Download IVF Lab Key - UF Animal Science

ANS 3319C Reproductive Physiology and Endocrinology Lab
Take Home Message: In vitro Fertilization & Embryo Transfer (25 pts: Spring, 2014)
Name: _________________________________
Lab Day & Time: _____________
1) Please list the four general steps involved in In vitro production of embryos (2 pts)?
a) Collection of immature oocytes c) Fertilization of mature oocytes b) Maturation of immature oocytes d) Culture of embryos 2) In vitro production of embryos for most mammalian species is similar to the methods used in farm
animals. Please answer the following questions as they relate to in vitro embryo production.
a) When collecting oocytes from live and (or) dead bovine donors, several methods are used to
collect oocytes. Please indicate what they are for both the live and dead donors (1 pt).
Oocytes can be collected from live animals either surgically or by transvaginal aspiration. Oocytes can be collected from the ovaries of dead animals either by aspiration or slashing the surface. b) In question 2a you indicated how & when oocytes are collected in the bovine; in contrast, when
would you collect oocytes in the human and horse relative to the step of in vitro fertilization (1 pt)?
It is more common to allow the oocytes to mature in vivo so the oocytes would not be collected until they are ready to be fertilized. c) What type of items would you add to the in vitro maturation media to assist the oocytes in their
development (2 pts)?
The typical maturation medium will include several components including nutrients, gonadotrophins and other hormones, and antibiotics. d) What general process must sperm cells be assist with in before fertilization of oocytes can occur?
A similar process that happens in vivo (1 pt)?
Capacitation e) What process is used as an alternative to in vitro fertilization when sperm quality is poor (1 pt).
Intra-­‐cytoplasmatic sperm injection (ICSI) f)
Although most domestic embryo culture occurs in vitro up to the blastocyst stage (day 7 of
development) before transfer into a recipient, why are human embryos transferred earlier? What
is the disadvantage to this (2 pts)?
They are transferred earlier to avoid the negative effects that prolonged in vitro culture has on the embryo (growth arrest, improper genome activation, large offspring syndrome). The drawback of this procedure is the need to transfer multiple embryos to increase the chances of establishing pregnancy which increases the chance of causing a multiple pregnancy (twins, triplets, quadruplets or more). Continued on the back!!!!!
3) Transfer of embryos into a recipient is the last step of an in vitro embryo production program. In vitro
production of embryos is not always the most practical option when trying to generate multiple
embryos from a live donor. Superovulation is used in conjunction with embryo transfer when live
animals are used.
a) What is the primary purpose of superovulation in the bovine? What is the drug of choice used?
What is the general administration schedule for this drug (2 pts)?
The purpose of superovulation is to generate as many ovulations and eventually embryos from a live donor. The drug of choice is FSH. The FSH is typically given twice a day over a four day period. b) Why do you think that superovulation is not a commonly used practice in horses (2 pts)?
Horses ovulate embryos from a single location on the ovary, which is the ovulation fossa. Therefore, it would be difficult to ovulate multiple embryos from an ovary.
4)
Please list the five basic steps in sequential order from start to finish of a bovine superovulation and
embryo transfer program that produces embryos for freezing and eventually direct transfer to
recipients. Additionally, answer the following questions and place your answers in the appropriate
step you have listed below: 1) Please list the cryoprotectant of choice that would be used in the
program and indicate what its primary function is. 2) What day of the estrous cycle are embryos
collected and what is the name of the device used to non-surgically collect them? 3) Embryos are
evaluated based on two different categories, please list them. Additionally, indicate which embryos
would be candidates for freezing based on the assumption that the embryos are classified as
morulas and blastocysts. 4) What day of the estrous cycle are embryos transferred into recipients
and where are the embryos placed in the reproductive tract (10 pts)?
a) Superovulation of the donor b) Flushing embryos from the donor The embryos are typically flushed on d 6 or 7 with a foley catheter c) Evaluation of embryos Embryos are classified on stage of development and graded based on their quality. Only embryos that are excellent or fair are typically frozen. d) Freezing embryos The cryoprotectant of choice is ethylene glycol and it is used to dehydrate the embryos to prevent ice crystal formation. e) Transfer of embryo into a recipient. Embryos are typically transferred on day 7 of the estrous cycle and are placed 1/3 the way up the uterine horn on the same side as the corpus luteum. 5) What are the success rates, based on pregnancy rates, for transfer of frozen bovine embryos and
transfer of fresh non-surgical derived embryos in horses (1 pt)?
Cows (Frozen only) 50-­‐70% and Horses (Fresh only) 45-­‐65%