IN VITRO EMBRYO PRODUCTION

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IN VITRO EMBRYO PRODUCTION
Dr. R. A. Siddique M.V.Sc. PhD Scholar N.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
Slaughtered animal
Ovum pick-up
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 an
irregular and dark ooplasm
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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
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Sperm treatment and Capacitation

• 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 Capacitation media

• Bovine serum albumin
• Heparin - glucoseaminoglycans
• Caffeine cyclic nucleotide phosphodiesterase
  inhibitor

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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
38.5C
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Important consideration for IVMFC

• Water quality
• Buffering system and Osmolarity
• Flux culture
• Effect of maturation time

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Other consideration..

• Antibiotic cover mineral oil covering
• Light environment
• Temperature and gas phase
• Storage and maintenance of media

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Culture supplements

• Protein
• Hormones and growth factors
• Culture vessel

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In vitro culture system
Complex medium with somatic cells
Simple medium
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Culture in complex media

• Oviductal epithelia cells
• Granulosa cell
• VERO
• BRL
• STO
• 3T3

• TCM-199
• F-10
• MEM
• RPMI

Somatic cells
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Culture in simple media
No somatic cells

• mCRaa1
• mCRaa2
• mSOFaa
• KSOMaa
• G1/G2

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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

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Evaluation of embryo development

• 2-16 cell stages
• Morula Embryo with gt32 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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Chronology of embryo development
Cell stage Estimated age in days Estimated age in days
Cell stage Cattle buffalo
Morula 5 4
Compact morula 6 5
Early blastocyst 7 6
Blastocyst 7 6
Expanded blastocyst 8 7
Hatched blastocyst 9 7.5 to 8
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Stem cell
Transgenic animal
Cloning
Genome reprogramming
Application of IVEP
Early developmental gene
Cryopresevation
Embryo sexing
Drug testing
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THANX