in vitro maturation and in vitro fertilization
TRANSCRIPT
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In vitro Maturation and In vitro Fertilization
ByAsad Ullah Baber
2013-ag-740
M.Phil. Theriogenology
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IMMATURE EGGS ARE RETRIEVED FROM OVARY AND MATURE IN LABORATORY
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Collection of oocyte Transportation
In vitro Maturation
In vitro Fertilization
Overview
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Collection of oocytes
Live animal
Ovum pick-up
Slaughtered animal
Aspiration
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Collection of oocyte
• Slicing or aspiration • Transvaginal ultrasound-guided ovum pick-up• laparoscopic oocyte recovery (LOR)
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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)
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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
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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
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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
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Media Supplementation
• Buffalo calf serum• Fetal calf serum• Bovine serum albumin
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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
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Washing of oocyte
• Oocytes washed three times in TL-Hepes medium
• Wash with in vitro maturation medium before transferring to the drops
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• 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
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• 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)
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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
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In vitro fertilization is a process by which an egg is fertilized by sperm outside the body
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Sperm treatment and Capacitation
• Through washing and exposure in media
• Enhance motility of spermatozoa
• Express the acrosome reaction
• Enhance successful fertilization of the oocytes
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Components of Capacitation media
• Bovine serum albumin• Heparin • Caffeine
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In vitro Fertilization Media
• TALP • IVF-TALP • Ca2+ free Tyrode medium
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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
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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
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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
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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
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