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Introduction/ Literature review:

Reproduction is the backbone of animal production. The possibility of enhancing reproductive potentials through the use of certain biotechnological techniques has recently been explored by different researchers (Da Ros, Maldera et al., 2008; George and Doe, 1989; Habsah, 2005; Wan Khadijah, 2008). Cryopreservation is another important development facilitating conservation of valuable animal genetic, this captured the attention of many researchers(Kaneko, Yamamura et al., 2006; Wan Khadijah, 2008). Several proteins such as OTP-1, bTP-1 and Interferon secreted by the developing embryo have been identified and purified, these substances were used as fertility promoters and markers of conception in different animal and human research activities(Srivistava A.K, 2005).

It is now possible to reduce the generation interval by producing more number of offspring from a female within a short period of time using IVF and embryo transfer technology. Recent development in Öocytes maturation and invitro fertilization in farm animals have given a new dimension to animal production. Using such techniques it is possible to produce a large number of embryos in the laboratory. Such embryos have a large demand in upstream areas of research in embryo biotechnology. Brackett et al. (1982) produced the first IVF calf using ovulated Öocytes, while Lu et al. (1987) reported birth of calf from total in vitro procedures ( in vitro maturation, fertilization development and culture of embryos). These reports generated worldwide interest in science of invitro fertilization in livestock.

In IVF matured Öocytes are coincubated with in vitro capacitated spermatozoa, which results in fertilization and formation of zygote. The time of sperm-egg interaction, medium utilized, temperature and sperm co-incubation play important role in fertilization, and was studied by many researchers(Brackett, 1982; George and Doe, 1989; Habsah, 2005; Heyers, Sousa et al., 2000; Kaneko et al., 2006; Koshimoto, Gamliel et al., 2000; Miao, Shi et al., 2007; Ozgunen, Erdogan et al., 2001; Zavos, Correa et al., 1994). It was observed that 3 hours after the release of first polar body is the best time for coincubation. Early as well as delayed incubation leads to destruction of Öocytes, failure of pronuclei fusion and cleavage(Srivistava A.K, 2005).

Major steps involve in invitro fertilization are collection of Öocytes, in vitro maturation of Öocytes, in vitro capacitation of spermatozoa, in vitro fertilization and invitro embroygenic development.

Success of IVF in any species depends primarily on recovery of good quality Öocytes. Ovulated Öocytes can be collected by laparoscopy; in vivo collection of Öocytes has been expensive time consuming and not practically possible (Srivistava A.K, 2005). Ovaries obtained from slaughter house may be an alternative, easy and cost effective method for collection Öocytes; however, in Malaysia, this may not be applicable, as most of the mature and reproductive female animals

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are kept in the farm for production, those taken to slaughter house are either too old not in their prime reproductive age or are too young for their reproductive system to produce the required Öocytes to harvested. Once taken from the slaughter house ovaries are brought in normal saline at 35oC-38oC and Öocytes are harvested immediately. The ovaries can be kept at 20-24oC for 8-10 hours after the slaughter without the reduction in fertilizing ability of Öocytes and their subsequent development(Srivistava A.K, 2005).

Another issue beside Öocytes recovery is sperm capacitation, which is the process by which spermatozoa undergo certain morphological and biochemical changes before taking part in the process of fertilization. Capacitation occurs as a result of decreased negative surface charges, loss of membrane cholesterol and an influx of calcium between the plasma and outer acrosomal membrane. In this practical study capacitation was done by incubating the collected sperm in TYH/BSA medium in 5% CO2 incubator for 1 hour at 37oC

Embryo Transfer(ET) is one of the techniques of reproductive biotechnology where embryos flushed from donor females are transferred to the recipients, which serve as foster mother for their development throughout the period of pregnancy. W. Heape in 1890 made the first successful embryo transfer in rabbit(Srivistava A.K, 2005). However systematic research on embryo transfer wasn’t started till after the birth of Wilmut’s calf; first calf embryo transfer out of frozen embryo(Rowson, 1973). The production of embryos can be increase manifold through superovulation, not only does it influence the embryo production, but it help to exploits the use of germ pool of ovarian reserve to the maximum level. Hormones like

Pregnant mare’s serum gonadotrophin (PMSG), human chorionic gonadotropin (hCG), human menopausal gonadotropin (hMG), follicle stimulating hormone (FSH) and equine chronic gonadotrophin (eCG) are among the most commonly used superovulatory hormones.

In general, the technology of Artificial insemination has the potential application in conservation of germplasm line, disease control and salvaging the reproductive problems.

Objective:

The aim of this practical study was:

To be able to demonstrate and apply what we have learnt in class lecture in techniques of IVF

To learn how to use the equipment in dissecting both male and female mouse and recover the spermatozoa and Oocytes respectively.

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To observe and learn the different reproductive tract of the mouse putting emphasis on their size and appearance when ovulated or the regions that have the sperm and their position for applying the knowledge in future IVF work.

To learn the techniques employed in storing the recovered sperm and Oocytes using different invitro culture media and the techniques of using CO2 incubator

To learn how to make invitro fertilization and incubate the fertilized oocytes using the CO2 incubator.

To learn the technique employed in embryo recovery from copulated female mouse To observe any short comings during the process and make possible suggestions.

Materials and Methods:

Animal Samples: laboratory Male mouse and Superovulated female mice (12 hours postovulation) sacrificed humanely

Medium:

TYH (Toyodo Yokoyama Hoshi) medium , WM (Whitten Medium), HWM (Hepes buffered Whitten medium),BSA (Bovine serum Albumen)

Equipment:

4”, 5” forceps, 4” dissecting scissors, 6” dissecting scissors, dissecting board, aluminum foil, gauze, 4-well culture dish, glass micropipette, hand gloves, stereomicroscope

Reagents:

70% ethanol

Methodology:

1.0 Sperm Recovery:

We were provided by a matured male mouse that spent some period isolated from females . We killed the animal by cervical dislocation using edge of a rubber cup in the laboratory sink. The cup-edge was placed at the base of the skull, little pressure was applied while twisting the edge on the neck and pulling the tail backward, at start the mouse was struggling, then after about two minutes we observed the mouse to be like in a quite sleep, by then noticed that it has been killed.

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We laid the mouse on its back, we wiped the entire abdominal region with gauze that was soaked in 70% ethanol, to disinfect the abdominal skin and reduce the risk of contamination of the dissection with the mice hair. We used forceps to held and 6” dissecting scissors to pinched up the skin at the lower abdominal cavity of the mouse, we then used the forceps to hold the skin from above and below; the skin on the top of the seizure was pulled toward the head region and that at the bottom of the seizure was pulled backward toward the lateral side of the mouse opposite thighs, this exposed the abdomen completely, while the fur was left away from the exposed abdomen.

Using the forceps and 4” dissecting scissors, we cut the peritoneum wall, the reproductive and digestive tracts of the mouse were exposed. We then pushed the intestinal tract away and located the epididymis, which look like spiral narrow-tube. We located the Caudae epididymis we pinched it with a sterile needle and the sperm was collected into equilibrated TYH+BSA medium in 4-well culture dish that contained 400µl TYH medium (100µl per each well)overlaid with silicon oil, this medium serves as a holding as well as suspension medium. We observed the sperm under stereomicroscope moving in the suspension like small tadpoles in a pond.

We used sony ericsson xperia camera to captured the image.

The 4-well culture dish and its content were preincubated in 5% CO 2 incubator at 37oC for 1 hour for the sperm to be capacitated while attend to the Öocytes to be recovered.

Figure I: enhanced Image of Recovered sperm from caudae epididymis of male mouse using stereomicroscope at 40X

Of a dissected mouse

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2.0 Öocytes Recovery:

We are provided with a superovulated female mouse at 12 hours post-ovulation; the female

was stimulated to superovulation by injection 10iu Pregnant mare’s serum gonadotrophin (PMSG) at day 1, followed by 10iu injection of human chorionic gonadotropin (hCG) at day 3, it was then kept away from male partners for successful development of ripe oocytes without been fertilized.

We killed the mouse using cervical dislocation as mentioned in item 1.0 above. After the mouse was killed, we laid the animal on its back and wiped its abdominal region with gauze that was soaked in 70% ethanol, to disinfect the abdominal skin and reduce the risk of contamination of the dissection with the mice hair. We used forceps to held and 6” dissecting scissors to pinched up the skin at the lower abdominal cavity of the mouse, we then used the forceps to hold the skin from above and below; the skin on the top of the seizure was pulled toward the head region and that at the bottom of the seizure was pulled backward toward the lateral side of the mouse opposite thighs, this exposed the abdomen completely, while the fur was left away from the exposed abdomen.

We used the watchmaker’s forceps and 4” dissecting scissors; we cut the peritoneum wall, the reproductive and digestive tracts of the mouse was exposed. We pushed the coiled intestine away and we located the horns of the uterus, we used the forceps to grasped the upper end of each of the uterine horns we gently pulled it out up to the position that the mesometrium membrane was revealed. We carefully removed the paragenital fats away, we observed the uterus, oviduct and the ovaries of the both sides to look like a string with a head knot (ovary) at the upper region and a small inter node between (oviduct) below it continuous the long string (uterus). We pulled the oviduct and the ovary while we removed the fat pad taut; we used the forceps to held the uterine region and placed the fine dissecting scissors at the margin between the ovary and the oviduct and we cut the oviduct, we then carefully repositioned the forceps to held the oviduct, we placed the fine dissecting scissors in between the oviduct and the uterus but a little ahead in the uterine region we cut the oviduct free.

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We immediately transferred each oviduct and the attached uterine segment into 35mm petri dish containing 100µl TYH medium overlaid with silicon oil (30µl of TYH overlaid with silicon oil, followed by 70µl TYH medium overlaid with another silicon oil on top) at room temperature, this served as the holding as well as incubation medium. We used 26G needle and a forceps to hold and pinched the cumulus Öocytes complex there by releasing the Öocytes into the medium. We observed the Öocytes under 40X magnification of stereomicroscope, we used sony ericsson xperia camera to capture the image as shown below:

Figure II: pictures of Oviduct dissection in superovulated female

uterus

Figure III: enhanced 40X Stereomicroscope image of Mouse Oocyte recovered from superovulated female mouse (12 hours post ovulation)

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3.0 Insemination of the Öocytes:

Immediately, after Öocytes were recovered, the 4-well culture dish that contained the capacitated sperms was taken from the CO2 incubator; 10µl of the sperm culture was taken using micro pipette and ejected into the petri dish containing the recovered Öocytes, the petri dish and its contents was then incubated in a CO2 incubator for 6 hours.

The petri dish was removed from the incubator after six hours. We used glass hand pipette to wash the fertilized Öocytes by 3-times HWM medium serial wash followed by 2-times WM medium serial wash to remove the sperm; before been transferred to fresh 100µl WM culture medium containing silicon oil.

Figure IV: Serial washing of IVF oocytes using HWM+BSA and WM+BSA media

The dish with the invitro culture medium of WM+ BSA was incubated in 5% CO 2 incubator at 37oC for 24 hours after which the first cleavage was viewed using 40X stereomicroscope.

It is worth mentioning here that why do we used WM medium to culture not HWM, the reason could probably be due the presence of toxic material in the Hepes buffered Whitten medium which when heated by incubation kill the embryo.

4.0 Embryo Flushing:

A female mouse that was copulated by first stimulated through hormone manipulation by

injection 10iu Pregnant mare’s serum gonadotropin (PMSG) at day 1, followed by 10iu

WM media wash

Transferred to WM + BSA culture mediumTo HWM

medium wash

TYH culture IVF oocytes

HWM medium

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injection of human chorionic gonadotropin (hCG) at day 3, it was then allowed to mate a male partner to effect copulation. After 5 days post mating period the mouse was killed by cervical dislocation as mentioned in item 1.0 above. We laid the animal on its back, we used gauze soaked in 70% ethanol to disinfect the abdominal skin and reduce the risk of contamination of the dissection with the mice hair. We used forceps to held and 6” dissecting scissors to pinched up the skin at the lower abdominal cavity of the mouse, we then used the forceps to hold the skin from above and below; the skin on the top of the seizure was pulled toward the head region and that at the bottom of the seizure was pulled backward toward the lateral side of the mouse opposite thighs, this exposed the abdomen completely, while the fur was left away from the exposed abdomen.

We used the watchmaker’s forceps and 4” dissecting scissors; we cut the peritoneum wall, the reproductive and digestive tracts of the mouse were exposed. We pushed the coiled intestine away and we located the horns of the uterus, we used the forceps to grasp the upper end of each of the uterine horns we gently pulled it out up to the position that the mesometrium membrane was revealed. We carefully removed the paragenital fats away, we observed the uterus, oviduct and the ovaries of the both sides to look like a string with a head knot (ovary) at the upper region and a small inter node between (oviduct) below it continuous the long string (uterus). We pulled the oviduct and the ovary while we removed the fat pad taut; we used the forceps to held the uterine region and placed the fine dissecting scissors at the margin between the ovary and the oviduct and we cut the oviduct, we then carefully repositioned the forceps to held the oviduct, we placed the fine dissecting scissors in between the oviduct and the uterus but a little ahead in the uterine region we cut the oviduct free.

Using the forceps we transferred the oviduct into 35mm petri dish that contained 100µl HWM+BSA medium as the holding medium at room temperature before the infundibulum was located.

The petri dish was placed on the stereomicroscope stage, viewing at 40X magnification we located the infundibulum, which look like a funnel shape having a mouth opening. We inserted flushing syringe needle that contained 0.1ml HWM medium via the opening of the infundibulum and flushed out the embryo into the medium, we were able to flush out a 2-cell embryo into the HWM+ BSA medium.

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Glass micro pipette was used to transferred the embryo into a washing medium that contained HWM+BSA and WM+ BSA medium and was subjected to wash as shown in figure IV above, this was done to get rid of the debris and other ovarian cells around the embryo. The embryo was then transferred to culture medium containing WM+BSA covered with silicon oil and incubated in 5% CO2 incubator at 37oC.

Result and Discussion:

Invitro fertilization has been a technique employed in animal transgenesis as reported by many literature(Habsah, 2005; Lu, 1987; Miao et al., 2007; Srivistava A.K, 2005), several research papers published detailed procedure for this technique, following the procedure documented in the IVF protocols, dissection techniques was used to collect the Sperm from a Male mouse, during the cause of dissection and collection we noticed that the appearance and position of caudae epididymis and the sperm content of the caudae epididymis is similar to what was reported; however, though the sperm was collected but due to time constraints we were un able to use haemocytometer to measure the sperm concentration. Instead we used the recovered sperm directly without measuring its concentration.

We observed in female oocytes recovery, due to the presence of matured oocytes the ampulla is much enlarged and was easily spotted under 40X magnification of the stereomicroscope. After we tear the oviduct with 2G needle we observed a cumulus mass that consist of some

Figure V: 2-cell mouse embryo as observed using 40X stereomicroscope; picture was taken using sony ericsson xperia camera

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oocytes surrounded by the cumulus cells. The oocytes were clearly visible using the stereomicroscope as depicted in figure III above.

During embryo flushing we expect to get an embryo that is 1 to 4 cells since we are using oviduct to flush the embryo, and to our expectation the embryo was at 2-cell stage embryo.

Therefore, in this study we were able to learn the techniques of sperm recovery, oocyte recover, IVF and embryo flushing, we learnt the hands-on procedure which I believe will not be something new to me in future.

References

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Brackett, B.G., Bousaquet, D., Boice N.L., Dona, W.,W.,J., Evans, J., F. and Dressel, M.,A. (1982) Normal development following invitro fertilisation in the Cow. Biol. Reprod., 27, 147-158.

Da Ros, V.G., Maldera, J.A., Willis, W.D., Cohen, D.J., Goulding, E.H., Gelman, D.M., Rubinstein, M., Eddy, E.M., and Cuasnicu, P.S. (2008) Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1). Developmental Biology, 320(1), 12-18.

George, M.A., and Doe, B.G. (1989) The influence of handling procedures during mouse oocyte and embryo recovery on viability and subsequent development in vitro. Journal of Assisted Reproduction and Genetics, 6(2), 69-72.

Habsah, B., W. E. Khadijah, R. B. Abdullah and K. Musaddin. (2005) IVF performance of low quality oocytes harvested from Malaysian cattle. Malaysian Journal of Animal Science, 10(2), 95-102.

Heyers, S., Sousa, M., Cangir, O., Schmoll, F., Schellander, K., van der Ven, H., and Montag, M. (2000) Activation of mouse oocytes requires multiple sperm factors but not sperm PLC[gamma]1. Molecular and Cellular Endocrinology, 166(1), 51-57.

Kaneko, T., Yamamura, A., Ide, Y., Ogi, M., Yanagita, T., and Nakagata, N. (2006) Long-term cryopreservation of mouse sperm. Theriogenology, 66(5), 1098-1101.

Koshimoto, C., Gamliel, E., and Mazur, P. (2000) Effect of Osmolality and Oxygen Tension on the Survival of Mouse Sperm Frozen to Various Temperatures in Various Concentrations of Glycerol and Raffinose. Cryobiology, 41(3), 204-231.

Lu, K., H., Gordon, S., Gallagher, M. and McGovern, H. (1987) Pregnancy established in Cattle by the Transfer of embryos derived from invitro fertilisation of oocytes Mature in vitro. Vet. Rec., 121, 259-260.

Miao, Y.-L., Shi, L.-H., Lei, Z.-L., Huang, J.-C., Yang, J.-W., OuYang, Y.-C., Sun, Q.-Y., and Chen, D.-Y. (2007) Effects of caffeine on in vivo and in vitro oocyte maturation in mice. Theriogenology, 68(4), 640-645.

Ozgunen, K.T., Erdogan, S., Mazmanoglu, N., Pamuk, I., Logoglu, G., and Ozgunen, T. (2001) Effect of gonadotrophin dose on oocyte retrieval in superovulated BALB/c mice. Theriogenology, 56(3), 435-445.

Rowson, W.a. (1973) Experiments on the low temperature preservation of cow embryos. Vet. Rec., 92, 686-690.

Srivistava A.K, S.R.K., Yadav M.P. (2005) Animal Biotechnology. New Delhi: Oxford and IBH.Wan Khadijah, W.E., K. Asmad and R.B. Abdullah. (2008) Cryoperservation of sperm in red

tilapia (Oreochromis niloticus). Catrina, 3(1), 27-30.Zavos, P.M., Correa, J.R., and Zarmakoupis, P.N. (1994) Improvements and short-term viability

of mouse epididymal spermatozoa recovered through the Spermprep(TM) filtration method. Theriogenology, 42(6), 1035-1042.