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7/30/2019 invitro embryo production
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IN VITRO EMBRYO
PRODUCTIONDr. R. A. Siddique
M.V.Sc. PhD ScholarN.D.R.I. Karnal (Haryana)
INDIA
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In vitro embryo production(IVEP)Involves:
Collection of oocytes
In vitro maturation (IVM)
In vitro fertilization (IVF)
In vitro culture (IVC)
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Collection of oocytes
Live animal
Ovum pick-up
Slaughtered animal
Aspiration
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Collection of oocytes
ovaries collected from
slaughterhouse
Transport to lab in
saline containing
antibiotics at 32-37C
Aspiration of follicles (2-8 mm) in
aspiration medium (TCM-199 + 10% FBS)
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Grading of oocytes
Grade A: Compact cumulus-oocyte-complexes (COCs)with an unexpanded cumulus mass having 4
layers of cumulus cells, and with homogenous
evenly granular ooplasm
Grade B: COCs with 2-3 layers of cumulus cells and a
homogenous evenly granular ooplasm
Grade C: Oocytes partially or wholly denuded or with
expanded or scattered cumulus cells or with anirregular and dark ooplasm
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In vitro fertilizationFrozen 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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Sperm treatment andCapacitation
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 Capacitationmedia
Bovine serum albumin
Heparin - glucoseaminoglycans
Caffeine
cyclic nucleotide phosphodiesteraseinhibitor
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In vitro culture of embryos
Presumptive zygotes washed with IVC media
Placed in 50-100 l 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 38.5C
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Important consideration forIVMFC
Water quality
Buffering system and Osmolarity
Flux culture
Effect of maturation time
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Otherconsideration..
Antibiotic cover & mineral oil covering
Light environment
Temperature and gas phase
Storage and maintenance of media
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Culturesupplements
Protein
Hormones and growth factors
Culture vessel
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In vitro culture system
Complex medium
with somatic cellsSimple medium
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Culture in complexmediaTCM-199
F-10MEM
RPMI
Oviductal epithelia cells
Granulosa cell
VERO
BRL
STO
3T3
Somatic cells
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Culture in simple media
mCRaa1
mCRaa2mSOFaa
KSOMaa
G1/G2
No somatic cells
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Criteria to evaluate thequality of embryoExcellent : Spherical,Symmetrical with cells of
uniform size
Good : Few extruded blastomeres, irregelarshape
Fair : Extruded blastomeres, few degenerated
cells
Poor : Numerous extruded blastomeres,
degenerated cells
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development
2-16 cell stagesMorula : Embryo with >32 cell stage
Compact morula : Compaction of blastomeres
Early blastocyst : Formation of blastocoel had just
started
Blastocyst : Well definrd blastocoel & start
differentiation
Expanded blastocyst : Expanded with zona thinning
Hatched blastocyst : partly or completely come out
from ruptured zona
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rono ogy o em ryodevelopmentCell stage Estimated age in days
Cattle buffalo
Morula 5 4Compact morula 6 5
Early blastocyst 7 6
Blastocyst 7 6Expanded blastocyst 8 7
Hatched blastocyst 9 7.5 to 8
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Application ofIVEP
Stem cell Transgenic animal
Cloning
Cryopresevation
Embryo sexingDrug testing
Early
developmental
gene
Genome
reprogramming
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