IN VITRO EMBRYO PRODUCTION In vitro embryo production

IN VITRO EMBRYO PRODUCTION

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 Ovum pick-up Slaughtered animal Aspiration

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)

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

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

Sperm treatment and Capacitation üThrough washing and exposure in media üEnhance motility of spermatozoa üExpress the acrosome reaction üEnhance successful fertilization of the oocytes

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

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 CO 2 incubator (5% CO 2 in air, 90 -95% humidity) at 38. 5°C

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

Other consideration. . ØAntibiotic cover & mineral oil covering ØLight environment ØTemperature and gas phase ØStorage and maintenance of media

Culture supplements Protein Hormones and growth factors Culture vessel

In vitro culture system Complex medium with somatic cells Simple medium

Culture in complex media §TCM-199 §F-10 §MEM §RPMI Somatic cells §Oviductal epithelia cells §Granulosa cell §VERO §BRL §STO § 3 T 3

Culture in simple media m. CRaa 1 m. CRaa 2 m. SOFaa KSOMaa G 1/G 2 No somatic cells

Criteria to evaluate the quality of embryo üExcellent : Spherical, Symmetrical with cells of uniform size üGood : Few extruded blastomeres, irregelar shape üFair : Extruded blastomeres, few degenerated cells üPoor : Numerous extruded blastomeres, degenerated cells

Evaluation of embryo development ü 2 -16 cell stages üMorula : Embryo with >32 cell stage üCompact morula : Compaction of blastomeres üEarly blastocyst : Formation of blastocoel had just started üBlastocyst : Well definrd blastocoel & start differentiation üExpanded blastocyst : Expanded with zona thinning üHatched blastocyst : partly or completely come out from ruptured zona

Chronology of embryo development Cell stage Morula Compact morula Early blastocyst Blastocyst Expanded blastocyst Hatched blastocyst Estimated age in days Cattle buffalo 5 4 6 5 7 6 8 7 9 7. 5 to 8

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