lecture 28: manipulating domestic animal reproduction 2 Flashcards
1
Q
What is a typical oestrus syncrhonisation programme?
A
- 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
Q
What is superovulation?
A
- 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
Q
What is a common superovulation schedule?
A
- 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
Q
What is non-surgical collection of cattle embryos?
A
- 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
Q
What is the superovulation ET process - 1?
A
- 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
Q
What is superovulation-ET process - 2?
A
- 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
Q
What is the history of reproductive technologies?
A
- 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
Q
What is artificial reproduction: cloning?
A
- two procedures
- embryo splitting or cloning
- multiple copies of OFFSPRING
- somatic cell nuclear transfer (SCNT)
- multiple copies on an INDIVIDUAL
- embryo splitting or cloning
- rationale and efficiency
- outcomes and implications on phenotype?
9
Q
What is embryo cloning by splitting?
A
- embryo splitting → 2 (or more) genetic clones
- maximises offspring from high genetic value embryos
10
Q
What is embryo cloning and transfer?
A
- 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
Q
What is the cloning efficiency by embryonic stage?
A
- fertilised egg, 1 cell → 34%
- two cell → 28%
- four cell → 21%
- eight cell → 5%
- compacted eight-cell early morula → 0%
12
Q
What are hypothetical restriction points in cell reprogrammability?
A
- % 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
Q
What is somatic cell nuclear transfer (SCNT)?
A
- 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
Q
What is multiplying valuable breeding animals?
A
- 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
Q
What is resurrection of breeding?
A
- “resurrection” of valuable genetics for desirable phenotypes
18
Q
What is continual pregnancy loss with bovine clones?
A
- % 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
22
Q
What is seen post-mortem in clones?
A
- 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
24
Q
What are transgenic animals?
A
- through molecular biology and embryology we can “humanise” other mammalian species in 1980s
- genetic modification (mutation, insertion or deletion)
25
Q
What is an example of a protein used in transengenic animals?
A
- green fluorescent protein (GFP)
- female embryos exhibit green fluorescence at !480nM from 2 cell stage
