Experiment No. 5 was a bioassay to determine unknown concentrations of oxytocin using isolated rat uterine horns through an interpolation bioassay. Rat uteri were isolated and mounted in an organ bath containing De Jalon solution. A concentration response curve was established for a standard oxytocin solution and responses to test solutions were recorded. The test responses were selected such that they fell on the linear portion of the standard curve. The height of contractions were measured and used to calculate the unknown oxytocin concentrations by interpolating the test responses on the standard curve graphically. The unknown concentrations were determined to be [value 1] μg/ml from one method of calculation and [value 2] μg/ml from the other method.

1. Experiment No. 5
Bioassay of oxytocin using rat uterine
horn by interpolation method
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
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2. Overview of Discussion
 Objective
 Principle
 Requirements
 Experimental specifications (conditions)
 Preparation of oxytocin standard solution
 Preparation of De Jalon solution (PSS)
 Procedure
 Kymograph recording of contractions
 Observation table
 Graphical presentation of DRC
 Calculation
 Result and interpretation
2

3. Objective
To determine unknown concentration(s) of oxytocin using
isolated rat uterine horn by interpolation bioassay.
3

4. Principle
In this type of bioassay, a concentration response curve (CRC)
or dose response curve (DRC) of a standard substance is
first established. Then record 2-3 responses due to test
substance. The selection of test responses should be such
that they lie on the linear portion of the concentration
response curve (CRC) of the standard drug.
The precision and reliability of this bioassay is much better as
compared to matching bioassay as the sensitivity of
preparation is assessed prior to testing the unknown or test
substance.
Interpolation Bioassay
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5. Principle
Oxytocin is a hormone secreted by posterior pituitary. The
rat uterine preparation is commonly used for the
bioassay of oxytocin. The sensitivity of uterus to oxytocin
is depends upon the estrus/ oestrus cycles. The various
stages of estrus cycle can be identified by preparing the
vaginal smears and observed under microscope.
Preparation of Vaginal smears
Cotton swab made with tooth-picks is moistened with saline
and gently inserted, and slightly rotated within the
vagina. The swab is then pressed. A drop off saline is put
on a slide and examined under the microscope to confirm
the actual stage of estrus cycle.
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6. Principle
Estrus cycle
The cycle makes its appearance at puberty at the age of 2-3
months, and the whole cycle lasts for about 4-5 days, being
divided into 4 stages according to the cell types found in
the vaginal smear.
Estrus It is characterized by sexual receptivity when the
female will allow copulation. During this period, there are
increased running activity, quivering of the ears and
lordosis in the presence of another rat. The vaginal smear
shows 100 % cornified epithelial cells. It lasts for 9-15 hours
and ends with the ovulation.
Metestrus It follows estrus and occurs shortly after ovulation.
The vaginal smear is characterized by many leucocytes with
a few cornified cells. It lasts for 15-18 hours.
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7. Principle
Diestrus It is the longest of the phases and vaginal smear
consists of mainly leucocytes.
Proestrus It follows diestrus in a preparatory phase preliminary
to the next estrus phase. The original smear is characterized
by nucleated epithelial cells. It lasts about 12 hours.
If the rat is not in estrus, it can be induced by the
administration of stilbestrol (0.1 mg/kg. s.c., 24 hours
before).
Estrus uterus is highly sensitive to oxytocin and hence
preferred for the bioassay. However high spontaneous
contractions of the uterus in estrus stage may pose difficulty
in carrying out the experiment. In contrast, diestrus uterus
is relatively less sensitive to oxytocin. The spontaneous
activity of uterus can be reduced by proper adjustment of
composition and pH of PSS and bath temperature. 7

8. Photograph of Various Phases of Estrus Cycle
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9. The rat uterus is duplex, i.e. it comprises two uterine horns that join
together and open into the vagina via two separate cervices.
The uterine horns are the points where the uterus and the fallopian
tubes meet. The basic histological organisation of the uterus is shown
below:
Rat uterus
Uterine horn (rat, H&E x10, longitudinal section) 9

10. Rat uterus
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11. Oxytocin receptors
The OXTR protein belongs to the G-protein coupled receptor family,
specifically Gq, and acts as a receptor for oxytocin. Its activity is
mediated by G proteins that activate several different second
messenger systems.
Oxytocin receptors are expressed by the myoepithelial cells of
the mammary gland, and both the myometrium and endometrium of
the uterus at the end of pregnancy. The oxytocin-oxytocin receptor
system plays an important role as an inducer of uterine contractions
during parturition and of milk ejection.
Oxytocin receptors are also present in the central nervous system. These
receptors modulate a variety of behaviors, including stress and anxiety,
social memory and recognition, sexual and aggressive behaviors,
bonding (affiliation) and maternal behavior.
In some mammals, oxytocin receptors are also found in
the kidney and heart.
The mechanism of uterine contraction induced by oxytocin through-
 depolarization of muscle fibres and influx of Ca2+ ions, and
 phosphoinositide hydrolysis and IP3 mediated intracellular release of
Ca2+ ions. 11

12. Requirements
Animal: Female rat (120-150 g)
Physiological solution: De Jalon solution.
Drug- Oxytocin (Standard solution: 10 μg/ml), Stilbesterol
(0.1 mg/kg. s.c.)
Chemical- Fixing solution.
Instruments: Sherrington recording drum , Student organ
bath, Aerator, Insulin or tuberculin syringe to inject drugs
in small fractions, Dissecting board and various dissecting
instruments. Simple straw lever or frontal writing lever
and stand, Pipette, Stop watch etc.
Miscellaneous: Kymograph paper, plasticin, clips, and
thread.
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13. Experimental specifications (conditions)
 Isolated tissue- Isolated rat uterine horn preparation
 Drug- Oxytocin (Stock solution- 1 mg/ml) (Standard solution: 10
μg/ml), Stilbesterol (0.1 mg/kg. s.c.)
 Physiological salt solution (PSS)- De Jalon solution.
 Time cycle- Total- 3 minutes, Base line- 30 seconds, Contact
time- 30 seconds, Washing period- 2 minutes
 Applied load/ tension- 0.5 g
 Bath capacity- 40 ml
 Bath temperature- 30- 32°C
 Speed of rotation of drum- 0.25 mm/ second
 Magnification value (Mf) = d (F-W)/ d (F-T)
 Aeration- Normal air (1- 2 bubbles/ second)
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14. Preparation of oxytocin standard solution
Stock solution- 1 mg/ml or 1000 μg/ml
 Dissolve 1 mg of oxytocin in 1 ml of distilled water →
1000 μg/ml
Standard solution
 Dilute 0.1 ml of 1000 μg/ml solution up to 10 ml with
distilled water → 10 μg/ml
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15. Preparation of De Jalon solution (PSS)
 Prepare 1 litre of De Jalon solution by dissolving NaCl (9.0
g), KCl (0.42 g), NaHCO3 (0.5 g), and glucose (0.5 g) in
distilled water.
 CaCl2 (0.06 g) should be dissolved separately in distilled
water to avoid chances of precipitation of salt.
 Mix CaCl2 solution to the higher volume of PSS.
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16. Procedure
 Examine the vaginal smear under microscope to know about
the proper stage of estrus cycle. If the rat is not in estrus
stage, inject 0.1 mg/kg of stilbestrol s.c. and wait for 24 hours
(vaginal smear is prepared by taking a drop of the vaginal
wash and putting on the slide glass).
 Sacrifice the animal by a blow on the head and carotid
bleeding.
 Cut open the pelvic region and expose both the horns of
uterus. Separate them gently from the surrounding fatty
material and transfer them to a dish containing De Jalon
solution (PSS). When the rat is in estrus stage, the uterus is
fleshy and pink in colour. Two separate pieces (2-3 cm) of
uterine preparation can be made for experimental use.
 Mount the uterine preparation in the organ bath containing De
Jalon solution at 30- 32°C. Apply a tension of 0.5 g and allow
the tissue to equilibrate for 30 minutes. 16

17.  Record the contraction due to oxytocin (10 μg/ml) using
frontal writing lever. Contact time of 30 seconds and 5
minutes of total time cycle may be followed for recording of
response of each dose of standard concentration.
 Record the responses with an increased doses of oxytocin std.
till the ceiling point or plateau stage is observed i.e. till the
maximum response is achieved if one gets same or slightly less
response with a higher concentration or dose.
 Record responses due to 0.1, 0.2 or 0.4 ml of test oxytocin
solution. See that these responses would fall on the linear
portion of CRC/ DRC of oxytocin standard (10 μg/ml) [ i.e.
select the test doses in such a way that their % responses
should lie on the linear portion of sigmoidal CRC/DRC of
standard].
 Properly label and fix the kymograph recording of responses
with the help of fixing solution.
 Measure the height of the response (cm/ mm) of standard
doses and test doses of oxytocin. 17

18.  Put all the values in observation table (as shown).
 Plot a CRC/ DRC graphically. Read and calculate the corresponding
concentration of oxytocin test from standard curve.
 Describe the shape and various parts of CRC/ DRC and interpret
the result.
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19. Kymograph recording of contractions
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Oxytocin std.
(10 μg/ml)
0.1 0.2 0.4 0.8 1.6
Submaximal Dose
Ceiling Dose
Supramaximal Dose
Fig. Dose Response Curve (DRC) of oxytocin std. and
response of test oxytocin doses
T1 T2
Test Doses
1 ml
Dose Response Curve (DRC) of Oxytocin std. (10 μg/ml)

20. Observation table- Example
20
Sr.
No.
Drug
(Conc.)
Dose
(ml)
Dose
(μg)
log
(dose)
Response in
Conc. of oxytocin in
40 ml organ bath
Height
(cm/mm)
%
1
Oxytocin
(10 μg/ml)
0.1 1 0 0.3 16.67 0.25
2 0.2 2 0.3010 0.5 27.78 0.5
3 0.4 4 0.6021 1.4 77.78 1
4 0.8 8 0.9031 1.8 100.00 2
5 1.6 16 1.2041 1.8 100.00 4
6 Test T1 1 --- --- 0.6 33.33 ---
7 Test T2 1 --- --- 1.3 72.22 ---

21. Graphical presentation of DRC
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22. Calculation
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Magnification value (Mf) = d (F-W)/ d (F-T)
Where-
d (F-W) → distance between fulcrum and stylus (writing tip)
d (F-T) → distance between fulcrum and point of attachment of tissue
Unknown concentration of oxytocin in T1 and T2
Graph I) % Response Vs log (dose) = Antilog (x)
=.........μg/y ml
= .........μg/ml
Graph II) % Response Vs concentration of oxytocin in 40 ml of organ bath
=........X 40
=.........μg/ml

23. Result and interpretation
 The concentration response curve (CRC) or dose response curve
(DRC) of standard concentration of oxytocin (10 μg/ml) by using
isolated rat uterine horn preparation was recorded and interpreted
graphically as sigmoid or S-shape curve.
 The magnification value was found to be- .........
 The unknown concentrations of oxytocin by interpolation bioassay
were found to be-
Graph I) % Response Vs log (dose) = .........μg/ml
Graph II) % Response Vs concentration of ACh in 40 ml of organ bath =
.......μg/ml
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Thank You...