(04) Assisted Reproductive Technologies

Question 1

(1)

(Embryo Transfer)

Generally refers to an in vivo technique for removal of embryos from one female (“donor” → recovery) and placing them into another (transfer → “recipient”). The actual transfer bit is also used for embryos produced in vitro. This is a well established technique in cattle and horses in North America and is used to a lesser extent in small ruminants. Swine programs exist but generally are restricted to seed stock companies. There is little interest in dogs, slightly more in cats; the interest for cats is in using them as models for endangered felid conservation programs. Also used in deer farming.

Question 2

(2)

(Embryo Transfer)

Why ET?
 Increase offspring from valuable/rare female
 Obtain offspring from female who can’t carry and deliver a pregnancy
 Transport genetics around the world
 Conserve genetics of a diseased herd or establish disease-free herds
o Most pathogens can’t penetrate the z.p. and you can wash off most of those stuck on the outside

Question 3

(3)

(Embryo Transfer)

(The Donor)

In species that generally only have 1 or 2 ovulations, superovulation of the donor with FSH or eCG (PMSG) is attempted to increase the number of ovulations and thus embryos that can be recovered at each flush attempt. Results in average of 5-6 transferrable quality embryos/cow flushed but 20-25% of cows flushed don’t respond and give no embryos.

1. Helps to start superovulation drugs when what?
2. Do horses respond to FSH from other species?
3. In Mares - superovulated give how many embryos?
4. To date, polytoccus species are generally not superovulated (you don’t pick up much in numbers).

Answer 3

1. here are no dominant follicles on the ovaries (inhibit follicle pool available to respond) but much variation appears genetic
2. no

(Horses don’t respond to FSH used in other species (porcine and ovine origin) and superovulation has not been successful till equine FSH became available in the last decade (of course now it’s off the market again).)

3. 1.8 (in comparison to 0.5 avg non)

Question 4

(4)

(Embryo Transfer)

1. Generally performed after embryos have entered what?
2. Non-surgical in cattle and horses with catheter placed through cervix, lavage uterus several times with fluid (in cows each horn is often done individually) and filter recovered fluid to retrieve embryos. Sheep, goats, pigs generally done surgically or by a laparoscopically assisted procedure. Collection catheter introduced into uterine horn via stab incision and flush solution introduced through smaller separate catheter either fed down oviduct or introduced more proximally in uterine horn.

Answer 4

1. the uterus (but can be done with them in the oviduct when using surgical techniques.)

Question 5

(5)

(Embryo Transfer)

(Embryo Handling)

Embryos identified under dissecting microscope at ~10x then transferred to holding media and examined at ~50x for developmental stage and morphology (scored on a 1-4 or 5 scale). Embryos are washed by serial transfer through several dishes of holding media and prepared for on-site transfer (all species), cooled for shipping for transfer elsewhere (equine), or frozen for storage and later transfer/sale/export (common in cattle, increasing in others). They can also be sexed by taking a biopsy (few trophoblast cells) and performing PCR.

Question 6

(6)

(Embryo Transfer)

(Recipients)

Quality of recipients is key to success of a program and they need to be not only healthy systemically and reproductively but also synchronized with the donor in terms of their estrous cycle, especially the day of ovulation; this is necessary for their uterus to be at a suitable stage to accept the embryo. For ruminants the CL is identified and the embryo is transferred to the uterine horn on this side (ipsilateral - for maternal recognition), non- surgically in cattle, surgically in sheep and goats. In horses transfer is non-surgical and site of deposition in the uterus is not critical.

Question 7

(6)

(Embryo Transfer)

(Embryo Splitting)

1. only performed commercialy in what?
2. Usually done with a microsurgical blade and inverted microscope to split embryo in half at what stage?
3. Split demi-embryos don’t have to be placed back in a zona pellucida prior to transfer. In cattle have 50% pregnancy rate for each demi embryo which = 100% for each original embryo split (on average).

Answer 7

1. cattle (an then fairly rarely)
2. morula or blastocyst stage (for latter have to split inner cell mass)

Question 8

(7)

Gamete Intrafallopian Transfer (GIFT)

1. Involves taking the oocyte from a donor animal that was either matured in vivo (taken out just before ovulation) or in vitro; and placing it, along with sperm, into what?
2. The recipient has her oocyte removed (aspiration of the dominant follicle) or a hormone-treated anestrus animal is used. No significant commercial use as such, may have problems with what?
3. Oocyte transfer is a subset of GIFT that is used commercially in horses. Used in mares in what three situations?

Answer 8

1. oviduct of a synchronized recipient
2. polyspermia (hard to get just the right number of sperm.)
3. with chronic uterine infections/oviductal issues, undiagnosed infertility and mares with repeated failure at establishing a pregnancy through ET

(Oocyte taken from donor mare generally using transvaginal ultrasound guided aspiration and transferred to a recipient (treated as above) but the recipient is bred by regular means (AI) either once before oocyte transfer or both before and after.)

Question 9

(8)

(In Vitro Fertilization)

This is often used as a blanket term but usually consists of 3 processes that are performed sequentially:

1. In vitro maturation (IVM) = ?
2. In vitro fertilization (IVF) = ?
3. In vitro culture (IVC) = ?

Answer 9

1. of immature oocytes to metaphase II
2. mature oocyte incubated with sperm (capacitated in vitro)

3 resultant zygoe is cultured through several division (often up to blastocyst stage)

Question 10

(8)

(In Vitro Fertilization)

(Oocyte Source)

1. Oocytes can be recovered repeatedly from donor cows and mares using ultrasound guided transvaginal aspiration of antral follicles (widely termed ovum pick-up OPU). This is often done on valuable infertile animals and usually without superovulation in cattle where it can be done repeatedly at 4-5 day intervals. These oocytes require what to mature?
2. In mares a similar system can be used (with OPU performed less frequently) and IVM employed; but often only mature follicles are aspirated just prior to ovulation so that the oocyte is matured in vivo prior to the aspiration. In small ruminants and pigs surgical or laparoscopic techniques are used.

3 Oocytes can also be recovered from ovaries removed at surgery, following euthanasia or at slaughter houses

the first two are used to do what?

latter used for?

Answer 10

1. culture
2. preserve genetics from valuable animals with terminal injuries or illness

used for research or to provide oocytes for cloning
purposes

(In these cases oocytes are aspirated from follicles with either syringe and needle, by ovarian slicing and/or by scraping the lining of the follicle with something like a bone curette – technique depends on species (how tightly the cumulus-oocyte complex is bound to the follicle wall) and lab preference.)

Question 11

(11)

(In Vitro Fert)

(In Vitro Maturation)

1. Oocytes recovered from immature follicles (anything other than a mature follicleimmediately before ovulation) require what prior to attempted fertilization?

Answer 11

1. IVM to metaphase II

(Success rates, culture conditions and time required vary with species but the process generally takes 24-48 hours and uses tissue culture media supplemented with FSH, LH, estradiol and serum with incubation taking place in an atmosphere of reduced oxygen (~5% O2, similar to IVC).)

Question 12

(12)

(In Vitro Fert)

(In Vitro Fertilization)

1. Matured oocytes are incubated with what?
2. Success varies widely across species with the horse and dog having very poor results. At least in the horse part of the problem has been defining repeatable techniques for what?
3. The other problem with successful IVF is what?

which animal has big problem with polyspermy? due to what?

Cattle are the best studied species and here there is considerable variation between bulls in the optimal sperm number needed to achieve fertilization but avoid polyspermy.

4. Several techniques have been used to aid fertilization of the egg by helping sperm penetrate the zona pellucida or bypass it when “regular” IVF isn’t working. These include what three things?

Answer 12

1. in-vitro capacitated sperm (they need capacitating because they are not exposed to the female tract)
2. sperm capacitation
3. getting right amount of sperm (too few = failed fert, too many = polyspermy)

pig; delayed development of zona hardening

4. Zona drilling – hole is made in the zona either mechanically with a microneedle or by local application of an enzyme like trypsin

Subzonal injection (SUZI) – sperm is injected through the zona into the perivitelline space

Intracytoplasmic Sperm Injection (ICSI) – sperm is injected directly into the oocyte cytoplasm (see later)

Question 13

(13)

(In Vitro Fert)

(In Vitro Culture)

Media for culturing embryos following fertilization are classified as:

1. Defined = ?
2. semi-defined = ?
3. non-defined = ?
4. In domestic animals, embryos are generally cultured to what stage prior to transfer or freezing?
5. The entire IVP process is only widely used in cattle where: of immature oocytes starting the IVP process 30-40% make it to the blastocyst stage (get through IVM, IVF and IVC). IVP embryos have fewer cells in the inner cell mass than comparable in-vivo derived embryos and an increased percentage of polyploid cells.
6. Following transfer into recipients, pregnancy rates for IVP embryos are lower or higher than with in vivo embryos?

Answer 13

1. all ingredients are chemically defined - no serum or bovine serum albumin (BSA).
2. : BSA included but all other ingredients are chemically defined (BSA is more defined than serum).
3. serum (undefined because contains hormones, growth factors etc in undefined amounts and may be virally contaminated)

or

co-culture systems with cells like oviductal cells or use of media “conditioned” by inclusion of the products of these cells.

4. blastocyst
5. lower

Question 14

(14)

(Intra Cytoplasmic Sperm Injection (ICSI))

1. What occurs in this technique?
2. In some cases this injection is sufficient to activate the oocyte from its second arrest (metaphase II) but in some cases additional treatments are used to ensure oocyte activation (generally something that alters calcium).
3. Following injection the zygote is generally cultured at least as far as what stage?

then what happens?

4 This is the technique for overcoming what?

Answer 14

1. single sperm is injected into the oocyte cytoplasm (of matured oocyte)

(The sperm is generally “immobilized” before injection (read: held down, beaten up and tail generally partly chopped off).)

3. 2-4 cell stage

is then either surgically transferred to the oviduct of the recipient or is continued in culture to the blastocyst stage before transfer

4. g infertility due to problems with the male (“male factor infertility”) and they don’t even need to produce viable sperm in the ejaculate

(Pregnancies have been obtained using immature sperm aspirated from the epididymis and even from spermatids and spermatocytes aspirated form the testes. It also overcomes failure of fertilization in conventional IVF programs without risking polyspermy (it’s starting to be used commercially in horses where conventional IVF generally fails).)

Question 15

(15)

Somatic Cell Nuclear Transfer (SCNT) a.k.a. Cloning

FUCK IT - just read the rest - start at page 232