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

Slaughtered animal

Ovum pick-up

Aspiration

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)

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 COCs with 2-3 layers of cumulus cells and a homogenous evenly granular ooplasm Oocytes partially or wholly denuded or with expanded or scattered cumulus cells or with an irregular and dark ooplasm

Grade B:

Grade C:

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

Through washing and exposure in media

Enhance motility of spermatozoa

Express the acrosome reaction Enhance successful fertilization of the oocytes

Components of Capacitation media

Bovine serum albumin

Heparin - glucoseaminoglycans
Caffeine cyclic nucleotide phosphodiesterase inhibitor

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

Important consideration for IVMFC

Water quality
Buffering system and Osmolarity Flux culture

Effect of maturation time

Other consideration..
Antibiotic cover & mineral oil covering Light environment Temperature and gas phase Storage 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 3T3

Culture in simple media

mCRaa1 mCRaa2 mSOFaa KSOMaa G1/G2

No somatic cells

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

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

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 7 6 8 7 9 7.5 to 8

Stem cell

Transgenic animal

Genome reprogramming Early developmental gene

Cloning Application of IVEP Cryopresevation

Drug testing

Embryo sexing