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REPRO > Assisted reproductive technologies > Flashcards

Assisted reproductive technologies Flashcards

1
Q

Define In vitro Fertilisation

A

The process by which an oocyte is fertilised by a sperm outside teh body

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2
Q

Why might IVF be used in animals?

A
  • To produce embryos to be used in breeding programs (genetic improvement)
  • Conservation projects to produce embryos from endangered species
  • In all animals for research
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3
Q

Describe the IVF procedure

A
  • Oocytes and sperm collection
  • Mix oocytes and sperm in culture dish
  • Allow fertilisation to occur
  • Monitor embryo development
  • Transfer embryos in surrogate recipients
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4
Q

Describe how oocytes are collected for IVF in different species (specifically humans, ruminants, wild animals)

A
  • Human: following superovulation
  • Bovine and ovine: ovum pick:up during follicular selection (oocyte maturation required)
  • Wild animal oocytes: OPU or post-mortem
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5
Q

Describe intra-cytoplasmic sperm injection

A
  • Procedure involving injecting sperm into an oocyte thus bypassing normal fertilisation
  • Normally in human medicine, used in animals for research
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6
Q

Define embryo transfer

A

Transfer of an embryo, derived from mating of genetically proven, valuable parents into a fertile but less valuable recipient (host) female who carries pregnancy to term and offspring to weaning

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7
Q

What are the advantages of embryo transfer?

A
  • Maximise reproductive efficiency oh high quality animal, esp females
  • Speed up genetic improvement of a breed
  • Circumvents female infertility due to uterine tract abnormality
  • Enables breeding from injured/aged females
  • Enables athletic females to remain working
  • Lowers risk of disease spread by movement of live animals
  • Recipient’s offspring acquire colostral immunity against indigenous pathogens
  • Role in conservation of endangered species
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8
Q

What are the disadvantages of embryo transfer?

A
  • Expensive
  • Potential narrowing of gene pool
  • Enhanvement of genetically linked undesirable traits accidentally
  • Breed society permission required
  • Technically complex
  • Reliant on fertility of sire and donor female, number and quality of embryos recovered and quality of recipient
  • recipient needs ot be at same stage of cycle as donor
  • Skilled persons required
  • Dioestrus uterus susceptible to microorganisms
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9
Q

What are important characteristics of donors for embryo transfer?

A
  • Fertile semen donor
  • Fertile female donor
  • Female needs to be responsive to superovulation
  • Multiple embryos produced
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10
Q

What are important characteristics of the embryo transfer recipient?

A
  • Cycle synchronised with donor
  • Reproductively sound
  • Ideally same size or bigger than donor
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11
Q

Describe superovulation

A
  • Stimulation and ovulation of multiple oocytes rather than just one or two
  • Common procedure in cow
  • Not possible in horse currently
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12
Q

Describe the method for superovulation in cows

A
  • D0 = first day of oestrus
  • D9-14: gonadotrophin e.g. eCCG administerd
  • 28-72 hours later: administer PG, mid cycle CL regresses
  • 40-56 hour later, in oestrus
  • Inseminate at least twice, 12-8 hours apart
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13
Q

Describe the gonadotrophins used for superovulation in cattle

A
  • eCG or pregnant mare serum gonadotrophin (PMSG used)
  • LH and FSH biological activity
  • Longer half life in cow
  • Single injection
  • Pituitary follicle stimulating hormone porcine, ovine and equine
  • Human menopausal gonadotrophin
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14
Q

What are the limitations of gonadotrophins for superovulation?

A
  • LH activity in eCG and crude FSH preoparations can lead to premature maturation of oocytes
  • Ovulation of existing fillicles
  • Deficiency in sperm transport
  • Compromise in embryo transport from oviduct to uterus
  • Improved purity of FSH preparations will assist
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15
Q

What are the possibilities and limitation of superovulation in the cow?

A
  • Donor repeatedly superovulated at 6-8 week intervals

- Ovarian response of individual highly variable

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16
Q

Describe the process of embryo collection and transfer

A
  • Selection of genetically superior male and female donors
  • Superovulation of donor female (not horse)
  • Insemination fo donor female with semen
  • Recovery and identification of viable embryo from donor female
  • Synchronisation of recipients with donor female
  • Transfer of embryos into synchronised recipients
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17
Q

What stage of the cycle should the recipient be in relation to the donor for embryo transfer

A
  • A few days behind is ideal
  • Ok to be same stage as donor
  • Should not be in advance of the donor as embryo will not be developed enough, uterus may also be starting to lyse CL
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18
Q

Describe the method for cycle synchronisation of recipient to donor

A
  • Animal should be cycling well
  • Progesterone suppresses HPO axis, therefore LH and FSH
  • Withdrawal allows rebound effect, stimulatin follicular development then ovulation
  • Will not work in deep anoestrus
  • Can adminsiter PGF2a to lyse CL, reduce progesterone and so return to oestrus
  • Most methods involve P4 to mimic dioestrus
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19
Q

Describe the use of PGF2a for syncrhonisation of recipient and donor cycles

A
  • Lyses CL
  • IM injection
  • PGF2a in donor, then in recipient a day later
  • Recipient should therefore be ~24 hours behind donor
  • Need to know where animals are in their cycle
  • Only effective after day 6 of cycle, when CL sensitive to PGF2a
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20
Q

Describe the insemination process for embryo transfer

A
  • Semen deposited in tuerin body or horn (mare and cow) to maximise chances of reachign ociduct and thus fertilisation
  • Sperm accumulate in oviduct
  • Inseminate before ovulation
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21
Q

Describe embryo collection for transfer

A
  • Will enter uretotubal junction ~d4 post ovulation
  • Enters as morula/early blastocyst
  • Non-surgical, trans-cervical flush of uterus via catheter on days 6, 7, 8
  • Unattached, spherical, small, intact ZP offering physical protection
  • Recovered through small catheter with minimal damage
  • Medium filtered and small volume retained
  • Transferred to recipient, embryo continues to grow and implant
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22
Q

What is the disadvantage of embryo collection for transfer in the cow later than 8 days post-ovulation?

A
  • Embryo will be bigger and elongated

- More fragile once hatched from ZP

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23
Q

Describe embryo searching

A
  • Embryos need to be located
  • Graded under dissecting microscope
  • Warm room
  • Warm slide
  • Selected based on quality
  • Transferred to recipient or chilled/frozen
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24
Q

Describe the physical transfer of an embryo into the recipient

A
  • Oestrus cycle closely synchronised to within 24 hours
  • Transfer to uterine horn on same side as CL
  • Surgical or non-surgical
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25
Q

What is the techniques for surgical transfer of an embryo?

A

Flank incision, local anaesthetic, blunt puncture of horn

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26
Q

What is the technique of non-surgical transfer of an embryo?

A
  • Aseptic technique
  • Transfer using fine pipette or catheter
  • Highly skilled to avoid trauma to uterine endometrium (as would trigger release of PGF2a)
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27
Q

Outline the key points in embryo survival in embryo transfer

A
  • Embryo develops and hatches from ZP in recipient
  • Maternal recognition of pregnancy signal synthesised by embryo
  • Recognised by recipient
  • Nutrition e.g. uterine milk/histotroph/haemotroph essential for survival, produced by recipient
  • Placenta forms and implantation proceeds
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28
Q

What are the key points for a successful embryo transfer?

A
  • Fertile donor and quality recipients
  • Quality embryos
  • Cleanliness
  • Gentle manipulation of cervix
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29
Q

Give examples of embryo manipulation

A
  • Splitting (identical siblings produced)
  • Sexing
  • IVF
  • Cloning from somatic cells
  • Cooling and cryopresevation
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30
Q

Define stem cells

A

Unspecialised cells with capacity to self-renew for long periods

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31
Q

Define self-renewal

A
  • Ability of stem cells to divide and maintain undifferentiated state
  • Not just proliferation
  • Make copy of selves to produce stem cell or differentiated cell
32
Q

Describe embryonic stem cells

A
  • From embryo

- Can form any type of cell in body, but not capable of developing into new organism

33
Q

Describe tissue stem cells

A
  • Derived from foetal or adult tissues

- Sustain turnover and repair throughout life in some tissues

34
Q

Where can foetal stem cells be derived from?

A
  • Cord blood
  • Amniotic fluid
  • Foetal liver
35
Q

Where can adult stem cells be derived from?

A
  • Neural
  • Haematopoietic
  • Spermatogonial
  • Mesenchymal
  • Epidermal
36
Q

Define totipotent

A

Ability to form an entire organism e.g. embryo up to morula stage

37
Q

Define pluripotent

A

Able to form all body’s cell lineages, including germ cells e.g. inner cell mass cells of blastocyst

38
Q

Define multipotent

A
  • Able to form multiple lineages that consitute an entire tissue or tissues
  • E.g. haematopoietic stem cells, neural stem cells
39
Q

How are embryonic stem cells generated?

A
  • Derived from inner cell mass cells
  • Derived from epiblast
  • Derived from primordial germ cells
40
Q

List the potential medical and veterinary uses of stem cells

A
  • Cell based therapies
  • Drug developing and screening
  • Disease models
  • Study of early development
  • Development of new gene therapy methods
  • Mesenchymal stem cells for therapies
41
Q

Outline the use of mesenchymal stem cells for therapies

A
  • Stem cells derive from bone marrow or fat
  • Treatment of tendon and ligament injury in horses and dogs
  • Osteoarthritis
42
Q

What is the major risk in non-autologous stem cell technologies?

A
  • Immune rejection
  • Possible solution is reprogramming
  • Process of reversal of differentiation of somatic cells into autologous pluripotent cells
43
Q

Describe autologous cell therapies

A
  • Take cell from patient, remove nucleus
  • Implant into nucleus, transfer cell to oocyte = embryo
  • From embryo harvest stem cells to put into patient
  • No rejection implications
44
Q

What is somatic cell nuclear transfer?

A

Cloning
- Reconstruction of an embryo by the transfer of genetic material from a donor cell to a recipient egg, from which the genetic material has been removed

45
Q

Describe the process of somatic cell nuclear transfer

A
  • Need mammary cell donor and egg cell donor
  • Culture mammary cells
  • Remove nucleus from ovary
  • Fuse cells to put nucleus from mammary cell into oocyte
  • Grown in culture to form early embryo
  • Inplanted into uterus of third sheep (surrogate mother)
  • Embryonic development and birth in surrogate
46
Q

What are the problems with cloning?

A
  • Very difficult
  • Problems in health (lack of understand of consequences of gene manipulation, some immune deficiencies )
  • Problems in growth (calves too big, late offspring syndrome, obesity)
  • Premature ageing
  • Lack of knowledge
  • Foetal abnormalities: hydroallantois, extended gestation, kidney, brain, cardiovascular, muscle, skeletal
47
Q

Outline the difference between an identical twin and a clone

A
  • Twin: same genetic material

- Clone: similar genetic material i.e. same nuclear DNA but different mitochondiral DNA

48
Q

Compare therpeutic and reproductive cloning

A
  • Therapeutic: aim is to derive stem cells from cloned embryo
  • Reproductive: aim is to produce cloned embryo to develop into cloned animal
49
Q

Describe induced pluripotent stem cells (iPS cells)

A
  • Expression of pluripotecy genes induces direct reprogramming of somatic cells to pluripotency
  • Bypass cloning add genes to cells important for developmental process
  • Deliver exogenous restriction factors important for stem cells and cloning
50
Q

Give advantages of induced pluripotent stem cells

A
  • Overcomes need of generating embryos for deriving pluripotent cell lines
  • Can be used in a variety of species
  • Arcs of iPS cells from endangered species - banked and can stimulate them to become gametes and produce more of that species
51
Q

Describe transgenesis

A
  • Genetically modified animal containing a gene from another species
  • A gene or segment of DNA containing a specific gene that is transferred by a genetic engineering or genetic modification from one organisms to another
52
Q

List methods of transgenesis

A
  • Pronuclear injection
  • Embryonic stem cells or iPSC-chimeras
  • Somatic cell nuclear transfer
  • CRISPR technology (gene editing)
53
Q

Outline the process of pronuclear injection

A
  • Fertilised egg with pronuclei present
  • Inject DNA into male pronucleus
  • Transfer to oviduct of pseudopregnant female to implant
  • Produces transgenic offspring
54
Q

Outline the process of gene targeting of embryonic stem cells of iPSC

A
  • iPSC in a dish, modify cells and put into recipient to form a chimera
  • Through genetic inheritance will produce some transgenic animals
55
Q

Outline the use of CRISPR/Cas9 in gene technology

A
  • Prokaryotic system that confers resistance to foreign genetic elements e.g. plasmids, phages
  • Relies on single protein, generates double-stranded breaks at a site homologous to guide RNA (sqRNA)
  • Very efficient and rapid
  • Gene editing (not transgenics)
  • ## Can be programmed to chop DNA wherever we want
56
Q

List the agricultural/veterinary benefits of generating transgenic animals

A
  • Research
  • Biotechnology applications
  • Biopharmaceuticals
  • Xeno-transplantation
  • Nutraceuticals
57
Q

What is meant by biopharmaceuticals?

A
  • Production of pharmaceutical products in fluids of transgenic animals
  • Human anitbodies in blood of transgenic rabbits or cattle for example
58
Q

Give an example of xeno-transplantation?

A

Generation of alpha-1,3-galactosyltransferase knock-outs for using animal organs in transplantation

59
Q

What is meant by nutraceuticals?

A
  • Altering composition of animal traits such as milk, or muscle, to have increased nutritional value or tolerance for some patients
  • E.g. milk without lactose, increased omega 3-fatty acids in muscle of pigs
60
Q

Define gene

A

A discreet unit of DNA which codes for a protein

61
Q

Define locus

A

The position of a gene on a chromosome

62
Q

Define allele

A

An alternative form of a gene that produces a distinguishable phenotypic effect

63
Q

Define genotype

A

Alleles contained in an organisms cells

64
Q

Define phenotype

A

The characteristics of an organism determined by its genotype and its environment

65
Q

Define homozygous and heterozygous

A

Homo: An organism which possesses 2 identical alleles of the same gene
Hetero: an organism which possesses 2 different allels of the same gene

66
Q

Dominant allele

A

Allele which is always expresssed in the phenotype of present in the genotype

67
Q

Recessive allele

A

Allele only expressed in phenotype when dominant allele not present

68
Q

Define codominance

A

When both alleles affect the phenotype of a heterozygous individual

69
Q

What are Mendel’s laws?

A
  • Law of segregation

- Law of independent assortment

70
Q

Define the law of segregation

A

2 alleles for a heritable character separate (segregate) during gamete formation end up in different gametes

71
Q

Define the law of independent assortment

A
  • Each pair of alleles segregates independently of each other pair of alleles during gamete formation
  • Applies only to genes on different, non-homologous chromosomes
  • Genes located near each other on same chromosome tend to be inherited together
72
Q

In what situations may inhertiance of characteristics by a single gene deviate from simple Mendelian patterns?

A
  • When alleles are not completely dominant or recessive (codominance/incomplete dominance
  • When a gene has more than 2 alleles
  • When a gene produces multiple phenotypes
73
Q

What is the effect of incomplete dominance/codominance?

A

Phenotypes of hybrids is between teh phenotypes of 2 parental varieties

74
Q

Describe an example of multiple alleles in a gene

A
  • Human blood groups determined by 3 alleles for enzyme I
  • Attaches A or B carbohydrates to red blood cells
  • Can be A, B, AB or O
  • Enzyme encoded by I(a) allele adds A carbohydrate, enzyme encoded by I(b) allele adds B carbohydrate, enzyme encoded by I adds neither
  • A and B are codominant hence can also have AB
75
Q

Describe the interaction between loci

A
  • Mendelian genetics for 2 or more genes
  • Gene at one locus alters phenotypic expression of gene at second locus
  • e.g. coat colour in mice
  • One gene determines pigment colour (B for black, b for brown) other determines whether the pigment will be deposited in the hair (C for colour, c for no colour))
  • i.e. BC = black coat
  • Bc = white coat
76
Q

Explain the effect of linkage and crossig over on the phenotypic ratios from dihybrid cross over

A
  • Genes located on same chromosome that tend to be inherited together are called linked genes
  • Crossing over between linked alleles can produce recombinant individuals
  • E.g. fruit flies carry linked genes for body colour (grey or black) and wing size (normal or vestigial)
  • Can end up with grey normal, grey vestigial, black normal and black vestigial

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