L11: Breeding, Conservation and ART Technologies Flashcards
Define ruminant species:
Ruminants are hoofed mammals, including cattle, sheep, and goats, with a unique digestive system that allows them to better use energy from fibrous plant material when compared with other herbivores.
Motivations for using breeding technologies:
- Able to access top quality genetics at low cost (cow semen: £10/straw, bull: >£10,000)
- Hygiene and disease control (studs kept in biosecure premises)
Give the 3 semen collection methods in cattle:
- Artificial vagina
- Electroejaculation
- Massage of ampullae
Methods for artificial insemination by species: (3 examples)
- Steel catheter, through cervix into uterus (cattle and pigs)
- Vaginal application (sheep)
- Insemination into uterine horns by laparoscopy (sheep and deer)
Barrier to conservation attempts in marsupials:
- Not possible to effectively cryopreserve marsupial sperm even though many are endangered
Issues with sperm cryopreservation in animals:
- Impairs plasma membrane (~50%)
- Shortens lifespan
- Freeze-thawing can also impair organelles and DNA
- Not succesful in a lot of species (e.g. marsupials) -> moving towards preservation of fibroblast cells instead for conservation purposes
Conception rate per cycle in cattle:
- 50-90%
FACS technique: Method and application in animal breeding
- Used for cell sorting
- Label sperm with nucleic acid stain (greater staining in X than Y chr.)
- Sort with fluorescence activated cell sorter
- Assigns a positive or negative charge depending on fluorescence
- Separate into female and male populations
Why is FACS used in cattle but not pigs?
- Lower number of sperm required in cattle vs pigs
- Efficiency of technique also depends on degree of separation between DNA content of X and Y in a given species (bull>boar)
Problems with use of sex sorting in sperm:
- Exposure to high pressure stress
- Needs immediate use if not frozen
- Damage of freeze/thaw process
- Illegal in humans except in case of sex-linked genetic conditions
What are the two methods for embryo/oocyte cryopreservation:
- Slow freezing
- Vitrification (no ice formation)
Penetrating vs non-penetrating cryoprotectant medium:
- Penetrating: Glycerol and DMSO (for slow freezing)
- Non-penetrating: sucrose/glucose/fructose/trehalose
Procedure for slow cooling method:
- Load embryos or oocytes into plastic straws
- Cool to between -5 and -7 degrees
- Seed straws to initiate ice formation
- Cool slowly (0.3 - 0.5 degrees/min to about -65 degrees and plunge into liquid nitrogen)
Procedure for vitrification:
- Cool very rapidly in droplets (thousands of degrees/min)
- Use high cryoprotectant concentrations
- Liquid nitrogen made into slush (negative pressure)
Why is cloning used in agriculture?
- Rapid multiplication of desired livestock (F1 crossbred or genetically superior)
Issues with cloned animals:
- Perinatal and postnatal mortality increased (13% birth rate vs 30-45% with in vitro produced embryos)
- Only 64% of cloned calves survive 3 months
- Low success rates in many species -> prevents cloning as a viable preservation technique (i.e. required population for likelihood of success would exceed existing captive population)
Basic cloning technique: (SCNT)
- Culture cells from genetically desirable male
- Recover oocyte from domestic species (surrogate) -> extract DNA (chromosomes and polar body)
- Inject cultured cell into enucleated oocyte
- Embryo develops with genetic material from endangered animal -> transfer into surrogate
- Issue: mitochondrial mixing will still take place -> hybrid male offspring -> breed F1 clone with female of endangered species
Potential alternative to cloning techniques:
- Biopsy somatic cells from infertile patients
- Generate autologous iPS cells
- Induce germ cells from iPS -> ART
- Historically, embryo splitting was used (before introduction of cloning)
Why might AI be useful in amphibians?
- External fertilisation (e.g. frogspawn) -> AI allows population management, without need for ART
- Amphibian fungal disease demands captive breeding programmes to preserve ‘clean’ populations in biosecure facilities
- Able to produce hundreds of thousands of tadpoles for release into the wild
Rationales for de-extinction: (x3)
- Ecological benefits e.g. restoring ‘mammoth steppe’ in northern siberia
- ‘Putting things right’
- Spectacle -> fame and financial reward
- Biobanks (aka ARK, frozen zoo) can be (and are!) kept in perpetuity until technology advances enough to ‘bring them back to life’
Rationales against de-extinction: (x3)
- Limited feasability of the approach (extremely low success rates, currently at ~1% in mammals, requirement for an appropriate surrogate animal e.g. Baur fetuses outgrowing domestic cow’s womb)
- Offspring, if successful, likely to have physiological abnormalities -> ethics of displaying a disabled animal in zoos
- Social species -> issue of behavioural development of a single animal without peers
Beyond preservation and de-extinction, what other potential applications do animal iPSCs have?
- Manufacturing animal tissue using iPSCs…
- Environmentally friendly meat
- Disease free -> no zoogenesis (e.g. salmonella), no MRSA (antibiotic resistance)
- Animal products (high value textiles, ivory etc.) -> increasing supply, preventing black-market supply of poached products -> protects endangered populations
Give 3 barriers in the use of animal iPSCs for economical/sterile means:
- Current protocol demands media containing animal products like PBS, BSA -> source of contamination, maintains demand for livestock
- Producing non-processed laboratory meat would require serious advancements in biological scaffold technology; products like steak require specific porosity and vascularisation to grow -> potentially would involved hydrogels made from algae
- Not currently cost effective, costing 10s of thousands of pounds per kilo -> bioreactors are being used to scale up production
