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IN VITRO EMBRYO PRODUCTION

IN VITRO EMBRYO PRODUCTION. Dr. R. A. Siddique M.V.Sc. PhD Scholar N.D.R.I. Karnal (Haryana) INDIA. In vitro embryo production (IVEP ). Involves: Collection of oocytes In vitro maturation (IVM) In vitro fertilization (IVF) In vitro culture (IVC) . Collection of oocytes. Live animal.

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IN VITRO EMBRYO PRODUCTION

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  1. IN VITRO EMBRYO PRODUCTION Dr. R. A. Siddique M.V.Sc. PhD Scholar N.D.R.I. Karnal (Haryana) INDIA

  2. In vitro embryo production (IVEP) Involves: Collection of oocytes In vitro maturation (IVM) In vitro fertilization (IVF) In vitro culture (IVC)

  3. Collection of oocytes Live animal Slaughtered animal Ovum pick-up Aspiration

  4. Collection of oocytes ovaries collected from slaughterhouse Transport to lab in saline containing antibiotics at 32-37°C Aspiration of follicles (2-8 mm) in aspiration medium (TCM‑199 + 10% FBS)

  5. 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 an irregular and dark ooplasm

  6. In vitro fertilization Frozen semen In vitro matured oocytes Thaw In vitro sperm treatment and Capacitation Incubation with 1-2 million/ml spermatozoa for 18 h Presumptive zygotes washed with IVC media

  7. Sperm treatment and Capacitation Through washing and exposure in media Enhance motility of spermatozoa Express the acrosome reaction Enhance successful fertilization of the oocytes

  8. Components of Capacitation media Bovine serum albumin Heparin - glucoseaminoglycans Caffeine – cyclic nucleotide phosphodiesterase inhibitor

  9. 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

  10. Important consideration for IVMFC Water quality Buffering system and Osmolarity Flux culture Effect of maturation time

  11. Other consideration.. Antibiotic cover & mineral oil covering Light environment Temperature and gas phase Storage and maintenance of media

  12. Culture supplements Protein Hormones and growth factors Culture vessel

  13. In vitro culture system Complex medium with somatic cells Simple medium

  14. Culture in complex media • Oviductal epithelia cells • Granulosa cell • VERO • BRL • STO • 3T3 • TCM-199 • F-10 • MEM • RPMI Somatic cells

  15. Culture in simple media No somatic cells • mCRaa1 • mCRaa2 • mSOFaa • KSOMaa • G1/G2

  16. Criteria to evaluate the quality of embryo Excellent : Spherical,Symmetrical with cells of uniform size Good : Few extruded blastomeres, irregelar shape Fair : Extruded blastomeres, few degenerated cells Poor : Numerous extruded blastomeres, degenerated cells

  17. Evaluation of embryo development 2-16 cell stages Morula : 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

  18. Chronology of embryo development

  19. Stem cell Transgenic animal Cloning Genome reprogramming Application of IVEP Early developmental gene Cryopresevation Drug testing Embryo sexing

  20. THANX

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