In vitro Maturation and In vitro Fertilization

ByAsad Ullah Baber

2013-ag-740

M.Phil. Theriogenology

IMMATURE EGGS ARE RETRIEVED FROM OVARY AND MATURE IN LABORATORY

Collection of oocyte Transportation

In vitro Maturation

In vitro Fertilization

Overview

Collection of oocytes

Live animal

Ovum pick-up

Slaughtered animal

Aspiration

Collection of oocyte

• Slicing or aspiration • Transvaginal ultrasound-guided ovum pick-up• laparoscopic oocyte recovery (LOR)

Transportation

• Most common medium» Physiological saline (0.9% sodium chloride )» 100 µg streptomycin/mL » 100 IU penicillin/mL) » 28 to 33ºC

• Storage of ovaries at 4ºC for 12 or 24 h significantly reduced the Developmental potential of oocytes.

• 6 h storage at 20°C, normal follicles were preserved. • Bovine ovaries stored for 24 h between 15 and 21ºC did not

influence the IVP of blastocysts(Schernthaner et al., 1977)

Grading of oocytes

• Grade A: Compact cumulus oocyte complexes ‑ ‑ (COCs) ≥4 layers of cumulus cells,

• Grade B: COCs with 2-3 layers of cumulus cells

• Grade C: expanded or scattered cumulus cells or with an irregular ooplasm

In vitro Maturation

• Oocytes are selected »compaction of cumulus-corona

investment »Homogeneity of the ooplasm

• Oocytes with compact and evenly granulated cumulus cells give higher maturation rates

Maturation Media

• Tissue Culture Medium (TCM) 199• Charles Rosenkran’s 1 amino acid (CR1aa), • Charles Rosenkran’s 2 amino acid (CR2aa),• Ham’s Nutrient mixture • Minimum Essential Medium and RPMI

(Roswell Park Memorial Institute) media

Media Supplementation

• Buffalo calf serum• Fetal calf serum• Bovine serum albumin

Protocol

• pH of media 7.3 - 7.4. • Drops of 200ul of maturation media prepare in

a Petri dish • Covered with mineral oil • 5% CO2 and 95% humidity

Washing of oocyte

• Oocytes washed three times in TL-Hepes medium

• Wash with in vitro maturation medium before transferring to the drops

• 10 - 20 oocytes transfer to the drops of TCM-199• The maturation drops covered with warm light

weight mineral oil and kept for 24 hr-26 hr in incubator

» 38.5ºC under 5% CO2 pressure » RH 90 to 95%.

• Maturation of oocytes evaluate by the cumulus cell expansion under stereo zoom microscope

• TCM 199 found best as it enhanced nuclear maturation if added

» 20% buffalo estrus serum, » FSH (0.5 µg/mL) and estradiol-17β(E2, 1

µg/mL) » But not when luteinizing hormone (LH)

(Totey et al., 1992)

Cont…

• Different growth factor also enhance the oocyte maturation rate

• Insulin-like growth factor-I and insulin-like growth factor-II increase cumulus expansion, nuclear maturation

In vitro fertilization is a process by which an egg is fertilized by sperm outside the body

Sperm treatment and Capacitation

• Through washing and exposure in media

• Enhance motility of spermatozoa

• Express the acrosome reaction

• Enhance successful fertilization of the oocytes

Components of Capacitation media

• Bovine serum albumin• Heparin • Caffeine

In vitro Fertilization Media

• TALP • IVF-TALP • Ca2+ free Tyrode medium

Media

• TALP Media should be pre warmed before use at 38.5 C in 5% CO2 or in the air

• TALP is used during purification of sperm by swim-up

Protocol

1. Collect oocytes after maturation, and wash 1-2 times in HEPES-TALP.

2. Place oocytes in IVF-TALP or other media3. Oocytes (5 - 10/drops) and sperm cells co-cultured4. Sperm suspension (dilute so concentration in drop

is ~1 x 106 /ml) 5. Incubate for 8-10 h at 39°C, 6. wash embryos 3-4 times with HEPES-TALP/ IVC

media and culture

In vitro fertilization

Frozen semen

Thaw

In vitro sperm treatment and Capacitation

In vitro matured oocytes

Incubation with 1-2 million/ml spermatozoa for 18 h

Presumptive zygotes washed with IVC media

In vitro culture of embryos

Presumptive zygotes washed with IVC media

Placed in 50-100 μl droplets of IVC media in 35 mm Petri dish in groups of 10-15

Cultured for 9 days in IVC medium in a CO2 incubator (5% CO2 in air,90-95% humidity) at 38.5°C