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Repro Bio 3 > Assisted reproductive technologies/techniques > Flashcards

Assisted reproductive technologies/techniques Flashcards

1
Q

what is embryo transfer

A
  • in vivo technique for removal of embryos from one female (donor –> recovery) and placing them into another (transfer –> recipient)
  • transfer bit is also used for embryos produced in vitro
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2
Q

reasons for embryo transfer

A
  • increase offspring from valuable/rare animals
  • obtain offspring from female who can’t carry/deliver pregnancy
  • transport genetics around the world
  • conserve genetics of a diseased herd or establish disease-free herds (most pathogens can’t penetrate ZP)
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3
Q

the donor

A
  • superovulation with FSH or eCG
  • results in 5-6 embryos/cow flushed, but 20-25% don’t respond and give no embryos
  • start superovulation drugs when no dominant follicles
  • horses less responsive
  • polytoccus species not superovulated
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4
Q

recovery (“the flush)

A
  • performed after embryos have entered the uterus (can be done surgically with them in oviduct)
  • non-surgical in cattle, horses with catheter through cervix - lavage uterus several times, filter recovered fluid to retrieve embryos
  • sheep, goats, pigs: surgically or laparoscopically assisted procedure
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5
Q

embryo handling

A
  • embryos identified under microscope, transferred to holding media, examined for developmental stage and morphology
  • washed, prepared for transfer, cooled, or frozen
  • can be sexed through biopsy or PCR
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6
Q

recipients

A
  • healthy and synched with donor in terms of estrous cycle (day of ovulation)
  • uterus needs to be at suitable stage to accept embryo
  • ruminants: CL identified, embryo transferred to horn on ipsilateral side
  • horses: site of deposition not important
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7
Q

embryo splitting

A
  • rare –> only commercially in cattle
  • split in half at morula or blastocyst stage
  • split demi-embryos don’t have to be placed back in ZP prior to transfer
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8
Q

what is gamete intrafallopian transfer (GIFT)

A
  • involves taking oocyte from donor animal and placing it, along with sperm, into the oviduct of a synched recipient
  • recipient has her oocyte removed (or hormone-treated anestrus animal used)
  • no significant commercial use
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9
Q

what is oocyte transfer

A
  • subset of GIFT used commercially in horses
  • oocyte from donor mare using transvaginal ultrasound guided aspiration, transferred to recipient
  • recipient bred by regular AI
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10
Q

3 processes performed in IVF (technically called in vitro production - IVP)

A
  • in vitro maturation (IVM): immature oocytes to metaphase II
  • in vitro fertilization (IVF): mature oocyte incubated with sperm (capacitated in vitro)
  • in vitro culture (IVC): resultant zygote cultured through several divisions (up to blastocyst stage)
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11
Q

oocyte removal (live animals)

A
  • cattle: ultrasound guided transvaginal aspiration of antral follicles (OPU)
  • cattle: without superovulation
  • mares: only mature follicles aspirated just prior to ovulation
  • small ruminants/pigs: surgical/laparoscopic technique
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12
Q

oocyte removal (dead/anesthetized animals)

A
  • oocytes removed from ovaries at surgery, following euthanasia, at slaughter houses
  • oocytes aspirated from follicles using various techniques
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13
Q

in vitro maturation

A
  • oocytes recovered from immature follicles require IVM to metaphase II prior to attempted fertilization
  • species variation: 24-48 hours, culture media with hormones and reduced O2
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14
Q

in vitro fertilization info and problems

A
  • matured oocytes incubated with in vitro capacitated sperm
  • problems with horse (capacitation issues), dogs
  • need correct sperm concentration (pigs have issues with polyspermy)
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15
Q

techniques to aid in egg fertilization by helping sperm penetrate ZP (3)

A
  • zona drilling
  • subzonal injection (SUZI)
  • intracytoplasmic sperm injection (ICSI)
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16
Q

zona drilling

A

hole is made in the zona either mechanically with microneedle or by local application of an enzyme like trypsin

17
Q

subzonal injection (SUZI)

A

sperm is injected through zona into perivitelline sace

18
Q

intracytoplasmic sperm injection (ICSI) definition

A

sperm is injected directly into the oocyte cytoplasm

19
Q

media for culturing embryos following fertilization are classified as (3)

A
  • defined (all ingredients chemically defined, no BSA)
  • semi-defined (BSA included but all other ingredients chemically defined)
  • non-defined (serum or co-culture systems with cells like oviductal cells)
20
Q

when are oocytes generally cultured to blastocyst stage

A

prior to transfer or freezing

21
Q

steps in intracytoplasmic sperm injection (ICSI)

A
  • single sperm is injected into matured oocyte cytoplasm
  • sperm immobilized before injection
  • zygote cultured at 2-4 cell stage then transferred or continued in culture to blastocyst stage before transfer
22
Q

reasons for using intracytoplasmic sperm injection (ICSI)

A
  • technique of choice for overcoming infertility due to problems with male (don’t even need viable sperm)
  • overcomes failure of fertilization in conventional IVF without risking polyspermy
23
Q

what is somatic cell nuclear transfer (SCNT)

A
  • one of several ways of cloning

- transfer of the nucleus from a fully differentiated cell (somatic cell)

24
Q

other types of cloning (3)

A
  • embryo splitting (creates identical twins)
  • blastomere dispersal (separates cells of embryo)
  • cloning by nuclear transfer of cell nuclei from undifferentiated animals
25
Q

why SCNT (4)

A
  • re-create pets (?)
  • create clone of valuable sterile animal, use clone for breeding
  • saving endangered animals/bringing back extinct ones
  • making transgenic animals (research)
26
Q

what is the difficulty with injecting transgene into a zygote (old way of making transgenic animals)

A

-you don’t know if it will be incorporated or expressed until after animal is born

27
Q

steps in SCNT

A
  • get donor cells
  • establish cell line in culture (and introduce transgene)
  • freeze cell lines
  • oocytes to receive donor nucleus are cultured to metaphase II
  • oocyte enucleated (cytoplast)
  • donor cell (karyoplast) nuclei inserted into cytoplast (direct injection of nucleus or whole cell placement)
  • activation of cytoplast with electrical stimulation
  • embryos cultured in vitro to blastocyst stage then transferred to recipient
28
Q

major consequence of ART in cattle/sheep

A

large/abnormal offspring

29
Q

in vitro causes of large/abnormal offspring

A

use of media containing serum or use of co-culture with other cells

30
Q

in vivo causes of large/abnormal offspring

A
  • placing embryo in the ligated oviduct of another species or a non-synchronized member of the same species
  • factors that alter the uterine environment (?)
31
Q

characteristics of large/abnormal offspring from ART

A
  • increased birthweight
  • prolonged gestation
  • respiratory difficulty
  • weakness
  • inability to stand
  • enlarged umbilical cord
  • contracted flexor tendons
  • hyper/hypothermia
  • reluctance to suckle
  • abnormal organ development
  • sudden death
  • increased morbidity and mortality up to 6 months of age (then ok)
32
Q

effects of carrying large offspring from ART on dam

A

increased risk of hydroallantois due to abnormal placental development and increased risk of dystocia due to increased fetal size

33
Q

what causes large offspring from ART

A

errors in genomic imprinting (similar to beckwith-wiedemann syndrome in children)

34
Q

genomic imprinting

A
  • similar to X-inactivation only single genes are involved (not entire chromosome)
  • one copy of gene is methylated (epigenetically modified), making it inactive
  • maternally imprinted gene –> only paternal copy expressed
  • paternally imprinted gene –> only maternal copy expressed
35
Q

genetic conflict/parental investment theory of genomic imprinting

A
  • females who can restrict the growth of each fetus will be able to raise more offspring on limited resources and also protect resources for themselves (more successful)
  • males benefit if their offspring are large and strong even if it happens at expense of mother
36
Q

maternal/paternal genes and genomic imprinting

A
  • maternally expressed genes Igf2R and Gnas curb fetal growth
  • paternally expressed genes Igf2 and Peg3 promote fetal growth
37
Q

theories why some species have issues with large offspring with ART and others don’t (4)

A
  • species differences (imprinting)
  • work in human/mouse ART uses more defined media
  • timing of transfer back to synched recipient
  • litter size in polytoccous species may limit potential for overgrowth

Repro Bio 3 (6 decks)

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