Embryo Splitting

Question 1

What is Embryo-splitting?

Answer 1

• Identical twins (or more) can be made by splitting a day-6 embryo (blastocyst before hatching from ZP) and placing the fragments into empty ZP shells and then transferring them into recipients (E.T.)

Question 2

What is the procedure for embryo splitting?

Answer 2

• Egg and Sperm are joined (naturally or in vitro)
• When embryo reaches about 8-cell stage (~6days post ovulation) it is split one or more times
  
  • At this stage embryo is a blastocyst and has not hatched from ZP
  • Only very good quality embryos are split
  • A micromanipulator scope, vacuum tips, and microblades are used
• 2 or more pieces of the original embryo are placed in empty ZP
  
  • empty ZP created by sucking out the contents of an egg or embryo (poor-quality or degenerating)
• Embryos are transferred into a recipient that is on the same day post ovulation as the donor (i.e. day 6 or 7 post ovulation)
• Survivability of split embryos is less than un-manipulated embryos - but not too bad
  
  • Overall success is greater than un-manipulated embryos (greater efficiency of E.T.)

Question 3

What is Cloning (Nuclear Transfer)?

Answer 3

• Multiple genetically identical animals can be produced by taking cells from an embryonic blastocyst and implanting them into enucleated oocytes which are transferred to a recipients

Question 4

When was Nuclear Transfer first done?

Answer 4

• 1986 - Steen Willadsen cloned lambs using cells from sheep embryos

Question 5

How is Nuclear transfer done?

Answer 5

• Remove DNA from unfertilized egg
• Remove one cell from a donor embryo
  
  • most often in blastocyst stage
  • could get ~60 clones from 1 embryo
• Transfer embryo cell to egg
• Fuse cells and transfer

Question 6

How is Somatic Cell Nuclear Transfer (SCNT) done?

Answer 6

• Donor cells: biopsy from ear of animal to be cloned (2N)
• Recipient cells: eggs collected from slaughterhouse ovaries (1N)

Question 7

What causes Cloned Calf Syndrome?

Answer 7

• SCNT Clone Pregnancies: 70% pregnancies lost in first trimester - up to 95% total loss
  
  • Natural - 1-3%
  • IVF - 5% cattle, 11% sheep
• Don’t fully understand why the embryos/fetuses/calves are not the same
  
  • Culture conditions may be involved
  • Placenta may not develop normally
    
    • inappropriate transition from yolk sac to allantoic nutrition?
  • Failure of normal placentome development

Question 8

What is Cloned Calf Syndrome?

Answer 8

• Calves that complete gestation:
  
  • increased birthweight (fetomegaly)
    
    • Abnormal placentas - placental edema
    • Few/huge placentomes
    • Normally, fetal growth is constrained by maternal and placental factors
    • ⇑ placental origin IGF-1 (overcome maternal constraint on fetal growth in late gestation?)
  • Respiratory Distress Syndrome
    
    • pulmonary hypertension
    • poor adrenal gland development/function
    • Low fetal cortisol leads to insufficient lung surfactant
    • Treatment of recipient dam with cortisol prior to delivery (aid final maturation of lungs) and oxygen therapy in the neonatal period
  • Pneumonia:
    
    • Predisposed to infectious disease?
    • Immune dysfunction?
• If calves survive the first couple weeks - they can grow to be healthy and have a normal life