lecture 28: manipulating domestic animal reproduction 2

Question 1

What is a typical oestrus syncrhonisation programme?

Answer 1

• time (days): event
  
  • 0: insert CIDR
  • 9: inject PGF2a (am + pm)
  • 10: remove CIDR + GnRH
  • 11: heat check am, pm
  • 12: heat check am, pm
  • → A.I or natural mating
  • → embryo transfer on day 7 post-oestrus
• many synchronisation programmes
  
  • depends on species and country of use (legislation)
  • depends on reason for synchronising

Question 2

What is superovulation?

Answer 2

• males not limited
• genetically superior females
• increase number of offspring
• multiple ovulation and embryo transfer (MOET)
• synchronisation of donor and recipient female cycles
  
  • PGF2a/CIDR
  • FSH 4-day step down decreasing doses
  • GnRH/hCG and AI
  • embryo recovery and transfer day 7

Question 3

What is a common superovulation schedule?

Answer 3

• typically 8-10 ovulations induced, producing ~5 transferable embryos
• Day 0:
  
  • donor female: PG
  • recipient females: -
• 12:
  
  • donor: PG
  • recip: -
• 14:
  
  • pre-synchrony heats
  • -
• 19:
  
  • insert CIDR
  • insert CIDR
• 25 am, pm:
  
  • FSH, FSH
• 26 am, pm:
  
  • FSH, FSH
• 27 am, pm:
  
  • FSH and PG, FSH and PG
  • inject PG
• 28 am, pm:
  
  • FSH, FSH, remove CIDR, GnRH
  • remove CIDR, GnRH
• 29:
  
  • heat check, AI
  • heat check
• 30:
  
  • heat check, AI
  • heat check
• 36:
  
  • flush day 7 embryos, embryo transfer or freeze
  • embryo transfers

Question 4

What is non-surgical collection of cattle embryos?

Answer 4

• identify genetically superior cow and bull
• synchronise donor and recipient cows
• superovulate donor
• flush and transfer embryos
• flushing fluid, Y-connector, foley catheter, bulb of foley catheter seals behind cervix

Question 5

What is the superovulation ET process - 1?

Answer 5

• superovulation of donor with hormones
• artificial insemination (5 days after initiating superovulation)
• non-surgical recovery of embryos (6-8 days after mating) using a Foley catheter
• foley catheter for recovery of embryos (cuff, air, flushing fluid)
• isolation and classification of embryos
• storage of embros indefinitely in liquid nitrogen or at room temperature for a few hours

Question 6

What is superovulation-ET process - 2?

Answer 6

• transfer of embryos to recipients surgically or non-surgically
• pregnancy diagnosis by palpitation through the wall 1-3 months after embryo transfer
• birth (9 months after embryo transfer)

Question 7

What is the history of reproductive technologies?

Answer 7

• animal IVF
  
  • AI (uptake and current use) - 1784 dogs, 1950s uptake
  • ET, IVF, and IVM in multiple species - 1890s and 1959 rabbit, 1935
  • 100,000s of cattle, 000’s sheep, pig, deer
  • sex selection via sperm - 1980s cattle
• human IVF (Bob Edwards founder)
  
  • basic process (1967 IVF, 1970s pregnancies)
  • IVM (1983) - still controversial (600+ babies)
  • sex selection - embryo biopsy not sperm (1990s USA)

Question 8

What is artificial reproduction: cloning?

Answer 8

• two procedures
  
  • embryo splitting or cloning
    
    • multiple copies of OFFSPRING
  • somatic cell nuclear transfer (SCNT)
    
    • multiple copies on an INDIVIDUAL
• rationale and efficiency
• outcomes and implications on phenotype?

Question 9

What is embryo cloning by splitting?

Answer 9

• embryo splitting → 2 (or more) genetic clones
• maximises offspring from high genetic value embryos

Question 10

What is embryo cloning and transfer?

Answer 10

• oocytes from abattoir ovaries
• mature eggs
• removal of zona pellucida and nucleus from cytoplasm
• donor embryo flushed from uterus 4-5 days after mating
• separate cells from elite embryo
• electrofusion
• new embro
• nuclear transfer embryo
• transferred

Question 11

What is the cloning efficiency by embryonic stage?

Answer 11

• fertilised egg, 1 cell → 34%
• two cell → 28%
• four cell → 21%
• eight cell → 5%
• compacted eight-cell early morula → 0%

Question 12

What are hypothetical restriction points in cell reprogrammability?

Answer 12

• % cloning efficiency vs decreasing donor cell potency
• blastomeres → ES cells → somatic stem cells → differentiated cells
• loss of totipotency between blastomeres and ES cells → 25% → 10%
• loss of pluripotency between ES cells and somatic stem cells → 10% to 0%

Question 13

What is somatic cell nuclear transfer (SCNT)?

Answer 13

• process
  
  • somatic cells = donor cell
  • oocyte enucleation
  • cell injection
  • cell electro-fusion
  • embryo activation
  • in vitro culture
  • day 7 blastocyst
  • embryo transfer
  • clones
• copy of individual, not multiple copies of its offspring (embryo cloning)
• very low success rate
• requires “reprogramming” of donor cell nucleus back to totipotency
• dolly = most famous sheep in the world
  
  • mature udder cell (starved so that it is in G1 phase of cell cycle) is put into unfertilised egg with nucleus removed (enucleated cell)
  • new ‘zygote’ placed in sheeps uterus
  • embryo develops into “Dolly” (the 1 out of 277 that worked!)
  • so why clone??

Question 14

What is multiplying valuable breeding animals?

Answer 14

• tool for the production of transgenic animals
• take a top Holstein-Friesian dairy sire in new zealand… → and generate 3 cloned bull calves with the same superior genetics for breeding

Question 15

What is resurrection of breeding?

Answer 15

• “resurrection” of valuable genetics for desirable phenotypes

Question 16

What is conservation of endangered breeds?

Answer 16

• last surviving cow of the Enderby Island breed
• south of new zealnd
• Dave Wells, in association with the New Zealand Rare Breeds Conservation Society

Question 17

What is the rationale and efficiency of cloning?

Answer 17

• why clone?
  
  • commercial applications
  • research knowledge
  • legality in different countries
• efficiencies and differences between
  
  • species
  • tissues
• bovine cloning
  
  • worldwide
  • AgResearch

Question 18

What is continual pregnancy loss with bovine clones?

Answer 18

• % embryo survival vs stage of development
• only 20% of SCNT make it to term vs 60% conventional
• the biggest risk for conventional is up to day 30 → drops to 60% and then remains steady
• for SCNT drops to ~40% and continues to drop over course of pregnancy

Question 19

What is the health and well being of bovine clones?

Answer 19

• bovine somatic cell nuclear transfer (NT) is associated with an increased incidence of abnormal placental and foetal development
• about 10% of transfered NT embryos result in a live calf and only 67% of these survive to weaning at 3 months of age
• health problems are commonly reported with neonatal NT calves
• NT calves that survive to weaning can appear healthy until exposed to external storessors or when examined at post-mortem
• lack of data on clone health - Public issue, USFDA risk assessment

Question 20

What is the hypothesis about clone cohorts?

Answer 20

• NT animals that survive past weaning have subtle abnormalities in their physiology that increase their susceptibility to diseases/disorders

Question 21

What are phenotype assessments?

Answer 21

• birth to maturity
  
  • survival rates decreased, growth parameters no difference
  • altered blood chemistry and haematologies (normal range)
• response to fasting
  
  • inability to regulate salt reabsorption (7 fold excretion)
  • basal metabolites decreased. Pertubed amino acid profiles
• response to hormonal challenges
  
  • pancrease - insulin production not different
  • kidneys - renal turnover higher
  • thyroid - increased thyroid hormone production
  • adrenals - stress tests
    
    • direct = delayed response but increased cortisol production
    • indirect = lack of secondary response, liver issues

Question 22

What is seen post-mortem in clones?

Answer 22

• organ morphology
  
  • differences in size and characteristics
• bone density
  
  • no change in weight
  • increased mineral density
  • lack of bone marrow
  • ‘flexor tendon’
• common abnormalities
  
  • kidney cysts
  • undescended testis
  • heart valve deficiencies
  • stomach lining
  • brain-grey:white matter

Question 23

What is an overall health summary of clones?

Answer 23

• clones that survive past weaning appeared normal and healthy
• however, physiological differences that may explain increased mortality post-weaning vs control animals
• common abnormalities vs donor cell or cohort specific
• possible mechanisms
  
  • cloning procedure → in utero changes to foetus
  • haematopoeitic stem cells - blood, increased organ size and BMD
  • dysfunctional adrenals (GCs) and thyroids- vital for brain, lung and bone development
• to clone or not to clone?
  
  • useful technique for the replication of transgenic animals

Question 24

What are transgenic animals?

Answer 24

• through molecular biology and embryology we can “humanise” other mammalian species in 1980s
• genetic modification (mutation, insertion or deletion)

Question 25

What is an example of a protein used in transengenic animals?

Answer 25

• green fluorescent protein (GFP)
  
  • female embryos exhibit green fluorescence at !480nM from 2 cell stage

Question 26

What are domestic transgenic animals?

Answer 26

• six classifications based on the intended purpose:
  
  • to enrich or enhance the animals’ interactions with humans (hypo-allergenic pets)
  • to enhance production or food quality traits (faster growing, more efficient pigs)
  • to improve animal health (disease resistance)
  • to research human diseases (develop animal models for these diseases)
  • to produce industrial or consumer products (fibre proteins for multiple uses)
  • to produce human therapeutics (pharmaceuticals or tissue for implantation)
• domestic GMOs
  
  • coagulation factor IX - haemophilia (herman), myelin based protein-cystic fibrosis
  • beta-casein, alpha-lactoglobulin in milk
  • organ transplants (human-histo-compatibility)
• commercial companies - PPL therapeutics, GTC biotherapeutics
• goal is germ line transmission

Question 27

What are stem cells?

Answer 27

• embryonic stem (ES) cells
  
  • derived from destroying embryos
  • specialist culture conditions
  • mouse and human
• adult stem cells and iPS cells
  
  • very new area
  • induced pluripotent stem cells (reprogramming throgh transcription factors)
  • could engineer sperm and eggs