In Vitro Fertilization - Anatomy & Physiology

Oocyte Collection

• Donor is superovulated to provide large numbers of mature follicles.
• Pre-ovulatory follicles are collected by a non-surgical procedure involving ultrasound guided aspiration of the follicular content.
• Cumulus-Oocyte-Complexes are matured and fertilized in vitro.

• Direct follicle aspiration:
  • A hyperdermic needle is inserted into the follicle.
  • Follicular fluid is aspirated.
  • Follicular fluid forcefully returned to the follicle.
  • Repeated 2-3 times to dislodge oocytes.
  • Alternatively, a constant vacuum (equivalent to aspirating 25 ml of water per minute through the aspiration needle) can be used for superovulated cows.

• Transvaginal Aspiration in the Mare:
  • Prior to the procedure, mares are injected with propatheline bromide (sedative) to relax the rectum.
  • Lubricated ultrasound transducer is inserted into the vagina and held in the fornix vagina.
  • Ovary transrectally positioned against the dorsal vaginal wall directly over the transducer head so that the follicle can be visualized.
  • Hypodermic needle is advanced through the vaginal wall into the antral follicle.
  • Follicular fluid containing the Cumulus-Oocyte-Complex is aspirated under constant vacuum. The association between the Cumulus-Oocyte Complex and the follicular wall is stronger in the mare compared with cows. It is advantageous to use double-lumen aspiration needle to flush the follicle while aspirating to increase recovery rates.

In Vitro Maturation

• • Oocytes are arrested in the diplotene stage of the first meiotic division (dictyate stage) and in mares and cows, are not capable of being fertilized at this stage. The Luteinizing Hormone (LH) surge induces germinal vesicle break down and assembly of the chromosomes into a metaphase plate (metaphase I). During telophase, half the chromosomal complement is extruded with hardly any cytoplasm as the first polar body. At this stage, the oocyte enters the second meiotic division (becomes a secondary oocyte). Cows and Mares ovulate oocytes in metaphase II.
  • Cumulus-Oocyte-Complexes of cows are usually aspirated from primary follicles prior to the LH surge and would require maturation before being able to be fertilized. In vitro maturation is less efficient in mares and thus mares can be given GnRH analogues to induce GV breakdown prior to aspiration to avoid having to go through the maturation step in vitro.

In Vitro Fertilization

• • In addition to nuclear maturation, cytoplasmic maturation is required before the oocyte is competent to be fertilized and develop normally. Final maturation is dependent on the close physical association between the oocyte and the inner layers of the cumulus granulosa cells surrounding the oocyte. Once the oocyte is mature, it is ready for fertilization.
  • Matured Cumulus-Oocyte-Complexes are placed in a culture vessel with capacitated and hyperactivated spermatozoa.
  • In vitro fertilization in mares is still being optimized but an alternative is to transfer the cumulus-oocyte-complex into an inseminated recipient animal. This is called Gamete Intrafallopian Transfer (GIFT). Alternatively, the semen can also be transferred to the recipient uterine tubule with the cumulus-oocyte complex.
  • IntraCytoplasmic Sperm Injections (ICSI) involves physically injecting a spermatozoa into the oocyte. Although technically different from IVF, ICSI produced embryos are not usually distinguished from true IVF produced embryos in human medicine. ICSI is a brute force insemination which is able to bypass spermatozoa-oocyte interaction and is useful for male causes of infertility and also an alternative to true IVF if the IVF conditions are not optimized in the facility for the species (such as mares).

In Vitro Culture of Embryo

• Embryos are incubated for a further period to the appropriate stage for transfer.
• Embryos transferred to recipient female.
• Can be transferred at the correct stage of the recipient's oestrous cycle.
• Embryos transferred around the blastocyst stage.
• Success rates are improving.
• Embryos can be frozen, eliminating the need for tight synchronization. This allows them to be shipped to other locations.
• Increases incidence of large offspring syndrome.