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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