ANIMAL

LESSON 11: IN VITRO FERTILIZATION AND EMBRYO TRANSFER

BIOTECHNO LOGY

Dear students, In last class we had been discussed about vaccines, interferons, monoclonal antibodies that theory it was given knowledge of immunization .today our class discussion on invitro fertilization and gene transfer, this technique manly used in hospitals in gynecology, those suffer with infertility that can be recovered by using this technique.

animals for a rapid multiplication of desirable genotypes of animals, and in cases of infertility of certain types in humans.

Objectives

Introduction to fertilization, Discussing about fertilization techniques, the invitro

and

How the egg transformation transplantation technique.

Definition
When the union between egg cell and sperm occurs outside the body in a culture vessel, it is known as in vitro fertilization. This involves collection of healthy ova and sperms from healthy females and males, respectively, and their fusion under appropriate conditions in vitro. In vitro fertilization and embryo transfer (IVF-ET) is the most basic Assisted Reproduction Technique and is designed to enhance the likelihood of conception in couples in whom other methods of fertility have been tried and are unsuccessful. IVF-ET involves multiple steps leading to insemination and fertilization of eggs in the laboratory. The embryos thus created are placed into the uterus for implantation. Each stage of the procedure has to be carefully managed and has its own specific risks.

Risks of Therapy
More chances of multiple pregnancy In IVF, ovaries are stimulated to produce several eggs rather than a single egg as in a natural cycle. Multiple eggs and multiple embryos, increase the number of embryos available for transfer and ultimately, the probability of multiple conception. The resulting zygotes may be cultured in vitro for a period of time to obtain young embryos, which ultimately are implanted in the uterus of healthy females to complete their development. The implementation of young embryos developed in vitro or obtained from the uterus of different donor females into the womb of selected females is termed as embryo transplantation. The techniques of in vitro fertilization and embryo transfer are being applied to

n Humans I n V it r o F e Collection of Oocytes r Oocytes for in vitro fertilization are, as far as possible, ti collected from the females desirous of having baby. li This presents no problem in cases (1) where the females z have normal functional ovaries but defective, damaged a ti or blocked fallopian tubes; as a result the oocytes o released from the ovary are unable to migrate to the n uterus causing infertility (tubal infertility). But when i sterility is due to the absence of or nonfunctional
ovaries, the oocytes have to be collected from donor

In vitro fertilization, coupled with embryo transfer, in humans is aimed to enable couples suffering from certain types of sterility to have children of their own. The different steps involved in the technique are: (/) collection of oocytes, (//) collection of sperms, (Hi) in vitro fertilization of the oocytes and (iv) implantation of the resulting zygotes in the uterus of would be mother.

females. In any case, the females who serve as donors of oocytes are admitted at the proper time for oocyte recovery, which may be done either during their natural or induced ovulation cycles.

Massaging the ovary to remove blood

Slashing of ovaries to recover oocytes. In panel A, a hemostat is attached to the base of the ovary to hold the ovary firmly in place. The excess tissue from the ovarian stalk is removed in

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panels B. Panels B and C shows how the ovary is held above the beaker and 2 3 mm incisions made in a a downward direction with a rapid but firm movement across follicles.

1.

Ovulation may be stimulated by the administration of clomiphene and/or human menopausal gonadotrophin (HMG). Human chronic gonadotrophin (hCG) is also administered to prevent the inhibition of LH surge by HMG.This approach yields several oocytes from a female which increases the chances of success of in vitro fertilization. However, it creates an abnormal environment

Harvesting of oocytes from slashed ovaries. In the left panel, the slashed ovary is being swirled in OCM. In the right panel, the ovary is being pressed against the side of the beaker to allow drainage of OCM.

Some instruments used to pick up embryos and oocytes. From left to right are 1) a 1 cc syringe with an extension of rubber tubing connected to a Unopette, 2) a wiretrol (from Drummond Scientific; we purchase from Fisher), 3) the same device as #1 without the rubber tubing extension and 4) a 5 ml Drummond Microdispensor The time of natural ovulation is determined by monitoring the rise in the level of luteinising hormone. (LH) either in urine or in blood, and the ova are recovered. This approach yields only one ovum per female/cycle.

f o r p r e g n a n c y i n t h e t r e a t e d f e m a l e s , a n d t h e r e i s a n i n c r e a s e d r i s

k of abnormal pregnancy. 2. Follicle development can be arrested at the optimum stage by administration of hCG so that the ova are not released. The oocytes can be collected by laparoscopy at a convenient time. But the chief difficulty of this approach is the determination of optimum time for hCG administration. A prolonged administration of clomiphene for stimulation of ovulation increases the chances of abortion in the treated females, while hMG treatment increases the cost. Therefore, often a combination of clomiphene and hMG is used. But hMG also produces some side effects like abdominal discom-fort, pleura! effusion etc. When hCG is to be administered, it is done when the follicles have reached the preovulation size of about 1.9 cm; this may be confirmed with the help of ultrasound imaging.
It is critical to determine the time of ovulation precisely particularly where laproscopy is to be done for oocyte recovery. If laproscopy is done after ovulation, no oocyte can be recov-ered, while if it is done 6 hr or more prior to ovulation, fertilization may not occur unless oocytes are incubated under specified conditions to allow them to mature. The time of ovulation is estimated on the basis of temperature chart of the donor female, change in cervical mucous score (which is related to follicle development), oestrogen level in blood or urine, level of luteinising hormone (LH) in blood or urine and determina-tion of the follicel size using ultrasound imaging. Plasma progesterone levels are also monitored since they begin to increase 24 hr prior to LH surge in stimulated ovulation cycles.

containing the oocyte and the necessary surgical manipulation of ovary sensors, laproscopic scissors and an aspirating apparatus inserted into the abdomen of the female via a suitable tube. This procedure involves minimum surgical intervention, causes very little damage to the ovary and is quite convenient.

ANIMAL BIOTECHNOLO GY

Collection of Sperms

Recovery of oocytes is the most convenient and efficient by a laproscopic equipment which permits the visualization of ovary through a monitor, aspiration (suction) of the follicular fluid
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The semen is collected about 60-90 minutes prior to fertilization, liquefied and centrifuged. The sperm pellet so formed is resuspended in culture medium, and incubated for 30-60 min at

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ANIMAL BIOTECHNOLO GY

37C. A sample of spermatozoa is taken from the surface of the medium for fertilization since the most active sperms are located there; The semen is collected from the prospective father. If this is not possible either due to a low sperm count of the semen (oligospermia) or due to a lack of motile sperms (azoospermia) semen may be collected from a suitable donor.

desired by many prospec-tive mothers. The embryo is transferred into uterus through

In Vitro Fertilization

Oocytes are identified by microscopic examination of the follicular fluid aspirated during laproscopy, and are incubated for 5-10 hrs depending upon the expected time of their maturation. One of the following media may be used for oocyte incubation, in vitro fertilization and the subsequent culture of the zygote: (i) modification of Hams F10 medium, (ii) Earls solution, (Hi) modified Whittens medium, and (iv) Whittinghams T6 medium. Fertilization is effected by adding 10,000-50,000 motile sperms to about 100 |al to 1 ml of culture medium in which the oocyte is being incubated. The oocyte is examined after 12-13 hours for the detection of following: (i) number of pronuclei, and polar bodies, (if) granulation of the oocyte and (Hi) the shape of oocyte. A normally fertilized oocyte (actually, now a zygote) contains two pronuclei and two polar bodies. Any departure from this and the presence of granulation and abnormalities in shape are indicative of abnormalities in the zygotes, and such zygotes are rejected. The first division in the zygote occurs about 24-30 hours after insemination, but each subsequent division takes about 10-12 hours. Therefore, if an oocyte fails to divide by about 30 hrs after insemination, it should not be used for implantation. 3.1.4. Embryo Transfer. In natural situations, fertilization and a limited embryo development (up to 8-16 cell stage) occurs in the fallopian tube. After this, the embryo migrates into uterus and establishes itself. In vitro fertilized embryos of 1-16 cells have been successfully transferred into uterus, but the best stage is perhaps the 2-4 cell stage. A prolonged in vitro culture of embryos reduces their survival rate, while younger embryos are less likely to survive in the uterine environment. Transfer of multiple embryos into a single uterus increases the chances of success, but it may lead to multiple pregnancy which may not be

c e r v ic al c a n al w it h t h e h el p o f a T e fl o n c at h et e r o

f specified dimensions. A small amount (about 10 LI!) of the culture medium is also transferred along with the embryo. The embryo transfer operations must be performed with extreme care to avoid fallopian tube pregnancy and the expulsion of transferred embryo from the uterus. It is necessary that the female receiving the embryo must be in the correct stage of her menstrual cycle so that her uterine environment is properly attuned to receive the embryo. In case the uterus of the female desiring to have the baby is either nonfunctional or absent, a surrogate mother may be used for the embryo transfer. The babies produced using this approach are popularly known as test tube babies. The first test tube baby was born on July 25, 1978 and was named as Loise Joy Brown. This technique is being widely used in many countries to provide the joy of having their own babies to couples suffering rom infertility. However, many ethical and social issues related to this development may need Resolution since the technique may be misused by unscrupulous elements.

Embryo Transfer in Cattle

Young embryos of cattle of superior genotype are collected r rior to their implantation in uterus, and are implanted in the uterus of other females of inferior genotype where

They complete development; this is called embryo transfer. The chief objective of embryo transfer is to obtain several progeny

per year from a single female of superior genotype. In a country like India, most cattle are of inferior genotype with rather low productivity, while superior genotype females are limited in number and of high price. Therefore, a programme of artificial insemination (AI) was widely used in an effort to improve our cattle breeds. In AI semen collected from superior males is suitably diluted, stored frozen in liquid nitrogen (-196C) if needed, and is used to inseminate females of inferior genotype when they are in heat. A limitation of AI is that superior genes (50%) are contributed by only the male side, while the female contributes (50%) inferior genes. In contrast, in embryo transfer technique the inferior females used as surrogate or substitute mothers do not contribute any genes to the progeny; they only serve as extremely sophisticated natural incubators for the normal development of young embryos which were obtained by mating between selected superior genotype males and females. As a result, the progeny obtained by embryo transfer are of superior genotype and are not limited by the genes contributed by inferior females as is the case in AI. The technology of embryo transfer in cattle may be briefly and simply summarised as follows.

3.

4.

The donor females are treated with appropriate doses 5. of the selected gonadotrophin, e.g., follicle stimulating hormone (FSH) or luteinising hormone (LH), to increase the number ova released at the time of ovulation; this is called super ovulation. Under optimum treatment conditions, a single female can provide up to 15 embryos in a single cycle. Super ovulation is ; induced in mares by equine pituitary extract, while antisera to steroids and gonadotrophins have similar effects in sheep. The chief objective of super ovulation is to greatly increase the number of embryos recovered per female in a single cycle. When the donor female is in heat (oestrus), it is artificially inseminated using semen from a genetically superior bull of top pedigree.

1. 2.

A genetically superior and high productivity female serves as the donor of embryos to be transferred. Healthy, young females of inferior genotype are selected to be the recipients of embryos to be transferred; these females are called surrogate or substitute mothers.

Th e fer tili ze d eg gs/ yo un g em br yo s are col lec ted by flu shi ng the ute rus of su per ior do no r fe ma les wit ha sp eci al nut rie nt sol uti on; thi s is do ne 7 da ys aft er the ins em

ination. The embryos are examined under a stereoscopic microscope and normal looking healthy embryos are selected. 6. The selected embryos are incubated in a special nutrient medium at 37C till their transfer into the surrogate mothers. Alternatively, they may be frozen and stored in liquid nitrogen for future use. 7. A single embryo is transferred into the uterus of each surrogate mother. It s important that the oestrus cycles of donor and surrogate mothers are Synchronized by administering prostaglandins. This is essential to provide the optimum uterine environment for survival, establishment and normal development of the young embryos transferred into the surrogate mothers.

ANIMAL BIOTECHNOLOGY

1.
A N I M A L

Applications of Embryo Transfer Technology

The embryo transfer technology offers several advantages which are manifest in its varied applications; these are briefly enumer-ated below. 1. This technology achieves a surprisingly rapid rate of multiplication of animals of the selected superior genotype. In natural course, a single female will produce a single progeny in about one year. But using superovulation and embryo transfer technology, it is feasible to collect around 36 embryos from one female in one year. A single female can be induced to ovulate, on an average, 6 times a year, and in each ovulation cycle about 6 healthy transplantable embryos are obtained. Assuming an average success rate of 50% in the embryo transfer, an average of 18 progeny can be derived from one superior female in one year. 2. Each young embryo can be split into 2-4 parts, each of which would develop into a separate progeny; this is called embryo splitting. By combining embryo splitting with superovulation and embryo transfer techniques, the rate of multiplication can be further increased. 3. The young embryos can be frozen and stored in liquid nitrogen for up to 10 years or more and used at a subsequent date. The frozen embryos are far more easier to transport, and present negligible quarantine problems as compared to the animals themselves. Freezing and storage of young embryos in liquid nitrogen (at -196C) is known as cryopreservation. The embryos are first treated with a suitable concentration of a compound like glycerol which protects them from injury during freezing and thawing; such compounds are called cryoprotectants. They are then cooled at a slow rate to -38C, ordinarily employing a programmable controlled rate freezer. These embryos are then plunged into liquid nitrogen and stored at -196C. The embryos are thawed at a very rapid rate by immersing the ampoule carrying them into a water bath maintained at 0C. 4. Superior course that are unfit to carry the foetus for full term can serve as donors of the young embryos.

2.

Limitations
The embryo transfer technology has some important limitations as follows. 3.

A hig h deg ree of exp erti se is req uir ed for an effi cie nt and suc ces sful ope rati on. Th e cos t of pro duc ing eac h pro gen y is sev eral fol d hig her tha n that fro m n at u ra l e v e nt s. Th

e used as donors of young embryos. do is therefore necessary to evaluate the gains from and the It nor of the technology before employing it on a commercial costs fe scale. The gains will be attractive in such cases where a ma relatively rare superior genotype is rapidly multiplied and les made available on a commercial scale. are Current Research Work on Animal Tissue re Culture mo ve d Students now we knew about animal tissue culture but we need practical oriented research work whats going in world, fro Hematoprotection and enrichment of transduced m pro cells in vivo after gene transfer of MGMTP140K du into hematopoietic stem cells cti Description: nature.com about npg nature on science update naturejobs for natureevents......CONTEXT: the ...Hematoprotection and enrichment of transduced cells in per vivo after gene transfer of MGMTP140K into iod hematopoietic stem cells Michael Jansen1, Ursula R the Sorg1, Susanne Ragg2, Michael Flasshove1, Siegfried y Seeber1, David A......http:// are www.nature.com/cgt/journal/v9/n9/full/7700490a.html

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