Topic 5 & 6

Question 1

What is CRISPR?

Answer 1

• Clustered Regularly Interspaced Short Palindromic Repeats

- Family of DNA sequences found in prokaryotes

Question 2

How does CRISPR work?

Answer 2

• Cas9 –> CRISPR-associated proteins –> nucleases that cuts DNA
• CRISPR is a collection of DNA sequences
• CRISPR tells Cas9 where to cut
• Cas9 is an enzyme that cuts DNA
• Involves changing the sequences which changes the messages –> exhibit new property/characteristic
• Microinjection of CRISPR/Cas9 embryos

Question 3

Function of CRISPR

Answer 3

• Genome editing –> fix alleles at existing trait loci, create new alleles/introduce alleles from other strains/species
• Enhance dz resistance in fish
• ↑ biomass/growth rate
• Achieve 100% sterility to prevent interbreeding w wild species (if escape)

Question 4

Ethical implications of genome editing/manipulation (Environment)

Answer 4

• Ecological risks if escaped
• Bioengineered organisms may have intraspecific/interspecific interactions w env/other org.
• Need to evaluate environmental risks in relation to pollution, energy, land use –> Environmental Impact Assessment (EIA)

Question 5

Drawbacks of genome-based biotechnologies (environmental issues)

Answer 5

• Similar to impacts of exotic/invasive species
• Fishes in open aquaculture system can interact w wild indivs. –> transfer of pathogen/diseases
• Escaped viable culture indivs –> ecological hazards
  1. Alteration of predation
  2. Resource competition
  3. Change in fitness of novel genotypes in the wild due to interbreeding –> breed w wild animals = affect genotype
• Genetically-engineered infertile aquatic species can reach maturity but X reproduce –> upset demography in small natural populations –> possible risk of local extinction

Question 6

Why is Broodstock nutrition important?

Answer 6

1. Nutrition (quality + quantity of diet) has profound effect on gonadal growth & fecundity –> affects broodstock reproductive success, spawning, hatching ability of eggs & survival rate of offspring
2. Quality nutrition = Maintenance & reproduction for commercially important fish species = ↑ production success (esp in less desirable culture practices) = ↑ income

Question 7

What does suboptimal nutrition/food restriction affect?

Answer 7

Reproductive potential

1. Time of first maturity (delayed)
2. No. of eggs produced (fecundity)
3. Egg size
4. Egg quality (measured by chemical composition)
5. Delayed spawning
6. Hatchability
7. Larval quality

Question 8

Energy Partitioning

Answer 8

Partitioned b/w

1. Maintenance
2. Growth
3. Reproduction

Maintenance requirements met then excess energy divided into growth & reproduction

Question 9

Essential nutrients in reproduction

Answer 9

1. Lipid + Fatty acids: HUFA affects fish maturation, (in some species:) ↑ fecundity, fertilization & egg quality; ↑ essential fatty acids = ↑ fecundity + hatchability
2. Vitamin E
3. Vitamin C
4. Amino acids
5. Trace minerals

Question 10

Lack Vitamin E

Answer 10

• Immature gonads + low reproductive performance
• Lower hatching rate & survival of offspring
• ↑ % of abnormal eggs
• ↓ fecundity in gilthead seabream

Question 11

Lack Vitamin C

Answer 11

• ↓ prod. of steroid
• Poor yolk formation
• Reduced sperm concentration

Question 12

Lack amino acids

Answer 12

• Indivs spawn at smaller size

- eggs w low hatchability & deformities

Question 13

Lack tace minerals

Answer 13

• lower % of both eyed & viable eggs

Question 14

Broodstock diet

Answer 14

1. Broodstock conditioning diet: optimized growout diet (high load of fatty acids)
2. Reproduction diet: before/during spawning –> meet needs for max reproductive performance (spawning success, fecundity, gamete quality) & vertical transfer of nutrients & biologically active substances to offspring
3. Recovery/maintenance diet: assist recovery from repro. exhaustion + recondition for next repro cycle

Question 15

What does stress in broodstock cause?

Answer 15

• Affect egg & larvae quality
• Impair fitness of progeny
• More difficult to rear small larvae

Question 16

What causes broodstock stress?

Answer 16

• Cannot acclimate in captivity
• Stocking density
• Water quality
• System design
• Handling processes: grading, sampling, tpt

Question 17

HPI axis

Answer 17

Hypothalamic-Pituitary-Interrenal Axis

1. Stress causes the hypothalamus to secrete Adrenocorticotropic hormone (ACTH) stimulating hormone
2. ACTH Stimulating hormone signals the pituitary gland to secrete ACTH
3. ACTH acts on the interrenal cells
4. Causes the interrenal cells to secrete cortisol
5. Cortisol acts on body cells –> loss of appetite, ↓ed mucus production, ↓ed resistance

Question 18

Stress indicators

Answer 18

Glucocorticoids (cortisol & corticosteroid) –> biomarker for stress quantification
- Consistently high cortisol level = inability to restore homeostasis
Results in
1. Chronic stress
2. Prolonged physiological response (from secretion of cortisol)
3. Detrimental effects of glucocorticoid mediated actions: ↓ed growth, ↓ reproduction, immune suppression, ↑ed mortality
4. Fish performance & welfare affected

Question 19

Stress & reproduction

Answer 19

Vertical transfer of maternal hormones (mother to egg)

• Developmental hormones (e.g. thyroxine)
• Reproductive hormones (e.g. testosterone)
• Metabolic hormones (e.g. cortisol)

Cortisol

• indirectly influence by altering allocation of vitellogenin (egg yolk precursor) & levels of developmental hormones during gametogenesis
• directly influences larval morphology: -ve correlation b/w level of cortisol in ovaries of breeding females & size of larvae produced (↑ed cortisol = smaller larvae)

Stressed broodstock = eggs w high level of cortisol = poor quality larvae

Question 20

Stress-free broodstock

Answer 20

1. High quality eggs
2. Larvae of greater fitness
3. Reduced larval mortalities
4. Stronger juveniles & healthier fish

Success in closing reproductive cycle of fish = no stress