EGG TRANSPLANTATIONBY

ADESEJI, WASIU ADEBAYO (B.Sc. Hons)

08/46KA006

ANA 816: ANIMAL BREEDING BIOTECHNOLOGY

DEPARTMENT OF ANATOMY,

UNIVERSITY OF ILORIN.

AUGUST, 2015.

Lecturer: DR G.O. OMOTOSO (PhD)

2 OUTLINE INTRODUCTION

OOCYTE RECOVERY

FERTILIZATION AND CULTURE

TRANSFER OF BLASTOCYST/EMBRYO TRANSPLANT

CONCLUSION

REFERENCES

3 Stages of Egg Transplantation

Egg transplantatio

n

Oocyte recovery

Fertilization and

culture

Embryo transplant

4Oocyte recovery

Process of oocyte collection before ovulation directly from the ovary.

Involves: Stimulation of ovary to produce numerous ova

Aspiration of the oocyte usually by ultrasound guided laparoscope

5 Fertilization and Culture After the eggs are retrieved, they

are examined in the laboratory for maturity and quality.

Mature eggs are placed in an IVF culture medium and transferred to an incubator to await fertilization by the sperm.

Sperm is separated from semen

Fertilization may be accomplished by insemination, where motile sperm are placed together with the oocytes and incubated overnight or by intracytoplasmic sperm injection (ICSI), where a single sperm is directly injected into each mature egg.

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COMPOSTIONS OF IVF CULTURE MEDIA

CaCl2

KC1

MgS04

NaCl

NaH2P04H20

Fe(N03)3-9H20

NaHCOj

Glucose

Amino acids

Arginine

Aspartic acid

Cystine

LysineMethioninePhenylalanineProlineSerineThreonineTryptophanTyrosineValineVitaminsBiotinCalcium pantothenateCholine ClFolie acidInositol

PABAPyridoxal HC1Pyridoxine HC1RiboflavinThiamineGlutamic acidGlutamineGlycineHistidineHydroxyprolineIsoleucineLeucine

8 EMBRYO TRANSPLANT

Embryo transfer takes place after eggs have been collected and fertilized in the laboratory.

Embryo transplant involves three process

Uterine preparation

Transplant proper

Follow up

9EMBRYO TRANSPLANT: Uterine preparation

In the human, the uterine lining (endometrium) needs to be appropriately prepared so that the embryo(s) can implant.

In a natural cycle the embryo transfer takes place in the luteal phase at a time where the lining is appropriately undeveloped in relation to the status of the present Luteinizing Hormone.

In a stimulated or a cycle where a "frozen" embryo is transferred, the recipient woman could be given first estrogen preparations (about 2 weeks), then a combination of oestrogen and progesterone so that the lining becomes receptive for the embryo.

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The time of receptivity is the implantation window. A scientific review in 2013 came to the conclusion that it is not possible to identify one method of endometrium preparation in frozen embryo transfer as being more effective than another (Groenewoud et al., 2013).

New evidences suggest better reception of the embryo if there is an imploratory reaction in the uterus prior to transfer (Nava et al., 2010).

Selection of embryos to be transferred

The embryo transfer procedure starts by placing a speculum in the vagina to visualize the cervix, which is cleansed with saline solution or culture media.

EMBRYO TRANSPLANT: Uterine preparation

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EMBRYO TRANSPLANT: Procedure

A fine tube (catheter) loaded with embryos is passed through the cervix, normally using ultrasound guidance. The embryos are passed down the tube into the uterine cavity.

After insertion of the catheter, the contents are expelled and the embryos are deposited.

The subject lie supine for few minutes after the transfer.

About two weeks after the embryo transfer, you will be given a pregnancy blood test. If it is positive, you will have a scan about two weeks later.

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14EMBRYO TRANSPLANT: Follow up

About two weeks after the embryo transfer, a pregnancy blood test is performed.

If it is positive, a scan will be done about two weeks later.

If pregnancy does not occur, the results at each stage of cycle will be evaluated such as response to the medications, the number and condition of eggs retrieved, whether they fertilized, and how the embryos grew in culture.

15IN ANIMALS

OOCYTE RECOVERY - COW

Laparoscopy. Food and water are withheld for 12-18 h before laparoscopy.

Laparoscopies are performed 24-25 h after hCG injection or estimated onset time of oestrus to obtain follicular oocytes as near as possible to ovulation. A recuperation time of 36 days (time for one natural oestrus) was allowed between two laparoscopies on the same animal.

Oocyte treatment - COW

Follicular oocytes are allowed to mature for 4—6 h in Brackett's defined medium before being transferred to the fertilization drops (0-2 ml defined medium under paraffin oil: Saybolt viscosity 125/135; Fisher Scientific, Fair Lawn, NJ, U.S.A.) for 12-20 h.

Sirard et al., 1985

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SEMEN RECOVERY - COW

Semen treatment. Fresh ejaculates were obtained from one single bull using an artificial vagina.

Undiluted semen samples are first incubated for 2-3 h at 34°C with penicillin-G and streptomycin (Flow Laboratories, Virginia, U.S.A.) at a final concentration of 100 i.u./ml and 100µg/ml, respectively.

After this incubation, 0-2 ml semen is diluted with 4-8 ml of high ionic-strength medium (Brackett, Evans, Donawick, Boice & Cofone, 1980), incubated for 5 min at 37°C, and then centrifuged for 5 min at 250g, (37 °C). Supernatants are removed and spermatozoa are resuspended in 0-8 ml defined medium (Brackett & Oliphant, 1975) for an incubation of 3-18 h under an atmosphere of 5% C02, 8% 02and 87% nitrogen (3 IO7 —9 IO7 spermatozoa/ml).

Spermatozoa are introduced in the fertilization drops at a final concentration of 1 106 cells/ml

Sirard et al., 1985

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EMBRYO TRANSFER- RABBIT

Female rabbits are induced to ovulate by an hCG injection (75 i.u. A.P.L.: Ayerst Laboratories) and 24-36 h later they are anaesthetized with pentobarbitone sodium (Nembutal, 50mg/ml: Abbott Laboratories, Montréal, Canada, H4P 1A5).

A midventral approach is used to expose the oviducts. First, the ovaries are checked for the presence of newly formed corpora lutea and then the oviducts are ligated at about 1 cm from the uterus and the embryos are injected inside (2-3 cm) via the ampulla, using a slightly curved fire-polished pipette.

After completion of the surgical procedure, the animals are individually caged for a mean time of 99 ± 5 h.

Sirard et al., 1985

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EMBRYO TRANSFER- COW

2-8-cell embryos are transferred to the oviduct of cows, using a standard paralumbar surgical approach. Surgery is done under local anaesthesia and embryos transferred to the oviduct ipsilateral to the corpus luteum. Synchronization was obtained by injection of 500 µg cloprostenol 24 h before the injection given to the donor.

Pregnancies can be confirmed at 50 days by rectal palpation.

Sirard et al., 1985

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REFERENCES

Groenewoud, E. R.; Cantineau, A. E. P.; Kollen, B. J.; MacKlon, N. S.; Cohlen, B. J. (2013). "What is the optimal means of preparing the endometrium in frozen-thawed embryo transfer cycles? A systematic review and meta-analysis". Human Reproduction Update 19 (5): 458–470.

Assisted Reproductive Technologies: A Guide for Patients (2011). AMERICAN SOCIETY FOR REPRODUCTIVE MEDICINE

Nava Dekel, Yulia Gnainsky, Irit Granot, and Gil Mor. (2010). Inflammation Implantation. Am J Reprod Immunol 63(1): 17-21

M. A. Sirard, R. D. Lambert, D. P. Menard, M. Bedoya. (1985). Pregnancies after in-vitro fertilization of cow follicular oocytes, their incubation in rabbit oviduct and their transfer to the cow uterus. J. Reprod. Fert. 75, 551-556

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