Lab Activity

MANUFACTURE AND EVALUATION OF VAGINAL SMEAR IN MICE

Day : Thursday

Date : September of 13th 2018

Name : Mellya Rizki Pitriani

Student ID : B1B017031
Group : VIII
Subgroup :1
Assistant : Nur Hidayati

LABORATORY OF ANIMAL STRUCTURE AND DEVELOPMENT

FACULTY OF BIOLOGY
JENDERAL SOEDIRMAN UNIVERSITY
PURWOKERTO
2018
 I. INTRODUCTION

A. Aims
the aims of this practical class are :
1. To know procedures to make the preparation of vaginal smear
2. To identify cell types in preparation of vaginal smear
3. To determine estrous phase in mice

B. Benefits
The benefits of this practical class are we can know procedures to make the
preparation of vaginal smear, identifying cell types in preparation of vaginal smear,
and determining estrous phase in mice.

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 II. MATERIAL AND WORK PROCEDURES

A. Material
The tools that used in this practical class are cotton bud, tissue, glass
object, light microscope and cover glass.
The materials that used in this practical class are methylene blue 1%,
alcohol 70%, NaCl 0,9% solution, aquades and mice.

B. Work Procedures
The work procedures that used in this practical class are:
1. The female mice that has been mature and not pregnant is prepared.
2. Female mice are held with the right hand by stretching them on the palm of
the hand while the nape is clamped by the thumb and the index finger is
clamped between the palm and the little finger.
3. The tip of the cotton bud was moistened with NaCl 0.9% solution and then
slowly inserted into the vagina of the mice as deep as ± 5 mm and slowly
rotated in two to three times.
4. Object glass is cleaned with alcohol 70% solution and dried.
5. The tip of the cotton bud that has been applied to the vagina is spread over
two or three spread lines in the same direction on the object glass.
6. The vaginal swab is dripped with 1% methylene blue solution while
occasionally tilting so that the dye is evenly distributed on the surface of the
review.
7. Excessive dyes are cleaned by rinsing with distilled water or running water
then covered with a cover glass.
8. Preparations are observed under a light microscope.

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 III. RESULT AND DISCUSSION

A. Result

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Details:
Figure (A): Microscopic Figure of Estrous Cycle of Mice in Estrous Phase
Magnification: 40x10
Figure details:
1. Cornified epithelial cell

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B. Discussion
Mice are one of the polyestrus animals, because mice experience the estrous
cycle many times throughout their lives, usually once a year. Estrous mice cycle
begins with the preparation of follicular development (proestrus), then continued
estrous, metestrus, and ends with diestrus (Nalley, 2011). At smear, the estrus phase
is marked with oval and core epithelial cells clearly visible and the presence of
leucocyte cells. The vaginal apus in the estrus phase is characterized by epithelial
cells that have been cornified, without nucleus and no visible leucocyte cells. The
metestrus phase is characterized by the results of vaginal swabs in the form of
cornified epithelial cells and the presence of leukocytes. The results of the vaginal
smear diestrus phase show nucleated epithelial cells, leukocytes and lenders (Taylor,
1994).
Based on observations that have been made, it can be seen from the cells that
are seen that the epithelium has been cornified and shows that the mice (Mus
muscullus) are undergoing the estrous phase. Rapid growth and cornification of the
vaginal epithelium during and at the end of estrous are caused by estrogen.
Increasing levels of estrogen in mice also contribute to a decrease in caloric levels in
mice (Mus muscullus) (Frisch, 1975). There are several methods that can be done to
determine the estrous phase in mice. One of them is the vaginal smear method. The
method of vaginal smear is more widely used because it can show more accurate
results. This method uses epithelial cells and leukocytes as identification material.
Epithelial cells are cells located on the vaginal surface, so that in the event of
changes in estrogen levels epithelial cells are the earliest cells affected by these
changes. Leukocytes are antibody cells that are found in all individual parts.
Leukocytes in the vagina function to kill bacteria and germs that can damage the
ovum. Epithelial cells are oval or polygonal, whereas leukocytes are round nucleated
(Nalbandov, 1990).

The estrous cycle phase obtained in the test animals during the practicum is
the estrous phase. This phase is characterized by the presence of epithelial cells that
undergo cornification. This is the same as the reference obtained, according to Adnan
(2006), at the time of estrous, the vagina shows epithelial cells that gore. Vaginal
smear are usually made in laboratory animals, such as mice, before male and female
animals are put together, unification should be done at the time of initial estrous.
When estrous, the female animal's vulva is usually red and swollen. Fast growth and
cornification of

the vaginal epithelium during and at the end of estrus are known to be caused by
estrogen. If in a normal cycle, estrogen decreases after ovulation, or in castrated
females, estrogen injection is stopped, then the vaginal epithelium with cornification
begins to diminish, scales disappear and dominant leukocytes. The vaginal
epithelium changes histologically from the thick-coated squamous type because
estrogen to the thin low cuboidal epithelium that signifies the anesthetic phase of the
estrous cycle (Helena, 2006).

The estrous cycle is a series of events related to uterine preparation for ovum
reception and planting. The phases include proestrus, estrous, metestrus, and diestrus.
There is an addition of glandular and vessel glandularity in the endometrium and
vaginal mucosa in the proestrus phase. The uterine wall becomes thicker and
smoother. The follicular graft in the ovary has matured and produces estrogen and
progesterone hormones in the proestrus phase before ovulation occurs. This change
is caused by gonadotropic and pituitary hormones, FSH (Follicle Stimulating
Hormone). Estron production increases and ovulation occurs in the estrus phase. The
mucosa of estrus expands and contains a lot of blood, at this time female animals are
ready to accept male animals. The formation of the corpus luteum from follicular
cells occurs in the metestrus phase. The corpus luteum from the time of ovulation at
the end of the estrous cycle acts as an endocrine gland. The progestron is currently
very active in preparing the uterine wall for implantation of the ovum, whereas
estron is only present in the body. The anesthetic phase is a period of sexual rest, the
uterus again takes its original structure. Diestrus's phase is the period between the
completion of the next marriage preparation reshuffle (Djuhanda, 1981).

The estrous cycle is the distance between the estrus one to the next estruos.
Each animal has different estrous cycles, there are monoestrus animals (estrous once
a year), polyestrus animals (estrous several times a year), and seasonal polyestrus
animals (estrous only during certain seasons of the year). Estrous cycle or cycle
consists of four phases, namely proestrus, estrous, metestrus, and diestrus. The
estrous phase is different from the estrous cycle. The estrus phase is the phase where
eggs are ovulated from the ovary to the oviduct. This phase indicates that female
individuals have sex. The estrous

phase of each species is different and can be observed with the vaginal smear
method, but it cannot be observed if the female animal is not yet mature and is
pregnant. (Hafez, 1968).

Estrous is the most important phase in the lust cycle, because in this phase
female animals show specific symptoms for each type of animal and in this phase
females also want to accept males for copulation, the characteristic of estrous is
copulation, if animals refuse copulation, although the signs of estrus are very clearly
visible, the refusal gives a sign that the female animal is still in the missed estrous
phase. Other signs of the estrous phase for each type of livestock are different, but in
general they show signs of anxiety, appetite decreases or disappears altogether,
approaches the male and does not run if the male approaches (Partodiharjo, 1986).
This estrous cycle is controlled by the hormone estrogen. The estrogen receptor is
not only in the oviduct, but also in the liver. The estrogen hormone receptor in the
oviduct serves to synthesize egg protein. The estrogen receptor on the liver functions
to synthesize vitelogen (Rugh, 1962).

Two different types of cycles are found in female mammals. Humans and
many other primates have menstrual cycles, while other mammals or non-primates
have an estrous cycle. Both cases of ovulation occur at some time in this cycle after
the endometrium begins to thicken and flow with a lot of blood, because it prepares
the uterus for possible embryo implantation. One difference between the two cycles
involves the fate of both uterine layers if pregnancy does not occur. The endometrial
menstrual cycle will decay from the uterus through the cervix and vagina in bleeding
called menstruation. The endometrial estrous cycle is reabsorbed by the uterus, and
there is not much bleeding (Campbell, 2010).

By far the most popular method to determine phase changes in rodentia

animals is to use vaginal cytology. The technique is simple and relatively non-
invasive and allows you to carry out individual re-monitoring for a long time. A new
alternative, namely electrical resistance measurements of vaginal mucous membranes
has been carried out in various species of small animals, such as rabbits and rats. It is
clear that experiments can be done to define hormonal status in female mice needed
to identify the effect of physiological sex hormones using female mice aged 3, 15
and 20 months. This

study aims to determine the general characteristics of systemic estrogen changes and
examine the experimental utility of determining the estrous cycle (E) with vaginal
smears and / or comparing the relationship of concentration oscillations with
systemic estrogen measured in feces (Weixelbaumer, 2014). Female mice in the
proestrus phase have many nucleic and several new corneum epithelial cells. Some
leucosides may become new if females experience proestrus faster as a phase in the
advanced cycle to estrus, with many new epithelium. If the cycle is not separated by
pregnancy, pseudopregnancy, or other phenomena, metestrus will begin (Byers,
2012).
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IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result and discussion, it can be concluded that :

1. Procedure for making vaginal smear preparations that is insert a cotton swab
that has been moistened with NaCl 0.9% solution into the vagina of the
mouse then rotate slowly in the direction of the cotton bud as deep as ± 5mm
then the cotton bud is applied extending two or three spread lines in the same
direction on the object glass that has been given alcohol 70% then smeared on
the object glass given a 1% methylene blue solution then dry it for about 5
minutes then observe it on a microscope.
2. The cell type used to identify the phases in the estrous cycle are epithelial
cells and leukocyte cells. Epithelium is oval or polygonal, whereas leukocytes
are round and nucleated.
3. The phase in mice in this lab is the initial estrus phase can be known from
preparations containing several cornified epithelium.
B. Suggestion
1. It is better to make vaginal smears not only in mice, but in other mammals
that can be tested with the vaginal smear method.
2. We recommend that all practitioners try one by one to make vaginal smear,
so that all practitioners can make vaginal smear properly.
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 REFFERENCES

Adnan., 2006. Reproduksi dan Embriologi. Makassar: Jurusan Biologi FMIPA

UNM.

Byers SL Wiles MV, Dunn SL, Taft RA.,2012. Mousse Estrus Cycle Identification
Tool and Image. vol. 7(4).

Campbell, N. A., 2010. Biologi Edisi ke 8 Jilid III. Jakarta: Erlangga.

Djuhanda, T., 1981. Embriologi Perbandingan. Bandung: Armico.

Frisch, R.E., 1975. Body weight and Food Intake at Early Estrus of Rats on a
HighFat Diet, vol. 72, 4172-4176.

Hafez, E. S. E., 1968. Reproduction in Farm Animals. Philadelphia: Lea & Febiger.

Helena., 2006. Changes in α-estradiol receptor and progesterone receptor expression

in the locus coeruleus and preoptic area through out the rat estrous cycle.
Journal of Endocrinology (2006) 188, 155-165.

Nalbandov, A. V., 1990. Reproductive Physiology of Mammals and Birds. San

Fransisco: W. H. Freeman and Company.

Nalley, Wilmientje Marlene Mesang, Ristika Handarini., 2011. Penentuan Siklus

Estrus Berdasarkan Gambaran Sitologi Vagina dan Profil Hormon pada
Rusa Timor. Vol. 12 No. 2: 98-106.

Partodiharjo S., 1980. Ilmu Reproduksi Hewan. Jakarta: Mutiara.

Rugh, R., 1962. Experimental Emrbryology. Minnesota: Burger Publishing

Company.

Taylor, Pamela., 1994. Practical Teratology. London: Saunders Co.

Weixelbaumer, Katrin M., 2014. Shock: Strus Cycle Status Defined by Vaginal
Cytology Does Not Correspond To Fluctuations Of Circulating Estrogens In
Female Mice. Vol. 41, No. 2.
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Pocock, G. & Richards, C. D., 2006. Human Physiology: The Basis of Medicine. 3rd
ed. Oxford: Oxford University Press. 1,5 spasi

Choudhary, P., Sudhamani, S., Pandit, A. & Kiri, V., 2012. Comparison of Modified
1 spasi Ultrafast Papanicolaou Stain with The Standard Rapid Papanicolaou Stain in
Cytology of Various Organs. Journal of Cytology/Indian Academy of
Cytologists, 29(4), pp.241-245.

NOTES :
1. Times New Roman font size 12 except the title and chapter (14).
2. The margin left 4, right, down, up 2.5.
3. Spacing between chapters to Section 2 spaces, spacing between last word
in subbab to next subbab is 2 space, spaces between sentences to the first
paragraph of section 1,5 and the spacing between lines of 1.5 spaces.
4. Paper A4 80 grams
5. The background contains practical reason for the event, when quoting
from journals or books do not forget listed the author and that included
in the reference list.
6. Background consist of at least 3 paragraphs, and each paragraph
consists of at least 3 sentences.
7. In preparing the report using at least 5 text book and 2 journals last 5
years (2014-2018).
8. Discussion contains a comparison between the theoretical and the
practical results of existing research results in journals that are relevant
to practical events.
9. Conclusions based on the results and discussion that refers to the goal.
10.All theories taken from the quote should be in included in the reference
list.