L11: Breeding, Conservation and ART Technologies

Question 1

Define ruminant species:

Answer 1

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.

Question 2

Motivations for using breeding technologies:

Answer 2

• 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)

Question 3

Give the 3 semen collection methods in cattle:

Answer 3

• Artificial vagina
• Electroejaculation
• Massage of ampullae

Question 4

Methods for artificial insemination by species: (3 examples)

Answer 4

• Steel catheter, through cervix into uterus (cattle and pigs)
• Vaginal application (sheep)
• Insemination into uterine horns by laparoscopy (sheep and deer)

Question 5

Barrier to conservation attempts in marsupials:

Answer 5

• Not possible to effectively cryopreserve marsupial sperm even though many are endangered

Question 6

Issues with sperm cryopreservation in animals:

Answer 6

• 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

Question 7

Conception rate per cycle in cattle:

Answer 7

• 50-90%

Question 8

FACS technique: Method and application in animal breeding

Answer 8

• 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

Question 9

Why is FACS used in cattle but not pigs?

Answer 9

• 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)

Question 10

Problems with use of sex sorting in sperm:

Answer 10

• 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

Question 11

What are the two methods for embryo/oocyte cryopreservation:

Answer 11

• Slow freezing
• Vitrification (no ice formation)

Question 12

Penetrating vs non-penetrating cryoprotectant medium:

Answer 12

• Penetrating: Glycerol and DMSO (for slow freezing)
• Non-penetrating: sucrose/glucose/fructose/trehalose

Question 13

Procedure for slow cooling method:

Answer 13

• 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)

Question 14

Procedure for vitrification:

Answer 14

• Cool very rapidly in droplets (thousands of degrees/min)
• Use high cryoprotectant concentrations
• Liquid nitrogen made into slush (negative pressure)

Question 15

Why is cloning used in agriculture?

Answer 15

• Rapid multiplication of desired livestock (F1 crossbred or genetically superior)

Question 16

Issues with cloned animals:

Answer 16

• 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)

Question 17

Basic cloning technique: (SCNT)

Answer 17

• 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

Question 18

Potential alternative to cloning techniques:

Answer 18

• 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)

Question 19

Why might AI be useful in amphibians?

Answer 19

• 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

Question 20

Rationales for de-extinction: (x3)

Answer 20

• 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’

Question 21

Rationales against de-extinction: (x3)

Answer 21

• 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

Question 22

Beyond preservation and de-extinction, what other potential applications do animal iPSCs have?

Answer 22

• 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

Question 23

Give 3 barriers in the use of animal iPSCs for economical/sterile means:

Answer 23

• 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