Assisted reproductive technologies Flashcards
Define In vitro Fertilisation
The process by which an oocyte is fertilised by a sperm outside teh body
Why might IVF be used in animals?
- To produce embryos to be used in breeding programs (genetic improvement)
- Conservation projects to produce embryos from endangered species
- In all animals for research
Describe the IVF procedure
- Oocytes and sperm collection
- Mix oocytes and sperm in culture dish
- Allow fertilisation to occur
- Monitor embryo development
- Transfer embryos in surrogate recipients
Describe how oocytes are collected for IVF in different species (specifically humans, ruminants, wild animals)
- Human: following superovulation
- Bovine and ovine: ovum pick:up during follicular selection (oocyte maturation required)
- Wild animal oocytes: OPU or post-mortem
Describe intra-cytoplasmic sperm injection
- Procedure involving injecting sperm into an oocyte thus bypassing normal fertilisation
- Normally in human medicine, used in animals for research
Define embryo transfer
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
What are the advantages of embryo transfer?
- 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
What are the disadvantages of embryo transfer?
- 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
What are important characteristics of donors for embryo transfer?
- Fertile semen donor
- Fertile female donor
- Female needs to be responsive to superovulation
- Multiple embryos produced
What are important characteristics of the embryo transfer recipient?
- Cycle synchronised with donor
- Reproductively sound
- Ideally same size or bigger than donor
Describe superovulation
- Stimulation and ovulation of multiple oocytes rather than just one or two
- Common procedure in cow
- Not possible in horse currently
Describe the method for superovulation in cows
- 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
Describe the gonadotrophins used for superovulation in cattle
- 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
What are the limitations of gonadotrophins for superovulation?
- 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
What are the possibilities and limitation of superovulation in the cow?
- Donor repeatedly superovulated at 6-8 week intervals
- Ovarian response of individual highly variable
Describe the process of embryo collection and transfer
- 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
What stage of the cycle should the recipient be in relation to the donor for embryo transfer
- 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
Describe the method for cycle synchronisation of recipient to donor
- 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
Describe the use of PGF2a for syncrhonisation of recipient and donor cycles
- 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
Describe the insemination process for embryo transfer
- 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
Describe embryo collection for transfer
- 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
What is the disadvantage of embryo collection for transfer in the cow later than 8 days post-ovulation?
- Embryo will be bigger and elongated
- More fragile once hatched from ZP
Describe embryo searching
- Embryos need to be located
- Graded under dissecting microscope
- Warm room
- Warm slide
- Selected based on quality
- Transferred to recipient or chilled/frozen
Describe the physical transfer of an embryo into the recipient
- Oestrus cycle closely synchronised to within 24 hours
- Transfer to uterine horn on same side as CL
- Surgical or non-surgical
What is the techniques for surgical transfer of an embryo?
Flank incision, local anaesthetic, blunt puncture of horn
What is the technique of non-surgical transfer of an embryo?
- Aseptic technique
- Transfer using fine pipette or catheter
- Highly skilled to avoid trauma to uterine endometrium (as would trigger release of PGF2a)
Outline the key points in embryo survival in embryo transfer
- 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
What are the key points for a successful embryo transfer?
- Fertile donor and quality recipients
- Quality embryos
- Cleanliness
- Gentle manipulation of cervix
Give examples of embryo manipulation
- Splitting (identical siblings produced)
- Sexing
- IVF
- Cloning from somatic cells
- Cooling and cryopresevation
Define stem cells
Unspecialised cells with capacity to self-renew for long periods
Define self-renewal
- Ability of stem cells to divide and maintain undifferentiated state
- Not just proliferation
- Make copy of selves to produce stem cell or differentiated cell
Describe embryonic stem cells
- From embryo
- Can form any type of cell in body, but not capable of developing into new organism
Describe tissue stem cells
- Derived from foetal or adult tissues
- Sustain turnover and repair throughout life in some tissues
Where can foetal stem cells be derived from?
- Cord blood
- Amniotic fluid
- Foetal liver
Where can adult stem cells be derived from?
- Neural
- Haematopoietic
- Spermatogonial
- Mesenchymal
- Epidermal
Define totipotent
Ability to form an entire organism e.g. embryo up to morula stage
Define pluripotent
Able to form all body’s cell lineages, including germ cells e.g. inner cell mass cells of blastocyst
Define multipotent
- Able to form multiple lineages that consitute an entire tissue or tissues
- E.g. haematopoietic stem cells, neural stem cells
How are embryonic stem cells generated?
- Derived from inner cell mass cells
- Derived from epiblast
- Derived from primordial germ cells
List the potential medical and veterinary uses of stem cells
- Cell based therapies
- Drug developing and screening
- Disease models
- Study of early development
- Development of new gene therapy methods
- Mesenchymal stem cells for therapies
Outline the use of mesenchymal stem cells for therapies
- Stem cells derive from bone marrow or fat
- Treatment of tendon and ligament injury in horses and dogs
- Osteoarthritis
What is the major risk in non-autologous stem cell technologies?
- Immune rejection
- Possible solution is reprogramming
- Process of reversal of differentiation of somatic cells into autologous pluripotent cells
Describe autologous cell therapies
- 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
What is somatic cell nuclear transfer?
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
Describe the process of somatic cell nuclear transfer
- 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
What are the problems with cloning?
- 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
Outline the difference between an identical twin and a clone
- Twin: same genetic material
- Clone: similar genetic material i.e. same nuclear DNA but different mitochondiral DNA
Compare therpeutic and reproductive cloning
- Therapeutic: aim is to derive stem cells from cloned embryo
- Reproductive: aim is to produce cloned embryo to develop into cloned animal
Describe induced pluripotent stem cells (iPS cells)
- 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
Give advantages of induced pluripotent stem cells
- 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
Describe transgenesis
- 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
List methods of transgenesis
- Pronuclear injection
- Embryonic stem cells or iPSC-chimeras
- Somatic cell nuclear transfer
- CRISPR technology (gene editing)
Outline the process of pronuclear injection
- Fertilised egg with pronuclei present
- Inject DNA into male pronucleus
- Transfer to oviduct of pseudopregnant female to implant
- Produces transgenic offspring
Outline the process of gene targeting of embryonic stem cells of iPSC
- iPSC in a dish, modify cells and put into recipient to form a chimera
- Through genetic inheritance will produce some transgenic animals
Outline the use of CRISPR/Cas9 in gene technology
- 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
List the agricultural/veterinary benefits of generating transgenic animals
- Research
- Biotechnology applications
- Biopharmaceuticals
- Xeno-transplantation
- Nutraceuticals
What is meant by biopharmaceuticals?
- Production of pharmaceutical products in fluids of transgenic animals
- Human anitbodies in blood of transgenic rabbits or cattle for example
Give an example of xeno-transplantation?
Generation of alpha-1,3-galactosyltransferase knock-outs for using animal organs in transplantation
What is meant by nutraceuticals?
- 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
Define gene
A discreet unit of DNA which codes for a protein
Define locus
The position of a gene on a chromosome
Define allele
An alternative form of a gene that produces a distinguishable phenotypic effect
Define genotype
Alleles contained in an organisms cells
Define phenotype
The characteristics of an organism determined by its genotype and its environment
Define homozygous and heterozygous
Homo: An organism which possesses 2 identical alleles of the same gene
Hetero: an organism which possesses 2 different allels of the same gene
Dominant allele
Allele which is always expresssed in the phenotype of present in the genotype
Recessive allele
Allele only expressed in phenotype when dominant allele not present
Define codominance
When both alleles affect the phenotype of a heterozygous individual
What are Mendel’s laws?
- Law of segregation
- Law of independent assortment
Define the law of segregation
2 alleles for a heritable character separate (segregate) during gamete formation end up in different gametes
Define the law of independent assortment
- 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
In what situations may inhertiance of characteristics by a single gene deviate from simple Mendelian patterns?
- 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
What is the effect of incomplete dominance/codominance?
Phenotypes of hybrids is between teh phenotypes of 2 parental varieties
Describe an example of multiple alleles in a gene
- 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
Describe the interaction between loci
- 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
Explain the effect of linkage and crossig over on the phenotypic ratios from dihybrid cross over
- 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
