
ultimate destination – the tissues, the control of cardiac contractility is complex and represents a
balance of intrinsic (within the heart) and extrinsic (from outside the heart) factors. An isolated
heart beats regularly without any extrinsic stimulation from nerves or hormones. This mechanical
automaticity reflects the fact that the action potentials that are conducted throughout the heart (the
signals that lead to contraction) are generated spontaneously within the cardiac muscle itself. The
cells responsible for spontaneous action potential production are often referred to as pacemaker
cells because they determine the rate at which the heart beats. Several cell types in the heart are
capable of pacemaker activity, but in the intact organ it is the fastest pacemaker which drives the
rest of the heart
Normally the pace-making frequency is highest in a group of specialized cells called the sinoatrial
node (SN\SAN), which also has a different shape of action potential. These cells dictate the rate
of electrical events in the rest of the heart.
The Sino-atrial node in humans is located in the wall of the right atrium close to the superior vena
cava. In the frog or turtle heart, the pacemaker region is the sinus venous, an enlarged region
between the vena cava and the right atrium.
Purpose
To study frog heartbeat records and examine some of these intrinsic and extrinsic factors that make
the heart such a unique and versatile pump (To record the relative force and frequency of
contraction in a resting heart).
Materials/Chemicals
Gloves,
Frog, Formaldehyde (or any Anesthetic),
Kymograph,
Dissection tools,
Dissection dish,
Ringer's Solution
Procedure
1. In a clean aseptic work area double-pith a frog and fasten it to a frog board, ventral side
up.

15
 2. Use scissors to make a Longitudinal incision through the skin and body wall of the thoracic
region to expose the heart.
3. Hold the pericardium with forceps and carefully cut away the sac from the heart, using
scissors. From this point on make sure that the heart is periodically moistened with frog
ringer's solution.
4. Using forceps, gently lift the apex of the heart upward.
5. Insert a bent insect pin or small fish hook through the tip of the ventricle, being careful not
to puncture the ventricle.
6. Tie a thin thread to the hook and connect the ventricle to the transducer as you connected
the gastrocnemius muscle to the transducer in:
7. If a physiograph type of transducer is used, attach the thread to the transducer hook and
adjust the tension on the ventricle until the recording pen is raised slightly above the
baseline.
Results
Normal heartbeat
Obtain a recording of the normal cardiac rhythm, using a medium to fast paper speed so as to
distinguish the atrial and ventricular. Run a 1-second timeline while recording so that the duration
of systole and diastole of the ventricle can be determined.

Figure 4.2. A typical record of the frog heartbeat showing Sinus (S), Articular (A), and
ventricular (v) beats. (Drum speed 5mm/sec)
([Link]

16
1. Temperature
Reduce the kymograph speed to 2mm/sec and record the normal heartbeat.
Then drop warm (40°C) frog ringer's solution on the heart until significant changes are seen in rate
and contractility.
Record contraction at this time finally, drop cold (5°C) ringer's solution on the heart and record
when changes are observed.
Then rinse the heart with room temperature ringer’s solution to return the beat to normal before
continuing the experiments
2. Effect of Drug
1. Record the normal contraction for 30 seconds
Irrigate the preparation with the solution of acetylcholine.
Note the effects of a drug on
a) Heart rate
b) The strength of heart contraction

Figure 4.3: the effects of Acetylcholine

([Link]
Significance
This Practical will help students to understand the basics of cardiac activity by using the
amphibian model. Amphibians have played a prominent part in the success of using key

17
 species to discover new information about all animals. Many of the fundamental studies on
cardiovascular physiology were based on frog models.

18
 Practical No: 5
Measurement of blood pressure and effect of exercise on blood pressure

Learning Outcomes
 Students will be able to understand the physiological concept of blood pressure
 To measure blood pressure by using a sphygmomanometer
 To interpret blood pressure readings

Introduction
A sphygmomanometer is a device that measures blood pressure. It is composed of an inflatable
rubber cuff, which is wrapped around the arm. A measuring device indicates the cuff's pressure.
A bulb inflates the cuff and a valve releases pressure. A stethoscope is used to listen to arterial
blood flow sounds. As the heart beats, blood forced through the arteries causes a rise in pressure,
called systolic pressure, followed by a decrease in pressure as the heart's ventricles prepare for
another beat. This low pressure is called the diastolic pressure.
The sphygmomanometer cuff is inflated to well above expected systolic pressure. As the valve
is opened, cuff pressure (slowly) decreases. When the cuff's pressure equals the arterial systolic
pressure, blood begins to flow past the cuff, creating blood flow turbulence and audible sounds.
Using a stethoscope, these sounds are heard and the cuff's pressure is recorded. The blood flow
sounds will continue until the cuff's pressure falls below the arterial diastolic pressure. The
pressure when the blood flow sounds stops indicates the diastolic pressure.
Systolic and diastolic pressures are commonly stated as systolic 'over' diastolic e.g. 120 over
80. Blood flow sounds are called Korotkoff sounds.
Instrument required
 Sphygmomanometer

19
Figure 5.1: View of Sphygmomanometer
([Link]
mercury-free-sphygmomanometer )
Procedures
1. To begin blood pressure measurement, use a properly sized blood pressure cuff. The
length of the cuff's bladder should be at least equal to 80% of the circumference of the
upper arm.
2. Wrap the cuff around the upper arm with the cuff's lower edge one inch above the
antecubital fossa.
3. Lightly press the stethoscope's bell over the brachial artery just below the cuff's edge.
Some healthcare workers have difficulty using the bell in the antecubital fossa, so we
suggest using the bell or the diaphragm to measure blood pressure.
4. Rapidly inflate the cuff to 180 mmHg. Release air from the cuff at a moderate rate
(3mm/sec).
5. Listen with the stethoscope and simultaneously observe the sphygmomanometer. The
first knocking sound (Korotkoff) is the subject's systolic pressure. When the knocking
sound disappears, that is the diastolic pressure (such as 120/80).
6. Record the pressure in both arms and note the difference; also record the subject's
position (supine), which arm was used, and the cuff size (small, standard, or large adult
cuff).
7. If the subject's pressure is elevated, measure blood pressure two additional times,
waiting a few minutes between measurements.
Precautions
1. Use a larger cuff on obese or heavily muscled subjects.

20
2. Use a smaller cuff for pediatric patients.
3. For pediatric patients a lower blood pressure may indicate the presence of hypertension.
4. Don't place the cuff over clothing.
5. Flex and support the subject's arm.
6. In some patients the Korotkoff sounds disappear as the systolic pressure is bled down. After
an interval, the Korotkoff sounds reappear. This interval is referred to as the "auscultatory
gap." This pathophysiologic occurrence can lead to a marked underestimation of systolic
pressure if the cuff pressure is not elevated enough. It is for this reason that the rapid inflation
of the blood pressure cuff to 180mmHg was recommended above. The "auscultatory gap"
is felt to be associated with carotid atherosclerosis and a decrease in arterial compliance in
patients with increased blood pressure.

Effects of exercise on blood pressure

Aerobic activities such as swimming, cycling, and running put additional demands on your
cardiovascular system. Your muscles need more oxygen than they do when you’re at rest, so you
have to breathe more quickly. Your heart starts to pump harder and faster to circulate blood to
deliver oxygen to your muscles. As a result, systolic blood pressure rises.

Procedure

 Record the normal readings as discussed before and now ask the subject to exercise
(running, jumping, etc.) for about 30 minutes and record the blood pressure.
 Now check and record the differences in blood pressure during normal conditions and after
exercise. What is the difference you recorded and why is that so?

It’s normal for systolic blood pressure to rise to between 160 and 220 mm Hg during exercise.
Unless you’ve cleared it with your doctor, stop exercising if your systolic blood pressure surpasses
200 mm Hg. Beyond 220 mm Hg, your risk of a heart problem increases. Different factors can
influence how your cardiovascular system responds to exercise. Some of these factors include diet,
medical conditions, and medications. For instance, exercise hypertension is a condition that causes

21
an extreme spike in blood pressure during physical activity. People with exercise hypertension can
experience spikes in systolic blood pressure up to 250 mm Hg during exercise.

According to guidelines provided by the Centers for Disease Control and Prevention Trusted
Source, “normal” blood pressure is less than 120/80 mm Hg. This includes a systolic pressure
reading under 120 mm Hg (the top number) and a diastolic pressure reading (the bottom number)
under 80 mm Hg.

Significance

It’s important to get an accurate blood pressure reading so that you have a clearer picture of your
risk for heart disease and stroke.

22
 Practical No: 6
Video Study of brains in different animals in relation to complexity of functions.

Learning Outcomes
 Students will be able to understand basic functions of the brain in different animals
 To interpret different behaviors observed in animals and relate them to the brain structures
and functions

General Introduction
The complexity of brain functions in different animals varies greatly based on factors
such as evolutionary history, ecological niche, and social behavior. Brains have evolved to serve
specific functions that are advantageous for an organism’s survival and reproduction. Here is a
general overview of how brains differ in complexity across various animal species:

1. Simple Nervous Systems (Invertebrates): Many invertebrates, such as insects and

mollusks, possess relatively simple nervous systems. These systems consist of clusters
of nerve cells (ganglia) connected by nerve cords. While their brains are small and lack
the complexity of vertebrate brains, they are adapted for specific behaviors. For instance,
insects have specialized brain regions for processing visual and olfactory information,
which are crucial for navigation and finding food.

Figure 6.1: Invertebrates nervous system

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([Link]
zed_nervous_systems.php)

2. Fish and Amphibians: Fish and amphibians have brains that are more developed
compared to invertebrates. They possess structures like the forebrain, midbrain, and
hindbrain. These structures are involved in sensory processing, motor control, and basic
cognitive functions. Fish, for example, have well-developed sensory systems for
detecting movement, light, and chemicals in water.

Figure 6.2. Brain of Fish

([Link]

Figure 6.3: Brain of Frog

([Link]

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3. Reptiles: Reptiles have larger and more complex brains than fish and amphibians. They
exhibit improved sensory processing, spatial memory, and problem-solving abilities.
Some reptiles, like certain species of lizards, show advanced cognitive skills such as
learning and memory retention.

Figure 6.4: Lizard Brain

([Link]

4. Birds: Birds are known for their relatively large brains and high cognitive capabilities.
The avian brain, particularly the forebrain region called the pallium is responsible for
complex behaviors like problem-solving, tool use, communication, and spatial memory.
Birds like crows, parrots, and pigeons have demonstrated impressive problem-solving
skills in laboratory experiments.

Figure 6.5: Brain structure of Bird

([Link]

5. Mammals: Mammals possess some of the most complex brains due to their

25
 evolutionary history and the demands of nurturing their young. Mammalian brains are
characterized by a highly developed neocortex, responsible for advanced cognitive
functions such as decision-making, language processing, and complex social
interactions. Primates, including humans, have particularly large and intricate
neocortices, which are thought to be related to their advanced social behaviors and
cognitive abilities.

Figure 6.6: Brain Structure of Dog

([Link]
anterior-to-left-adapted-from_fig2_254887847)

In summary, the complexity of brain functions in different animals corresponds to their

evolutionary history, ecological needs, and social behaviors. The brains of animals have adapted
to serve various survival and reproductive strategies, leading to a wide range of cognitive
abilities across species.

26
 Practical No: 7
Video Study of human brain model and different areas eliciting behaviors.

Learning Outcomes
 Students will be able to understand and describe the anatomy of the human brain, including its
major structures, lobes, and specialized areas associated with specific behaviors and functions

Introduction
The brain is the most complex part of the human body. This three-pound organ is the seat of
intelligence, interpreter of the senses, initiator of body movement, and controller of behavior. The
brain is like a group of experts. All the parts of the brain work together, but each part has its own
special responsibilities. The brain can be divided into three basic units: the forebrain,
the midbrain, and the hindbrain

Figure 7.1: Parts of human brain

([Link]
Forebrain
The forebrain is the largest and most highly developed part of the human brain: it consists
primarily of the cerebrum and the structures hidden beneath such as the thalamus, hypothalamus,
pituitary gland, and limbic system.

27
When people see pictures of the brain it is usually the cerebrum that they notice. The cerebrum sits
at the topmost part of the brain and is the source of intellectual activities. It holds your memories,
allows you to plan, and enables you to imagine and think. It allows you to recognize friends, read
books, and play games. The cerebrum is split into two halves (hemispheres) by a deep fissure.
Despite the split, the two cerebral hemispheres communicate with each other through a thick tract
of nerve fibers that lies at the base of this fissure. Traditionally, each of the hemispheres has been
divided into four lobes: frontal, parietal, temporal, and occipital.
 Frontal Lobe: Responsible for executive functions, decision-making, impulse
control, and motor planning.

 Parietal Lobe: Plays a role in spatial awareness and sensation.

 Temporal Lobe: Important for auditory processing and memory.

 Occipital Lobe: Responsible for visual processing.

Although the two hemispheres seem to be mirror images of each other, they are different. For
instance, the ability to form words seems to lie primarily in the left hemisphere, while the right
hemisphere seems to control many abstract reasoning skills. The right cerebral hemisphere
primarily controls the left side of the body, and the left hemisphere primarily controls the right
side. When one side of the brain is damaged, the opposite side of the body is affected. For example,
a stroke in the right hemisphere of the brain can leave the left arm and leg paralyzed.
Midbrain
The uppermost part of the brainstem is the midbrain, which controls some reflex actions and is
part of the circuit involved in the control of eye movements and other voluntary movements.
Hindbrain
The hindbrain includes the upper part of the spinal cord, the brain stem, and a wrinkled ball of
tissue called the cerebellum. The hindbrain controls the body’s vital functions such as respiration
and heart rate.

The cerebellum coordinates movement and is involved in learned rote movements. When you play
the piano or hit a tennis ball you are activating the cerebellum.

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Experimental Design

Researchers design experiments to investigate specific behaviors. Participants are often exposed
to stimuli or tasks that elicit the desired behavior, while their brain activity is monitored using
neuroimaging techniques. Comparisons are made between brain activity during different
conditions to identify the areas associated with the behavior of interest.

 Prefrontal Cortex: This area, particularly the dorsolateral prefrontal cortex, is linked to
executive functions such as decision-making, impulse control, and working memory.

 Parietal Lobes: These are responsible for integrating sensory information, spatial
awareness, and attention.

 Temporal Lobes: These are involved in auditory processing, language comprehension,

and memory formation. The hippocampus, located within the temporal lobes, is crucial for
memory consolidation.

 Amygdala: This almond-shaped structure in the temporal lobe is associated with

processing emotions, particularly fear and threat detection

 Occipital Lobes: The primary visual cortex in the occipital lobes processes visual
information, allowing us to perceive and recognize objects and scenes.

 Striatum: Part of the basal ganglia, the striatum is involved in reward processing, habit
formation, and motor control.

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 

Figure 7.2: Human brain anatomy

([Link]

4. Limitations and Future Directions:

While neuroimaging studies provide valuable insights, they have limitations.
Correlations between brain activity and behavior do not imply causation. Additionally,
individual variability and the complexity of behaviors make definitive conclusions challenging.
In recent years, advancements in techniques like functional connectivity analysis and machine
learning have allowed researchers to uncover more nuanced relationships between brain regions
and behaviors. However, studying the brain and its relationship to behavior remains an ongoing
and complex endeavor.

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 Practical No: 8
Stages in estrous cycle (studies on the effects of hormones in breeding season)

Learning Outcomes
 Students will be able to understand different stages of the estrous cycle.
 To comprehend neuroendocrine regulation of the estrous cycle.
 To interpret behavioral changes that correspond to each stage of the estrous cycle.

Introduction
The estrous cycle or oestrus cycle is the set of recurring physiological changes that are induced by
reproductive hormones in most mammalian therian females. Estrous cycles start after sexual
maturity in females and are interrupted by anestrous phases or by pregnancies. Typically, estrous
cycles continue until death.
Stages of estrus cycle
A four-phase terminology is used in reference to animals with estrous cycles.

Figure 8.1: Stages of estrus cycle

([Link]

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1. In the proestrus stage one or several follicles of the ovary start to grow. Their number is
species-specific. Typically, this phase can last as little as one day or as long as three weeks,
depending on the species. Under the influence of estrogen, the lining in the uterus
(endometrium) starts to develop.
2. Estrus or oestrus refers to the phase when the female is sexually receptive. Under regulation
by gonadotropic hormones, ovarian follicles mature and estrogen secretions exert their biggest
influence. The female then exhibits sexually receptive behavior. Estrus is commonly seen in
mammalian species, including primates.
3. The metestrus or diestrus phase is characterized by the activity of the corpus luteum, which
produces progesterone. The signs of estrogen stimulation subside and the corpus luteum starts
to form. The uterine lining begins to appear. In the absence of pregnancy, the diestrus phase
(also termed pseudo-pregnancy) terminates with the regression of the corpus luteum. The
lining in the uterus is not shed but is reorganized for the next cycle.
4. Anestrus refers to the phase when the sexual cycle rests. This is typically a seasonal event and
is controlled by light exposure through the pineal gland that releases melatonin. Melatonin
may repress the stimulation of reproduction in long-day breeders and stimulate reproduction
in short-day breeders. Melatonin is thought to act by regulating the hypothalamic pulse activity
of the gonadotropin-releasing hormone.
After completion (or abortion) of a pregnancy, some species have postpartum estrus, which is
ovulation and corpus luteum production that occurs immediately following the birth of the young.
For example, the mouse has a fertile postpartum estrus that occurs 14 to 24 hours following
parturition. Estrous cycle variability differs among species, but cycles are typically more frequent
in smaller animals. Even within species significant variability can be observed, thus cats may
undergo an estrous cycle of 3 to 7 weeks. Domestication can affect estrous cycles due to changes
in the environment.

Endocrinology of the Estrous Cycle

Estrous cycles give females repeated opportunities to become pregnant throughout their productive
lifetime. The cycle is regulated by the hypothalamic-pituitary-gonadal axis, which produces

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hormones that dictate reproductive events. The reproductive axis is composed of the
hypothalamus, pituitary, and the ovary.
The sequence of hormonal release essentially begins with the synthesis and release of GnRH from
the hypothalamus. This polypeptide hormone is transported to the anterior pituitary through a
highly specialized capillary network called the hypothalamo-hypophyseal portal system. GnRH
functions to stimulate the anterior pituitary to produce and release FSH and LH. FSH and LH are
transported through systemic blood circulation to the ovaries, where they initiate a series of
morphological changes that lead to ovulation and pregnancy if fertilization occurs.

The primary hormones produced by the ovary are estrogen and progesterone. These hormones are
transported by the blood stream to "target" tissues to cause a reaction. Estrogen is produced by the
follicle, which is located on the ovary. As the follicle grows, more estrogen is produced. As
increasing amounts of estrogen are released into the bloodstream and travel to the anterior
pituitary, it acts in a positive feedback fashion, stimulating pulsatile LH release. It also affects the
nervous system of the cow, causing restlessness, phonation, mounting, and most importantly, the
willingness to be mounted by other animals. Estrogen causes the uterus to contract, allowing sperm
to be transported through the female reproductive tract more efficiently after insemination. Other
effects of high estrogen concentrations in the blood include increased blood flow to the genital
organs and the production of mucus by glands in the cervix and vagina. These characteristics are
all signs of estrus or sexual receptivity.

Progesterone produced by the CL prevents cyclicity by acting on the anterior pituitary in a negative
feedback fashion; therefore, decreasing the release of FSH and LH. It prepares the uterus for the
reception of fertilized ova and subsequent pregnancy. It also helps the cow maintain pregnancy by
suppressing uterine contractions and promoting the development of the uterine lining.

A fifth hormone important in female reproduction is prostaglandin F2a(PGF2a). This hormone is

secreted by the endometrium of the uterus and also affects structures on the ovary, helping to
initiate ovulation by causing the demise of the CL, which results in the withdrawal of
progesterone's negative feedback mechanism. High circulating concentrations of progesterone
stimulate the production and release of PGF2a from the uterus.

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Figure 8.2: Hypothalamic Pituitary Gonadal axis

([Link]

Significance
A sound understanding of the estrous cycle allows cattle producers to troubleshoot reproductive
problems. That understanding is also important when using estrous synchronization and other
reproductive technologies.

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 Practical No: 9
Oxygen consumption in fish and effect of temperature (by dissolved oxygen meter) and
terrestrial animal (mouse)

Learning Outcomes
 Students will be able to describe basic concepts of oxygen consumption and respiration in
fish and mouse.
 To acquire skill to measure dissolved oxygen concentration.
 To understand the effect of temperature on dissolved oxygen consumption

Background
Dissolved oxygen concentration (DO) is considered the most important water quality variable in
fish culture. In the broadest sense, however, dissolved oxygen concentration is no more important
than other environmental variables because any factor that is outside the range tolerated by fish
can cause stress or death. What makes dissolved oxygen concentration so important in intensive
fish culture is the speed with which it can change. Over a matter of hours, sometimes even minutes,
DO can change from optimum to lethal levels. No other critical environmental variable in fish
culture is so dynamic. The dynamic nature of dissolved oxygen results from the interaction of three
factors. First, oxygen is not very soluble in water so water has only a limited capacity to “hold”
oxygen. Second, the rate of oxygen use by fish, plankton, and organisms living in the pond mud
can be high. Third, oxygen diffuses very slowly from the atmosphere into undisturbed water. The
combination of these three factors—limited solubility, rapid use, and slow replenishment—can
cause rapid changes in dissolved oxygen concentrations. Dissolved oxygen levels can be managed
with aeration, but the response time for taking corrective measures is short. This makes it critical
to have a rapid and reliable method of measuring dissolved oxygen concentrations so that aeration
devices can be activated when needed.
There are a number of ways to measure dissolved oxygen concentration. Select a method based on
1) the number of ponds or tanks to be measured, 2) the level of accuracy required, and 3) the cost
of the measurement technique. The titration-based “drop count” method fairly rapidly assesses
whether or not there is sufficient dissolved oxygen in water. The drop count method is inexpensive
and appropriate if DO concentration is to be measured infrequently in a few ponds or tanks.

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However, on commercial fish farms or in any other situation where DO measurement of many
ponds or culture units is routine, a dissolved oxygen meter is an indispensable piece of equipment.
What is an oxygen meter?
An oxygen meter has two components—the sensor (sometimes called the probe) and the meter.
The sensor reacts with oxygen and an electrical signal is produced in proportion to the oxygen
concentration. The signal is then amplified, translated into concentration units, and displayed by
the meter. The meter circuitry also compensates the reading for changes in temperature, altitude
or salinity.
DO meters do not measure oxygen concentration directly, but measure a voltage that is produced
by the chemical reactions of oxygen with the various components of the sensor.
Materials
 Dissolved oxygen meter
 Beakers
 Thermometer
 Fishes and mouse
Procedure
Making dissolved oxygen measurements
The process of measuring dissolved oxygen concentration with an oxygen meter is simple,
although it involves a bit more than simply putting the sensor in the water and reading a number
on the display. Accurate measurement of DO concentration with polarographic sensors requires
moving the sensor in the water. Oxygen is consumed across the membrane, so failure to move the
sensor will create an oxygen-depleted microzone around the sensor tip and show a measurement
that is too low. Move the sensor up and down or side to side about 1 foot per second to prevent
this problem.
Dissolved oxygen measurements made near the pond bank are very different (usually lower) than
those made in open areas of the pond because of the effects of sediment respiration and pond bank
vegetation. The dissolved oxygen meter should not be turned off between measurements to avoid
depolarizing the probe. Recalibrate the meter when it is used again. If a large meter adjustment is
necessary during recalibration it may be an indication of problems with the membrane or sensor.
Dissolved oxygen monitoring in ponds
Dissolved oxygen concentrations in fish ponds vary with depth, from one side of a pond to the

36
other and particularly from one time of day to another. In general, dissolved oxygen concentrations
are lowest at dawn and highest at dusk; lowest near the bottom and highest near the surface; and
lowest near the upwind side and highest near the downwind side.
Effect of temperature on oxygen consumption
Take 2 beakers. 1st beaker contain cold water and 2nd contain warm water.
Now placed fishes in these beakers (one each).
First check oxygen consumption in 1 st beaker for 10 seconds.
Now check oxygen consumption in 2nd beaker for 10 second.
Now record your readings
Now check dissolved oxygen consumption again after 5 minutes for both beaker following
previous method.
In the same way use an enclosed glass chamber and record the oxygen consumption of the mouse.

Figure 9.1: View of dissolved oxygen meter

([Link]
Significance
Measuring the concentration of dissolved oxygen is important with such applications as live fish

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transport, corrosion control, wastewater treatment, and fish farming. This measurement can
provide you with a clear understanding of water quality and what treatments may be necessary.
For water treatment plants that test and treat water to make sure that the water is safe to drink,
many measurements will be taken during the treatment process. The exact amount of DO in the
water will dictate how the smell and taste of the water must be changed before the treatment
process is completed.

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 Practical No:10
Study through clinics data on the insulin and glycemia in type1 and type 2 diabetic
subjects.

Learning Outcomes
 Students will be able to develop an understanding of diabetes, its classification, and the
difference between both types.
 To gain the skill to measure the glucose level in the blood sample by using a glucometer
 To interpret clinical data on the glycemic level.

Introduction
Diabetes is a condition that happens when your blood sugar (glucose) is too high. It develops when
your pancreas doesn’t make enough insulin or any at all, or when your body isn’t responding to
the effects of insulin properly. Insulin is a hormone that controls the glucose level in blood. Most
forms of diabetes are chronic (lifelong), and all forms are manageable with medications and/or
lifestyle changes. There are two types of diabetes.
Type 1 diabetes:
The body’s immune system is responsible for fighting off foreign invaders, such as harmful viruses
and bacteria. In people with type 1 diabetes, the immune system mistakes the body’s own healthy
cells for foreign invaders. The immune system attacks and destroys the insulin-producing beta
cells in the pancreas. After these beta cells are destroyed, the body is unable to produce insulin.
Researchers don’t know why the immune system sometimes attacks the body’s own cells. It may
have something to do with genetic and environmental factors, such as exposure to viruses.
Research into autoimmune diseases is ongoing.
Type 2 diabetes:
People with type 2 diabetes have insulin resistance. The body still produces insulin, but it’s unable
to use it effectively.
Other genetic and environmental factors may also play a role. When you develop type 2 diabetes,
your pancreas will try to compensate by producing more insulin. Because your body is unable to
effectively use insulin, glucose will accumulate in your bloodstream.

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Glycemia
It refers to the concentration of sugar or glucose in the [Link] is also of 2 types.
1. Hyperglycemia
it occurs when blood sugar levels are too high. People develop hyperglycemia if their diabetes is
not treated properly. Its symptoms are following.
 Extreme thirst, drinking a lot and then urinating frequently as a result
 Unintentionally losing a lot of weight within a few weeks
 Noticeable loss of energy with muscle weakness, tiredness and generally feeling quite
unwell
 Nausea and stomach ache
 Trouble seeing
 Poor concentration
 Frequent infections (cystitis, thrush)
 Confusion and drowsiness, or even coma
If you or your child have these symptoms, you should see a doctor as soon as you can
2. Hypoglycemia
it sets in when blood sugar levels are too low. Low blood sugar is most common in people who
use insulin or take certain tablets to reduce high blood sugar. This is because things like
unplanned physical activity, eating meals later than usual, or drinking too much alcohol can
mean that you need less insulin than you thought, causing your blood sugar to drop very low.
Its symptoms include Racing pulse.

Signs that your blood sugar is too low may include:

 Racing pulse
 Cold sweats
 Pale face
 Headache
 Feeling incredibly hungry
 Shivering, feeling weak in the knees
 Feeling restless, nervous or anxious
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  Difficulty concentrating, confusion

These symptoms do not occur all at once. The signs of hypoglycemia not only depend on the blood
sugar level, but also vary from person to person. If you are not sure whether your blood sugar is
too low, you can measure it to make sure. Mild hypoglycemia doesn't usually have any harmful
effects. But it is important to react quickly enough and eat or drink something, such as dextrose
sugar or sugary lemonade.

People who have severe hypoglycemia may feel very drowsy and confused, and might even
become unconscious. If this happens, someone else can inject the hormone glucagon.

When is blood sugar considered to be too high or too low?

Slight fluctuations in blood sugar levels are completely normal and also happen on a daily basis in
people who do not have diabetes. Between around 60 and 140 milligrams of sugar per deciliter of
blood (mg/dL) is considered to be healthy. This is equivalent to blood sugar concentrations
between 3.3 and 7.8 mmol/L. “Millimole per liter” (mmol/L) is the international unit for measuring
blood sugar. It indicates the concentration of a certain substance per liter.

If type 1 diabetes is left untreated, people’s blood sugar levels can get very high, sometimes
exceeding 27.8 mmol/L (500 mg/dL). Blood sugar concentrations below 3.3 mmol/L (60 mg/dL)
are considered to be too low.
Material
 Glucometer
 Test strip
 Lancet
Procedure

1. Make sure the meter is clean and ready to use.

2. After removing a test strip, immediately close the test strip container tightly. Test
strips can be damaged if they are exposed to moisture.

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 3. Wash your hands with soap and warm water. Dry well. Massage your hand to get
blood into your finger. Don’t use alcohol because it dries the skin too much.
4. Use a lancet to prick your finger. Squeezing from the base of the finger, gently place
a small amount of blood onto the test strip. Place the strip in the meter.
5. After a few seconds, the reading will appear. Track and record your results. Add
notes about anything that might have made the reading out of your target range,
such as food, activity, etc.
6. Properly dispose the lancet and strip in a trash container.

Figure 10.1: View of Glucometer

([Link]
Result
A fasting blood sugar level of 99 mg/dL or lower is normal, 100 to 125 mg/dL indicates you have
prediabetes, and 126 mg/dL or higher indicates you have diabetes.

Method of Collecting Data:

This practical requires a questionnaire survey of diabetes patients (Clinics data on the insulin and

42
glycaemia in type1 and type 2 diabetic patients).

Results:
Compute your results in tabulated form.

Significance
Monitoring blood sugar helps to determine if you are meeting your glucose targets which helps to
reduce the unpleasant symptoms of high and low blood sugar, and avoid long-term diabetes
complications

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