MANUFACTURE AND EVALUATION OF VAGINAL SMEAR

OF MICE

By:
Name : Dion Satrio Pambudi
Student ID : B1B015018
Group : VII
Subgroup :4
Assistant : Kamilah Dwi Septiani

PRACTICAL REPORT OF ANIMAL DEVELOPMENT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY BIOLOGY
PURWOKERTO

2016
 I. INTRODUCTION

A. Background

Precise semen analysis is the most important test for Determining potential
fertility in the male, and the necessity for external quality control of this semen analysis
has been highlighted in numerous studies. In semen analysis, there exist many possible
sources of variability and Tus it is Necessary to control the factors that could influence
results. Reviews These may be physiological factors, methodological or related to the
operator's experience. The first two factors can be reduced by the Standardisation of
preanalytical and analytical procedures, while the third requires technical expertise,
procedural care, meticulous quality control, both internal and external and internal and
external participation in proficiency testing programs (Alvarez, 2005).
Some of the male reproductive system assessment methods have been
developed. One commonly used is the analysis of sperm quality. Analysis of sperm
quality can give us information about the fertility status of the male genital organ. In
addition to the necessary descriptive study about the image of an animal sperm, this
assessment is also used in the study of toxicology or pharmacology of a substance on
male fertility. This analysis may show an increase or decrease in the fertility of a test
animal. The purpose of the analysis was to assess the quality of sperm parameters
descriptive of the sperm sample test animals. The quality is generally rated is the
number of sperm (density / concentration of sperm), sperm morphology and motility
of sperm (Luthfi, 2012).
The effectiveness of IUI depends mainly on semen quality, which is assessed
by the total motile sperm count (TMSC) and sperm morphology. TMSC in the
ejaculate is the product of multiplying the semen volume by the sperm concentration
by the percentage of progressively motile sperms. The best results are achieved when
the number of TMSC exceeds a threshold of approximately 10 million (Nikbakht,
2011).
Sperm morphology is another factor that may influence the IUI result. Most
studies have found a strong correlation between sperm morphology and the IUI result.
In assessing sperms morphology by strict criteria, success rates with IUI are highest
when 14% or more of the sperm have normal morphology, like the results observed in
in vitro fertilization (IVF) cycles (Nikbakht, 2011).
B. The Aims

The purpose of the practice and lab activity this time is to perform the
procedure of making smear vagina, identify the types of cells in the preparations and
found the estrus phase in test animals.
 II. MATERIALS AND METHODS

A. Materials
The tools used in this lab is a light microscope, along with the cover glass
object, cotton bud and tissue.
The materials used in the vaginal smear lab is ready for mating adult female
mice (Mus musculus ) sex and not pregnant, 0.9% NaCl solution, a solution of 70%
alcohol, 1% methylene blue stain, and aquades.

B. Methods

1. Glass object was cleaned with 70% alcohol and air dried.
2. Female mice examined, is vested with the right palm. in the supine position on the
hands, neck clamped between thumb and forefinger, tail clamped between palms
and little finger.
3. The tip of a cotton bud moistened with a solution of NaCl 0.9% and slowly inserted
into the vagina of mice as deep as 5 mm, rotated clockwise two to three times.
4. Cotton bud tip is applied to the glass object spreadable three rows in the same
direction (parallel).
5. Reviews vagina on glass object etched with 1% methylene blue stain, in order to
shake the surface evenly spread. Left to stand for 5 minutes.
6. Glass object had been washed in running water with low discharge, air dried and
then covered with a cover glass.
7. Vaginal smears that have been so observed under a microscope with a magnification
of weak, then with strong magnification.
8. Overview cells in preparations with a standard compared to known phases of the
test animals and then take a picture the observation.
 III. RESULT AND DISCUSSION

A. Result

(A) (B)

Details:
Picture (A) : Microscopic Estrous Cycle Phase Proestrus Magnification 400.
Picture (B) : Schematic Estrous Cycle Phase Proestrus Magnification 400.
Details Picture:
1. Leukocyte Cell
2. Cornification Epithelial Cells
 B. Discussion

There are several methods that can be performed to determine the estrus phase
in mice. One of them with a vaginal smear method. Vaginal smear method is more
widely used because it can show more accurate results. This method uses the epithelial
cells and leukocytes as material identification. Epithelial cells are the cells located at
the surface of the vagina, so that when there is a change in estrogen levels, the
epithelial cells are the cells most affected by the beginning of the changes. Leukocyte
cell antibody is contained in all parts of the individual. Leukocytes in the vaginal
functioning kill bacteria and germs that can damage the ovum. Epithelial cells are oval
or polygonal, whereas globular nucleated leukocytes (Nalbandov, 1990).
Estrus stage is the stage where follicles are ripe and ready to ovulate. Not seen
leukocytes. More cornification of epithelial cells and some nucleated epithelial cells.
Estrus phase can be seen from the behavior of mice and mice vagina morphology. At
the time of estrus usually mice look preoccupied and more active, in other words, the
mice were in a state of seeking attention to male mice (Chakraborti, 2013). The
reproductive cycle is commonly called the estrous cycle has four stages: proestrus,
Estrus, Metestrus, and diestrus. Proestrus stage is an initial stage in which follicles
grow to stimuli and produce the FSH hormone estrogen. There are many nucleated
epithelial cells and some leucocytes and cornification epithelial cells (Xiao, 2014).
Female reproductive organs in a mammal in the form of ovarian form of a flat
in a resting state, but it is round, long, lumps on edges in the reproductive phase. Each
ovary is very close to a hole shaped like a funnel (osteum) at the distal end of the
uterine tubae (oviductus=oviduct) on the edge of the hole oesteum tufts are called
fimbria. Oviductus near the end oesteum somewhat dilated called the ampulla. Once
past this, especially after reaching the uterus, the egg cannot be fertilized by sperm
again. Oviductus mammal other than a street egg to the uterus also serves as a venue
for the fertilization process. The uterus serves as the venue for the development of the
embryo (giving place, protect and nourish (Soeminto, 2000).
 IV. CONCLUSION AND RECOMMENDATION

A. Conclusion

1. Vaginal smears are used to identify the types of cells in the vaginal smear and to
determine the phases of the estrous cycle that occurs the test animals. Cell types
that identified in vaginal smears are epithelial cells and leukocytes.
2. There are four stages of the estrous cycle is proestrus, estrus, and diestrus
metestrus.
3. The cell types in the estrus phase of the estrous cycle in female mammals are
their cornification epithelial cells.
4. Vaginal smear practicum obtained for this time of estrus in female mice phase
characterized by cornification epithelial cells and going for 18 hours.
B. Recommendation

1. Preferably in the manufacture of vaginal smear is not only done in mice, but in
many other mammals that can be tested by vaginal smear method.
2. All the students one by one try to manufacture the vaginal smear, so that all
students can make vaginal swabs properly.
 REFERENCES

Abidin, A.F. 2014. Laporan Vaginal Smear. Fakultas Biologi: Purwokerto.

Djuhanda, T. 1981. Embriologi Perbandingan. Bandung: Armico.
Chakraborty P, Roy SK. 2013. Expression of Estrogen Receptor in the Hamster
Ovary throughout the Estrous Cycle. Massachusetts: Sianuer Associates Inc.
Nalbandov, A. V. 1990. Reproductive Physiology of Mammals and Birds. San
Fransisco: Freeman and Company.
Soeminto. 2000. Embriologi Vertebrata. Unsoed: Purwokerto.
Xiao L, Zhang C, Li X, Gong S, Hu R, et al. 2014. Signaling Role of Prokineticin 2
on the Estrous Cycle of Female Mice. Massachusetts: Sianuer Associates Inc.