Assisted reproductive technologies/techniques Flashcards
what is embryo transfer
- 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
reasons for embryo transfer
- 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)
the donor
- 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
recovery (“the flush)
- 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
embryo handling
- 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
recipients
- 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
embryo splitting
- 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
what is gamete intrafallopian transfer (GIFT)
- 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
what is oocyte transfer
- subset of GIFT used commercially in horses
- oocyte from donor mare using transvaginal ultrasound guided aspiration, transferred to recipient
- recipient bred by regular AI
3 processes performed in IVF (technically called in vitro production - IVP)
- 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)
oocyte removal (live animals)
- 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
oocyte removal (dead/anesthetized animals)
- oocytes removed from ovaries at surgery, following euthanasia, at slaughter houses
- oocytes aspirated from follicles using various techniques
in vitro maturation
- 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
in vitro fertilization info and problems
- matured oocytes incubated with in vitro capacitated sperm
- problems with horse (capacitation issues), dogs
- need correct sperm concentration (pigs have issues with polyspermy)
techniques to aid in egg fertilization by helping sperm penetrate ZP (3)
- zona drilling
- subzonal injection (SUZI)
- intracytoplasmic sperm injection (ICSI)
zona drilling
hole is made in the zona either mechanically with microneedle or by local application of an enzyme like trypsin
subzonal injection (SUZI)
sperm is injected through zona into perivitelline sace
intracytoplasmic sperm injection (ICSI) definition
sperm is injected directly into the oocyte cytoplasm
media for culturing embryos following fertilization are classified as (3)
- 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)
when are oocytes generally cultured to blastocyst stage
prior to transfer or freezing
steps in intracytoplasmic sperm injection (ICSI)
- 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
reasons for using intracytoplasmic sperm injection (ICSI)
- 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
what is somatic cell nuclear transfer (SCNT)
- one of several ways of cloning
- transfer of the nucleus from a fully differentiated cell (somatic cell)
other types of cloning (3)
- embryo splitting (creates identical twins)
- blastomere dispersal (separates cells of embryo)
- cloning by nuclear transfer of cell nuclei from undifferentiated animals
why SCNT (4)
- re-create pets (?)
- create clone of valuable sterile animal, use clone for breeding
- saving endangered animals/bringing back extinct ones
- making transgenic animals (research)
what is the difficulty with injecting transgene into a zygote (old way of making transgenic animals)
-you don’t know if it will be incorporated or expressed until after animal is born
steps in SCNT
- 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
major consequence of ART in cattle/sheep
large/abnormal offspring
in vitro causes of large/abnormal offspring
use of media containing serum or use of co-culture with other cells
in vivo causes of large/abnormal offspring
- placing embryo in the ligated oviduct of another species or a non-synchronized member of the same species
- factors that alter the uterine environment (?)
characteristics of large/abnormal offspring from ART
- 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)
effects of carrying large offspring from ART on dam
increased risk of hydroallantois due to abnormal placental development and increased risk of dystocia due to increased fetal size
what causes large offspring from ART
errors in genomic imprinting (similar to beckwith-wiedemann syndrome in children)
genomic imprinting
- 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
genetic conflict/parental investment theory of genomic imprinting
- 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
maternal/paternal genes and genomic imprinting
- maternally expressed genes Igf2R and Gnas curb fetal growth
- paternally expressed genes Igf2 and Peg3 promote fetal growth
theories why some species have issues with large offspring with ART and others don’t (4)
- 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
