Assisted Reproductive Techniques

Question 1

what is assisted reproduction

Answer 1

• anything above what a natural breeding is
• or
• alter normal reproduction processes or function

Question 2

what are examples of assisted reproduction?

Answer 2

• hormonal management
• collection and preservation of gametes
• AI
• embruo transfer
• in vitro fertilization
• cloning
• intractyoplasmic sperm inj

Question 3

what is superovulation?

Answer 3

stimulation of development and ovulation of multiple follicles
- usually accompanied by adminsitration of gonadotropin hormones: FSH (pRSH, oFSH, eFSH) , eCG (PMSG)

Question 4

what is superovulation used for?

Answer 4

ruminants for embryo transfer; limited efficacy in horse

Question 5

how effective is superovulation in cattle?

Answer 5

may yield 10-15 embryos, of which 5-6 or more are suitable for transfer

Question 6

what is embryo transfer?

Answer 6

procedure in which an embryo is removed from the uterus of one female (donor) and transferred into the uterus of a surrogate female (recipient)

Question 7

what is so important about embryo transfer?

Answer 7

• first few steps have to happen!! need to get “a little bit pregnant”
• cow or horse has to grow 1+ follicles
• have to ovulate
• fertilization has to occur in the oviduct
• embryo has to transfer down oviduct and enter a uterus that has a healthy uterine environment!! all of that has to happen

Question 8

as a vet we don’t have access to the embryo until it

Answer 8

leaves the oviduct and enters the uterus: in cattle and horses this takes 6-8 days for the embryo to be removed from teh uterus

Question 9

fertilization and early embryonic development for embryo transfer takes place where?

Answer 9

in the “donor” oviduct and uterus

Question 10

what is the technique for embryo collection?

Answer 10

• non surgical transcervical uterine lavage
• filling uterus with fluid and lavaging it and recovering fluid

Question 11

what is short term storage of embryos?

Answer 11

• cooled
• decrease metabolism to prolong survival
• 24 hour storage

Question 12

what is embryo recovery technique?

Answer 12

• catheter passed thru cervix
• catheter pulled back against internal os
• fluid infused into uterus by gravity flow
• recovered thru a filter
• fluid in filter cup poured into petri dish
• filter cup rinsed
• drops of holding media added into petri dish
• embryo moved from drop to drop, changing straws between moves

Question 13

what is long term storage?

Answer 13

• frozen
• liquid nitrogen
• forever storage

Question 14

how common is surgical embryo transfer done?

Answer 14

rare. small hole placed thru uterus from a flank incision. pipette gets guided into lumen of uterus and deposits it there

Question 15

how does embryo freezing work?

Answer 15

• embryos collected from the uterus are placed in a cryoprotectant (glycerol or ethylene glycol) and stored in liquid nitrogen
• frozen thawed embryos are transferred at a later date (potentially years later)

Question 16

can you sex embryos?

Answer 16

• embryo biopsy can be used to determine sex. old school way is to slice thru embryo and using DNA in that slice to determine if it is male/female
• aspiration of blastomeres. or can use a micro manipulator and take a couple cells
• biopsy of blastomeres

Question 17

with cutting horses what sex is preferred?

Answer 17

fillies

Question 18

what is in vitro fertilization?

Answer 18

procedure by which an oocyte is fertilized outside the oviduct
- first calf born from IVF in 1982
- first foal born from IVF in 1990

Question 19

how is in vitro performed?

Answer 19

• oocytes collected by a transVAGINAL ultrasound probe and needle aspiration system. each large follicle gets aspirated. might get 30 oocytes from pair of ovaries!!
• oocyte and capacitated sperm are palced together in petri dish
• fertilization and embryonic development proceeds

Question 20

what is intracytoplasmic sperm injection? (ICSI)

Answer 20

• single sperm injected into the cytoplasm of an oocyte using a micromanipulator
• if cleavage and embryonic development continues, the embryo is transferred

Question 20

what are limitations of in vitro fertilization?

Answer 20

• not successful in all species
• “blocks” to embryonic development may occur at certain stages in various species

Question 21

what is the timeline with ICSI produced embryos?

Answer 21

after sperm injection, embryo cells divide every 12-24 hours. monitor to see if they continue to divide to form a blastocyst
- transfer at late morula to blastocyst stage into a “synchronized” recipient
- alternates: freeze, biopsy, ship, autotransfer

Question 23

what has more DNA content: X bearing sperm or Y bearing sperm?

Answer 23

X bearing sperm: burn brighter than males

Question 24

what is a clone?

Answer 24

cell or organism that is genetically identical to another cell or organism

Question 25

why might clone calves have different color markings?

Answer 25

the migration of cells that forms the melanin or color patterns don't migrate the same way in every individual. they are genetically identical, but not physically identical

Question 26

how is cloning performed?

Answer 26

transplanting the nucleus of one cell into an oocyte from which the nucleus has been removed. gets activated with some charge and then offspring produced

Question 27

if a genetically valuable male has a catastrophic injury or disease and is going to be put down, how do you save his sperm?

Answer 27

billions of sperm are stored in the tail of the epididymis and vas deferens - stallion or bull castrated leaving the testis and epididymis intact - as much of the vas deferens as possible is saved - rinsed and placed in a plastic bag - then shipped cooled (not frozen) to the referral center - need to be processed within 12-24 hours!! - sperm flushed from epididymis and counted - freezing extender is added, sperm frozen using standard techniques - can get 5-25 breeding doses

Question 27

from what part of the testicle is sperm harvested post mortem?

Answer 27

tail of the epididmysi

Question 28

can you salvage oocytes from mares postmortem?

Answer 28

yes- can be recovered from excised ovaries for transfer into recipients upon death of a valuable mare - ovaries removed, rinsed and placed in sleeve. - transported at a referral center - should be maintained at ambient temp- NOT frozen or cooled!!

Question 29

how are oocytes collected postmortem?

Answer 29

collected by aspiration, scraping the follicular wall or slicing the ovary. recovered oocytes cultured for 24-30 hours