Lab Activity I

MANUFACTURE AND EVALUATION OF VAGINAL SMEAR IN MICE

Day : Thursday
Date : September 13th 2018

Name : Nadhilla Haura W.

Student ID : B1B017017
Group : D1
Subgroup :3
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. Be able to make a preparat of vaginal smear
2. To identified type of cells
3. To determine estrous phase in mice

B. Benefits

The benefits of this practical class are know how to make vaginal smear speciment
and know about rodentia estrous cycle.
 II. MATERIAL AND WORK PROCEDURES

A. Material

The tools that used in this practical class are cotton bud, NaCl solution 0,9%,
alcohol 70%, methylene blue 1%, aquadest, object glass with cover, and light
microscope.
The material that used in this practical class is mice (Mus musculus).

B. Work Procedures
The methods that used in this activity are:

1. The female mice that has been mature and not pregnant is prepared.
2. The female mice is pinched by thumb and index finger at nape of neck then
stretched at the palm and held the tail using pinkie.
3. The object glass is cleaned up using alcohol 70% and dried it. The cotton bud
was dipped in NaCl 0,9% and put it in slowly into mice vagina as deep as 5
mm and was rotate for three times.
4. The cotton bud is smeared above the object glass with direction three parallel
and two row then dried it.
5. When speciment has been dried, it is dropped with methylene blue 1% and
waited for 5 minutes.
6. The speciment is washed with aquadest and then covered with cover glass.
7. Observed under microscope, be drawn, the phase is identified, and then be
recorded in work sheets.
 III. RESULT AND DISCUSSION
A. Result

1 2

Details
Figure (A): Microscopic Figure of Estrous Cycle of Mice in Metestrous
Phase
Magnification : 10x40
Figure details :
1. Cornified epithelial cell
2. Leucocyte
B. Discussion

The estrous cycle (British spelling, oestrous) is the main reproductive cycle of
other species females of nonprimate vertebrates, for example rats, mice, horses,
pig have this form of reproductive cycle. Estrous cycle is the reproductive system
in most mammals, reproductive hormones cause the cycles to start after puberty
in sexually mature females. The mean duration of the estrous cycle was 4-5 days
for 60%-70% of female rats. The short length of the estrous cycle of rats makes
them ideal for investigation of changes occurring during the reproductive cycle
(Aritonang et al., 2017). Estrous cycle in mice consists of four phase i.e. proestrus,
estrus, metestrus and diestrus. These phase occur repeatedly and forming a cycle.
This cycle easily observed by see the changes in cells of vaginal epithelial lining
with vaginal smear method. Result of vaginal smear that are nucleated epithelial
cell, cornified epithel, and leucocyte. Based on result of vaginal smear then there
are signs of each phase that is being experienced by female mice. Each phase in
the estrous cycle is characterized by each characteristic, especially in vaginal
epithelial cells. Proestrus is characterized by appearing gore cells. Estrus is
characterized by all epithelial cells experiencing cornification (gore) and no
leucocytes. Metestrus is characterized by the loss of gore cells and becoming
nucleated cells and leucocytes. Diestrus is characterized by all nucleated epithelial
cells (normal) and leucocytes (Novriyanti et al., 2014).
The stage of the reproductive cycle cannot easily be determined from the
appearance of the ovarian follicles in a species like the rat, which has a short
estrous cycle. Follicles in virtually all stages of development are generally present
within the ovaries at all phases of the cycle. However, the formation, progression,
and regression of the corpora lutea are somewhat synchronized, and they can be
used as an aid (Yuan and Foley, 2002).
In this practical lab activity the cell we observed is in metestrus phase.
Metestrus is the phase after ovulation where the corpus luteum begins to works.
The length of the metestrus can depend on the length of time the LTH (Lutetropic
Hormone) is secreted by adenohipofisis. During this period there are decrease in
estrogen and increased progesterone formed by the ovaries (Frandson, 1993).
Metestrus is a brief stage when the corpora lutea form but fail to fully luteinize
due to a lack of progesterone. The uterine lining will begin to slough and evidence
of this is seen in the form of cornified eipithelial cells and polymorphonuclear
leucocytes present in vaginal swabs. Some nucleated epithelia cells will also be
present in late metestrus (Byers et al., 2012).
Metestrus is characterized by a combination of anucleated keratinized
epithelial cells and neutrophils. In mice, nucleated cells may appear occasionally
throughout metestrus. In rats, the small and large nucleated cells of late estrus are
present in low to moderate numbers throughout the stage. In early metestrus,
neutrophils are interspersed with the epithelial cells and are sometimes tightly
packed together or clumped around the cells; the epithelial cells usually
predominate but may be in equal proportion to the neutrophils. As metestrus
progresses, neutrophils become very high in number—outnumbering epithelial
cells by as much as 10-fold and the smear will be highly cellular and dense.
Neutrophil and epithelial cell numbers decrease by late metestrus, with a decrease
in smear cellularity, before the transition to diestrus. The separation between
where metestrus ends and diestrus begins is not always obvious, as they (i.e., the
end of metestrus and start of diestrus) are very similar in appearance and are
defined by the same cell types. Early and mid metestrus, however, are easily
identified if and when sample collection happens to occur at these times. Some
investigators who are only interested in cycle length and number of days in estrus
and diestrus usually count metestrus as a ‘‘diestrus’’ day. While distinguishing
and recording metestrus is recommended, if it cannot be certain if a smear is (late)
metestrus or diestrus, then it is best to be as consistent as possible and
pragmatically ‘‘error’’ on the side of diestrus; there is minimal value in overly
scrutinizing a late metestrus/early diestrus smear (Cora et al., 2015).
Morphological changes during metestrus phase in vagina: The start of
metestrus is marked by the mid region of the vagina showing a complete
detachment of the cornified epithelium,generally with residual squames present in
the lumen. As noted above, some cornified epithelium may persist, particularly
adjacent to the vaginal opening. There is a continued desquamation of epithelial
cells throughout the stage,with a progressive loss of the stratum granulosum and
upper germinativum (stratum spinosum). There is an accompanying variable
leucocyte infiltration. As noted under diestrus, the end of metestrus/start of
diestrus is marked by the epithelium reaching its lowest level. The smear during
this stage shows leukocytes, a few cornified cells, and basophilic cells. In uterus:
 During metestrus, the uterine endometrial epithelium shows continued
vacuolar degeneration,but also a marked return of mitotic activity, so both are seen
together. There is also a variable leucocyte infiltration. In ovaries: Ovarian
corpora lutea may still contain a fluid-filled central cavity generally devoid of
fibrous tissue, and the cytoplasm of the new corpora lutea are somewhat less
basophilic than at estrus but smaller than that seen at diestrus (Westwood, 2008).
 IV. CONCLUSION AND SUGGESTION

A. Conclusion
Based on the result and discussion, it can be conclude that:
1. Vaginal smear is used to identify cell types in vaginal smear preparation
and to determine the phase of the estrous cycle that occur in test animal.
The cell types that identified in vaginal smear are epithelial cells and
leucocyte.
2. The estrous cycle has four stages proestrus, estrus, metestrus, and
diestrus.
3. Vaginal smear in this observation obtain metestrus phase that consists of
leucocyte between cornified epithelial cells.

B. Suggestion

1. It is recommended that in making vaginal smear not only bye done on

mice, but in the other mammals that can be tested with this method of
vaginal smear.
 REFERENCES

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Byers, S. L., Michael, V. W., Sadie, L. D., & Robert, A. T., 2012. Mouse
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