INVITROEMBRYOPRODUCTION

1、IN VITRO EMBRYO PRODUCTIONDr. R. A. SiddiqueM.V.Sc. PhD ScholarN.D.R.I. Karnal (Haryana)INDIA1In vitro embryo production (IVEP)Involves: Collection of oocytes In vitro maturation (IVM) In vitro fertilization (IVF) In vitro culture (IVC) 2Collection of oocytes Live animalOvum pick-upSlaughtered anima

2、lAspiration3Collection 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 (TCM199 + 10% FBS) 4Grading of oocytesGrade A:Compact cumulusoocytecomplexes (COCs) with an unexpanded cumulus mas

3、s 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 ooplasmGrade C: Oocytes partially or wholly denuded or with expanded or scattered cumulus cells or with an irregular and dark ooplasm5In vitr

4、o fertilization Frozen semen ThawIn vitro sperm treatment and CapacitationIn vitro matured oocytes Incubation with 1-2 million/ml spermatozoa for 18 h Presumptive zygotes washed with IVC media 6Sperm treatment and CapacitationThrough washing and exposure in mediaEnhance motility of spermatozoaExpres

5、s the acrosome reactionEnhance successful fertilization of the oocytes 7Components of Capacitation mediaBovine serum albuminHeparin - glucoseaminoglycansCaffeine cyclic nucleotide phosphodiesterase inhibitor8In vitro culture of embryosPresumptive zygotes washed with IVC media Placed in 50-100 l drop

6、lets of IVC media in 35 mm Petri dish in groups of 10-15Cultured for 9 days in IVC medium in a CO2 incubator (5% CO2 in air,90-95% humidity) at 38.5C 9Important consideration for IVMFC Water quality Buffering system and Osmolarity Flux culture Effect of maturation time10Other consideration.Antibioti

7、c cover & mineral oil coveringLight environmentTemperature and gas phaseStorage and maintenance of media11Culture supplementsProtein Hormones and growth factorsCulture vessel12In vitro culture systemComplex medium with somatic cellsSimple medium 13Culture in complex mediaTCM-199F-10MEMRPMIOviductal

8、epithelia cellsGranulosa cellVEROBRLSTO3T3Somatic cells14Culture in simple media mCRaa1 mCRaa2 mSOFaa KSOMaa G1/G2No somatic cells15Criteria to evaluate the quality of embryoExcellent : Spherical,Symmetrical with cells of uniform sizeGood : Few extruded blastomeres, irregelar shapeFair : Extruded bl

9、astomeres, few degenerated cellsPoor : Numerous extruded blastomeres, degenerated cells 16Evaluation of embryo development2-16 cell stagesMorula : Embryo with 32 cell stageCompact morula : Compaction of blastomeresEarly blastocyst : Formation of blastocoel had just startedBlastocyst : Well definrd b

10、lastocoel & start differentiationExpanded blastocyst : Expanded with zona thinningHatched blastocyst : partly or completely come out from ruptured zona17Chronology of embryo developmentCell stageEstimated age in daysCattlebuffaloMorula54Compact morula65Early blastocyst76Blastocyst76Expanded blastocyst87Hatched blastocys