lecture 28: manipulating domestic animal reproduction 2 Flashcards Preview

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Flashcards in lecture 28: manipulating domestic animal reproduction 2 Deck (27):
1

What is a typical oestrus syncrhonisation programme?

  • 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 

2

What is superovulation?

  • 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 

3

What is a common superovulation schedule?

  • 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 

4

What is non-surgical collection of cattle embryos?

  • 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 

5

What is the superovulation ET process - 1?

  • 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 

6

What is superovulation-ET process - 2?

  • 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) 

7

What is the history of reproductive technologies?

  • 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) 

8

What is artificial reproduction: cloning?

  • 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?

9

What is embryo cloning by splitting?

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

10

What is embryo cloning and transfer?

  • 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 

11

What is the cloning efficiency by embryonic stage?

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

12

What are hypothetical restriction points in cell reprogrammability?

  • % 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% 

13

What is somatic cell nuclear transfer (SCNT)?

  • 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??

14

What is multiplying valuable breeding animals?

  • 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 

15

What is resurrection of breeding?

  • "resurrection" of valuable genetics for desirable phenotypes 

16

What is conservation of endangered breeds?

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

17

What is the rationale and efficiency of cloning?

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

18

What is continual pregnancy loss with bovine clones?

  • % 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 

19

What is the health and well being of bovine clones?

  • 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 

20

What is the hypothesis about clone cohorts?

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

21

What are phenotype assessments?

  • 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

22

What is seen post-mortem in clones?

  • 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 

23

What is an overall health summary of clones?

  • 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 

24

What are transgenic animals?

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

25

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

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

26

What are domestic transgenic animals?

  • 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 

27

What are stem cells?

  • 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