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The International University Vietnam National University HCMC School of Biotechnology



Advisor: Dr. DO MINH SY Submitted by: L TH NG C NH BT070001

HCMC, Feb 15 - 2011




Today, advances in the society are not only to support human living but also to damage human health. Particularly, the high rate of people got problems related to the reproductive. However, the development of biotechnology opens a new century of high technologies. An illustration of this is the fertilization in glass . This is due to the fact that those wife/husband who has problems to their reproductive organs, still have strong ability to produce off-spring. Fertilization in glass or In vitro fertilization (IVF) , is commonly referred to as IVF. IVF is the process of fertilization by manually combining an egg and sperm in a laboratory dish. When the IVF procedure is successful, the process is combined with a procedure known as embryo transfer, which is used to physically place the embryo in the uterus. II. LITERATURE REVIEW 1. Why scientists inject PMSG and hCG to female mice? Inorder to control eggs to be ovulated, PMSG and hCG are two commonly chemicals used by scientist The hormone PMSG is commonly used in concert with progestogen to induce ovulation in livestock prior to artificial insemination. hCG (Human chorionic gonadotropin) is frequently called the "pregnancy hormone". hCG sends a message to the corpus luteum to keep the progesterone flowing. That's because progesterone supports and nourishes the endometrium lining where the embryo is implanted. If no hCG is detected by the corpus luteum, the levels of progesterone will decrease at the end of the second half of the menstrual cycle and a woman will have her period.

In fact, FSH has similar functions as PMSG and LH has common functions to hCG. Those FSH and LH also could be used in controlling the ovulated eggs. 2. Why people used to remove fat as much as possible while collecting the sperm? Because fat may restrict the activities of sperms, resulting in affecting the fertilized result. 3. Why we have to transfer sperms and eggs after collecting to PBS/PVA 1% medium? Phosphate buffered saline (abbreviated PBS) is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH Polyvinyl alcohol (PVA) has been reported to decrease the spontaneous acrosome reaction in mouse spermatozoa. In cattle, it has been reported that PVA added to medium containing heparin can stimulate sperm capacitation but barely support the fertilization of cumulus 4. How to detect the sperms density? Haemocytometer is the suitable solution Haemocytometer is a device originally designed for the counting of blood cells. It is now also used to count other types of cells as well as other microscopic particles.

Figure II.4: Hemocytometer grid

Counting cells + The smallest square has side 50 l x 50 l. The volume over the smallest square is 2.5 x 104 m3 = 2.5 x 104 l. + Number of smallest squares were counted = 16 x 5 = 80 spores +Thus, the volume of the 80 spores is equal to 80 x 2.5 x 104 l = 0.02 l + Therefore, the formula of counting cells per ml
X = A x 1000/0.02

Where: + X: number of cells per ml + A: number of cells per 80 counted spores An adult male mouse has from 63 to 71 million sperm per ml.



a. Samples: Mouse (1 male and 1 female) b. Equipments: Pipette 100 l, capillary pipes, tubes, microscope, gloves, etc.

c. Chemicals: PMSG and hCG 2. Methods Collecting sperms from cauda epididymis and oocytes from ovary/oviduct a. Collecting sperms: Sacrifice the male mouse Collecting caudae epididymides and removing fat as much as possible

Dissecting caudae epididymides and transferred into 100 l PBS/PVA 1% Tear epididymis and using haemocytometer to detect sperms density

b. Collecting oocytes from ovary/oviduct Inject 1iu PMSH and hCG to a female mouse one day before collecting eggs Next day, Sacrifice the female mouse pad Separate ovary from fat pad finding uterus, oviduct,ovary and fat Tearing Detect

cut oviduct and pick up ovary

and collecting oocytes-cumulus (COCs) complexes under microscope density of oocytes/mouse

using a needle to tear ampullae containing

oocytes transfer COCs into new dish In vitro fertilization Transfer 10 l sperm into 500 l fertilization medium resuspense sperms in the incubate

fertilization medium transfer oocytes clutches into fertilization medium fertilization dish in 37oC, 5% CO2 for 4-6hrs

wash oocytes to remove access of

sperm using a capillary transfer pipette to transfer the oocytes from the IVF drop to one of the drops of the medium in the wash dish transfer all viable oocytes to a second drop distributing oocytes among the remaining drops of culture medium.


RESULT 1. Result

Figure IV.1.a: The egg was collected from the ovary

Figure IV.1.b: The sperms were collected from the caudae epidymides

Figure IV.1.c: Sperms were detected by haemocytometter Table 1a : The density of sperms counted by haemocytometter (Unit: cells) Square 1 164 Square 2 150 Square 3 170 Square 4 169 Square 5 145

Total number of sperms per 80 smallest spores (is equal to the sum from square 1 to 5): 798 cells Therefore, + The total number of cells per 10 l sperm: 399, 000 cells + Total number of cells per ml: 39, 900, 000 cells

Figure 1.c: The picture shows the In vitro fertilization In fact, after transferring sperm to the medium fertilization, we have to work following next steps as below for testing the survival of embryo: + incubate fertilization dish in 37oC, 5% CO2 for 4-6hrs + wash oocytes to remove access of sperm +Using a capillary transfer pipette to transfer the oocytes from the IVF drop to one of the drops of the medium in the wash dish + Transfer all viable oocytes to a second drop. + Distributing oocytes among the remaining drops of culture medium. V. However, the limited experimental time due to ignoring those above steps. DISCUSSION AND CONSLUSION

There are many elements affect to the fertilization rate. Firstly, that is the quantity of sperms. A normal adult mouse has from 63 to 71 million sperms per ml. However, the experimented mouse has about 39.9 million sperms per ml. There are two main reasons. The first one is the young of the mouse lead to the little amount of sperm. The second reason is the high environmental temperature resulted in reducing the significant number of sperm. Secondly, that is the quality of sperm. The sperm quality could be decrease because of the high temperature. After collecting the sperm from the caudae epidymides, we used to test the availability of sperm by using the microscope. However, the light of the

microscope emitted the temperature radiation. This high temperature caused the death of a large number of sperm before the fertilization occurred. Thirdly, the purity of equipments is also the factor affect to the quality of the embryo. Because of the limitation of dishes in culturing, so when we would like to use them again, we did not wash them as clean as possible. So, the remaining chemicals in dishes were degradable to other substances. That was the reason why affected to the quality of sperm. In conclusion, when couples have difficulty conceiving children on their own, they often turn to fertility treatments. And in vitro fertilization, is considered a process in which egg cells are removed from the woman s nody, fertilized with the man s sperm, grow into blastocysts in a Petri dish, and implanted in the lining of the woman s uterus. However, there are also containing many risk elements affect to the quality of embryo such as: temperature, skills, etc. In general, the strength of IVF is impossible to be denied.

VI. REFERENCES 1. Song Sieu Am Cao Tang. [Internet]. [Cited: Feb 10, 2011]. Available at: http://www.mediplantex.com/Noidung/Thong_tin_huu_ich/song_sieu_cao_tan..../ 2. Animal Health. [Internet]. [Cited: Feb 10, 2011]. Available at:

http://www.ilri.org/InfoServ/Webpub/fulldocs/LivProd/chapter2.htm 3. Hemocytometer. Internet]. [Cited: Feb 10, 2011]. Available at:

http://en.wikipedia.org/wiki/Hemocytometer 4. Parviz Tajik, Wei-Hua Wang, Kiyoshi Okuda, and Koji Niwa. (1994). Internet]. [Cited: Feb 10, 2011]. Available at: