Topic 7 - Reproductive technology & immunology

Question 1

Define animal propagation…

Answer 1

selection & mating -> very successful

various ways to aid process or aid results

Question 2

Pros of artificial insemination (AI)…

Answer 2

• Allows genetically desirable animals to be bred more efficiently (Natural mating = 1 bull services few females)
• diluted semen sample from 1 bull -> 500-1000 heifers!
  
  • used in beef/dairy 40 years; some use in sheep; in horse (but not racehorse)

Question 3

AI semen collection…

Answer 3

• Collected from sires via electroejaculation, dummy or real female
• Can be diluted (extender) with egg yolk
  
  • Frozen in liquid nitrogen or used fresh

Question 4

AI uses…

Answer 4

• extending coverage of individual males
• human repro.
• improving genetic diversity in zoo pop’s
• animal conservation in many endangered species

Question 5

2 types of animal cloning…

Answer 5

Embryo splitting (ES)
Nuclear transfer (NT)

Question 6

Describe method of embryo splitting (ES)… (draw diagram)

Answer 6

1. collect eggs & fertilise with sperm in petri dish (IVF)
2. culture embryo to 8-16 cells
3. using microscope, separate cells
4. incubate each separately to early embryo stage
5. implant into surrogate mothers

Question 7

Uses of ES…

Answer 7

• 2 or more superior animals
• transgenic mice experiments (1 transgenic, 1 control)
• extended to humans (may cause defective embryos) to assist with IVF when mother cannot produce enough eggs after superovulation
• clone monkeys

Question 8

Cons of ES?

Answer 8

• unknown genetic worth (may not pass ‘good’ genes to offspring)
• may cause defective embryos in humans

Question 9

Describe nuclear transfer…

Answer 9

Replacing the nucleus of an egg cell (haploid) with a diploid cell to produce a viable embryo -> genetically identical individual of the donor nucleus
- can increase offspring from an animal 100’s to 1000’s

Question 10

1st successful cloning done by…? Animal? Year? Results?

Answer 10

nuclear transplantation in Leopard frogs in 1950’s

Results: Blastula best (nuclei totipotent)

Question 11

Commercial aspects of animal cloning… (include problems)

Answer 11

Only useful if traits are known e.g. superior milk yield, Melb cup winner, transgenic animals
Probs: genetic merit of organism cannot be established until adult; difficult, expensive

Question 12

The Dolly clone… everything! Sheep used, process, diagram, break thru etc. Use diagram for help!

Answer 12

Finn Dorset sheep
- used nucleus from differentiated (somatic) mammary epithelial cell of 6 yo ewe
Process: Differentiated cells cultured in vitro & starved of nutrients -> G0 phase (non-dividing state) -> enucleated egg cells from Scottish Blackface breed -> nuclei transferred by mixing eggs & mammary cells via electrofusion -> embryos cultured & implanted into ewes prepared with hormone treatments
Dolly born 1996 - 2003
Break thru: dolly was 1st cloned from differentiated nuclei
Uses: study environment on isogenic animals; multiplication of superior livestock

Question 13

First successful sheep cloning experiment via NT…?

Answer 13

1986, sheep eggs collected & enucleated via UV radiation -> each egg fused with cell from 16-32 cell embryo in petri-dish -> embryos cultured & implanted into surrogate ewes -> 32 cell embryo => 32 clones

Question 14

What process is important to ensure genetic diversity?

Answer 14

reproductive techniques in conservation biology via storage of (eggs & fertilised embryos) germplasm & frozen sperm banks

Question 15

What is MOET? Animals used in…

Answer 15

Multiple ovulation & embryo transfer

• Cattle industry
  
  • superovulate heifers with hormone treatments -> AI laproscopically with bull semen -> flush fertilised eggs & freeze in liquid nitrogen -> implant in surrogates
• used in zoos to make embryos mobile

Question 16

Name the 3 types of natural immunity…

Answer 16

1. Body surface
2. Non-specific responses
3. Immune responses

Question 17

Name some body surface barriers…

Answer 17

skin
mucuous membranes
tears & saliva (lysosome)

Question 18

What is meant by non-specific responses?

Answer 18

inflammation
WBC - neutrophils, macrophages, eosinophils, basophils
complement - blood clotting

Question 19

What are cytokines? Give examples… (remember viral figure)

Answer 19

antimicrobial agents

eg. Interferons, interleukins, lactoferrins, transferrins…

Question 20

What are interferons? (draw viral figure)

Answer 20

carbohydrate (peptide glycoprotein) molecules cytokines) important in viral infections produced by cells infected with virus

Question 21

What are interleukins? Properties…

Answer 21

cytokines that are produced by cells that attract phagocytic cells, induce inflammatory response, stimulate Ab prod. by B cells (plasma cells), and TNF
There are several…

Question 22

Lactoferrins, transferrins…

Answer 22

Iron binding proteins - bacteria require iron for growth
lactoferrin in milk, saliva, tears
transferrin in blood

Question 23

What is the complement system? What are the different ‘complement systems’? Remember diagram

Answer 23

30 different proteins in the blood that have multiple effects

1. opsonisation - increase phagocytosis by coating pathogen
2. inflammation - histamine -> increase blood vessel permeability and attracts phagocytes
3. cytolysis - pores in microbial plasma membrane -> inflow of extracellular fluid -> burst of microbe

Question 24

Phagocytic cells…

Answer 24

cells engulf bacteria, viruses… & digest them
contain hydrolytic enzymes (lysozymes, lipase…)
WBC - leukocytes & monocytes

Question 25

Aquired or adaptive immunity… eg. if errors occur

Answer 25

second line of defence
captures invading organism/tissue -> recognises as ‘foreign’ -> responds & creates memory
Errors -> allergies or over response (asthma, peanuts, shellfish)

Question 26

Antigens…

Answer 26

foreign material - proteins, lipids, glycoproteins, carbs, DNA & RNA
each bacteria has many different antigens, thus recognition is specific

Question 27

What is a polyclonal Ab? How does the process work? Uses? Problems?

Answer 27

Antibodies secreted by different B cell lines within the body.
Process works by injecting animal (mouse, rabbit, goat) with antigen -> induces B-lymphocytes -> IgG’s -> collect serum with Ab’s bound to many different epitopes.
Uses involve antigen detection - ABO, HIV infection, preg tests
Problem - not specific enough

Question 28

MABs… Limitations… (cannot vs can be cultured)?

Answer 28

monoclonal antibodies - monospecific Ab’s that are the same cos made by identical immune cells that are clones of a unique parent cell & have high specificity for single epitopes - recognise and distinguish between strains of bacteria or viruses
B cells or plasma cells producing Ab’s cannot be cultured
Cancer cell lines eg ‘myeloma’ derived from B cells can be cultured

Question 29

Producing monoclonals…(draw diagram)

Answer 29

slide 56…

Question 30

What are monoclonals used for?

Answer 30

Research:
Probes to cDNA libraries expressing proteins
immunoassays
used to purify proteins
Diagnostics:
detect molecules like hormones or viral proteins
preg tests
disease diagosis
anticancer therapeutics
organ transplants

Question 31

What is Western Blotting?

Answer 31

A process that detects particular proteins in mixtures eg. cell extracts based on weight & Ab binding specificity -> protein gel electrophoresis of cell extracts -> transferred to membrane -> probe with MAB to desired protein -> add goat anit-human Ab to detect protein to measure size, quantity & presence.
Used in microscopy for similar purposes

Question 32

What is ELISA? Process… Good diagram on slide 61…

Answer 32

Enzyme Linked Immunosorbent Assay
used to detect Ab’s in sera to an infective agent eg. HIV, hep B…
Bind Ag to bottoms of wells in microtitre plate -> add serum -> add goat anti-human Ab linked to an enzyme (eg. peroxide) -> add substrate which produces coloured product in the presence of enzyme -> colour indicates AB’s to Ag present
Quantitative