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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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