World Veterinarian Quiz 36

 

Steps involved in ETT

1.  Selection of donor

2.  Selection of recipient

3.  Estrus synchronization of donor and recipient

4.  Superovulation

5.  Artificial insemination of donor

6.  Embryo collection

7.  Evaluation of embryo

8.  Transfer of embryo/cryopreservation of embryo

 

1.  Selection of donor

⇨ Superior individual performance/high yielder

⇨ Good productive performance of offsprings

⇨ Regular cyclicity

⇨ Ovaries must be free (no adhesions)

⇨ Intact tubular genitalia (free from any sort of abnormalities)

⇨ Younger (4-8 yrs of age), should have completed at least one standard lactation.

⇨ Healthy and have good body weight

⇨ Must have calved at least 60 days back (best 90-100 days postpartum)

 

2.  Selection of recipient

⇨ Healthy, free from infection and have good body weight

⇨ Regular cyclicity Reproductive cycle length should be normal.

⇨ Intact genitalia (free from any sort of abnormalities)

⇨ Must have good cyclic CL of desired stage at the time of embryo transfer.

3.  Synchronization

4.  Superovulation

⇨ Superovulation is the procedure for increased ovulatory response by administration of hormones (gonadotrophins) to produce several ova instead of one which is normally produced at each estrus.

⇨ In the ewe, doe and cow, an average of 12 ovulations can be expected. In sows, the number of ovulation could be > 20.

⇨ In mares, reliable superovulation has not yet been achieved because ovulation occurs at one site on the ovary, the ovulation/ovarian fossa.

Time of superovulation?

⇨ For optimum response gonadotrophin treatment is initiated during mid-luteal phase i.e. on days 9-14 (if we consider day 0 as estrus) of a normal estrous cycle.

⇨ Donor cows can be superovulated repeatedly at approximately 6-8 weeks intervals with no adverse effect on subsequent fertility.

⇨ Some cows continue to respond up to ten treatments but most of the cows becomes refractory and give poor response.

⇨ The reason for this may be an immune reaction in which antibodies are formed against the foreign protein (peptide hormone) used for superovulation.    

Which Hormones are used for superovulation?

•         PMSG/eCG

•         FSH

 

PMSG/eCG

⇨ A potent placental gonadotrophin

⇨ Secreted by endometrial cups of pregnant mare uterus between day 40 – 140 of gestation.

⇨ Possessing higher contents of carbohydrates specially sialic acid.

⇨ Longer biological half life (50-120 hrs).

⇨ Possesses both FSH and LH biological activities but FSH like action is being dominant.

⇨ Used as a single IM injection during mid luteal phase of the cycle followed by IM administration of normal luteolytic dose of PGF₂ alpha 48-72 hr after the injection of PMSG.

⇨ Ovulation, embryo recovery rate and number of transferable embryo per donor using PMSG has been found low.

FSH

⇨ A gonadotrophin secreted by delta basophilic cells of the anterior-pituitary gland.

⇨ Has lesser sialic acid contents than PMSG, this accounts for its shorter biological half live (2-4hrs).

⇨ FSH stimulates growth and maturation of follicles by the initiation of granulosa cell mitosis, formation of antrum, follicular fluid and inducing granulosa cell sensitivity to LH by increasing LH receptors.

⇨ FSH rescues follicles to become atretic.

⇨ FSH is used in multiple doses, twice daily morning and evening for 3 or 4 days in constant or tapering dose schedule.

⇨ Treatment is initiated during mid luteal phase of the cycle.

⇨ Normal luteolytic dose of PGF2 alpha in injected 48-72 hours following start of 1^st injection of FSH.

⇨ Superovulation response and number transferable embryo recovery have been found good.

⇨ In general superovulation response and number of transferable embryo recovery has been lower in buffalo than cattle.

 

Superovulation protocol for cow

⇨ FSH

⇨ Day 9:

•         Day of estrus is considered as day 0.

•         Rectal palpation of donor to confirm presence of CL.

•         Donor must have a good corpus luteum prior to start of superovulation.

Day	Morning	Evening
Day 10	4.5 mg	4.5 mg
Day 11	4.5 mg	4.5 mg
Day 12	4.5 mg	4.5 mg
Day 13	4.5 mg + 25 mg PGF2 alpha IM	4.5 mg
Day 14	Detection of estrus
Day 15	Majority of donors will be in estrus. This day = Day 0 (Superovulatory estrus)
Day 1	AI
Day 6	Rectal palpation of donor ovary for assessing superovulation response. Handle the donor very carefully and examine very quickly.
Day 7	- Prepare fresh Dulbeccos Phosphate buffered saline (DPBS). - Flushing of donor for collection of embryo. - Expacted stages of embryos: Compact morula and early blastocyst.

 

⇨ PMSG

⇨ Day 9:

•         Day of estrus is considered as day 0.

•         Rectal palpation of donor to confirm presence of CL on any one of the ovary.

•         Donor must have a good corpus luteum prior to start of superovulation.

Day
Day 10	Inject 2500 IU eCG/PMSG IM in the morning.
Day 13	Inject 25 mg PGF2 alpha IM in the morning.
Day 14	Observe for the estrus symptoms.
Day 15	Majority of donors will be in estrus. This day = Day 0 (Superovulatory estrus)
Day 1	AI in the morning.
Day 6	- Rectal palpation of donor ovary for assessing superovulation response. - Handle the donor very carefully and examine very quickly.
Day 7	- Prepare fresh Dulbeccos Phosphate buffered saline (DPBS) – Flushing medium. - Flushing of donor for collection of embryo.

 

Superovulation protocol for buffalo

FSH

Day 9:

•         Day of estrus is considered as day 0.

•         Rectal palpation of donor to confirm presence of CL.

•         Presence of a good CL is mandatory for initiation of superovulation treatment.

Day	Morning	Evening
Day 10	5 mg	5 mg
Day 11	5 mg	5 mg
Day 12	5 mg + 25 mg PGF2 alpha IM	5 mg
Day 13	5 mg	5 mg
Day 14	Detection of estrus
Day 1	AI
Day 6	- Rectal palpation of donor ovaries for assessing superovulation response. -       Flushing of donor using DPBS for collection of embryo. -       Expected stage of embryos: Morula.

PMSG

Day 9:

•         Day of estrus is considered as day 0.

•         Rectal palpation of donor to confirm presence of CL on any one of the ovary.

•         Presence of a good CL is mandatory for initiation of superovulation treatment.

Day
Day 10	Inject 2500 IU eCG/PMSG IM in the morning.
Day 12	Inject 25 mg PGF2 alpha IM in the morning.
Day 13	Observe for the estrus symptoms.
Day 0	Donors will be in estrus. This day = Day 0 (Superovulatory estrus)
Day 1	AI in the morning.
Day 6	- Rectal palpation of donor ovaries for assessing superovulation response. -       Flushing of donor using DPBS for collection of embryo.

 

⇨ Ovulation rate, superovulation response, embryo recovery and their quality have been found low using PMSG/eCG than FSH.

5.  Insemination of donor

⇨ Confirm estrus on per rectal examination by observing relaxation of cervix,  Donor should be inseminated artificially 2-3 times at 10-12 hours interval, beginning at 8-10 after the onset of estrus. This is required because ovulation can occur over an extended time period.

⇨ Fresh semen is preferred.

⇨ If frozen semen- then use double insemination dose at each insemination.

6.  Embryo recovery

⇨ Embryo can be collected by following methods

–        Surgical method

–        Non-surgical method

–        Laparoscopy

Surgical collection of embryo

⇨ Embryos can be collected by surgical method from the oviducts or uterus.

⇨ The recovery rate of embryos are better using surgical method of collection of embryo.

⇨ This approach is the method of choice in sheep and goat, however, in larger animals, the non-surgical method of collection of embryo from the uterus is preferred.

⇨ Surgical method is most often used in sheep, goat and swine through mid-ventral incision under general anaesthesia.

⇨ The method can be performed on day 3-4 after estrus in sheep and goat (8 -cell embryo or less) and on 2-3 days after estrus in swine (4-cell stage).

⇨ Embryo/ova remain in the oviduct 3-4 days after estrus and ovulation and then they migrate to the uterus.

⇨ Therefore, if collection is aimed from oviduct operation is performed 72 hours following mating/AI.

⇨ Uterine embryos can be collected by flushing of uterus on day 5 after mating.

Surgical approaches

1. Flushing oviduct toward fimbria
2. Flushing oviduct toward uterotubal junction
3. Flushing uterus toward base of the uterine horn

  A. Flushing oviduct toward fimbria

⇨ Oviductal embryo/ova can be flushed from the fimbriated end of oviduct by putting plastic catheter from the fimbriated end of oviduct and flushing fluid is introduced using a syringe and needle from the uterotubal junction. This approach is usually preferred.

    B. Flushing oviduct toward uterotubal junction

⇨ Embryo from oviduct can also be collected in reverse way by introducing fluid from the fimbriated end of oviduct.

   C. Flushing uterus toward base of the uterine horn

⇨ Uterine horns are flushed by introducing flushing medium from the uterotubal junction and collection of fluid from the base of uterine horn.

Surgical collection in cattle & buffalo

⇨ Following laparotomy incision at flank, reproductive tract is exposed. 

⇨ A clamp or thumb and forefinger can be used to block the distal one-third of the uterine horn so that fluid (flushing medium i.e. PBS, 20 ml) injected in to that segment can be forced through the oviduct with gentle milking action and collected at the infundibulum.

⇨ An alternate approach is to occlude uterine horn at the body of uterus, with culture medium being introduced through a puncture at the uterotubal junction or through the oviduct until uterus is turgid.

⇨ The uterus is then punctured with a blunt needle attached to a flexible catheter. The pressure will cause the medium to gush through the catheter turbulence to carry the embryos into a collection tube.

Advantages

⇨ Animal is immobilized

⇨ Genitalia can be manipulated directly

⇨ Only 20-50 ml of medium is required

⇨ Procedure can be carried out prior to 5^th day of estrous cycle

Disadvantages

⇨ The surgical trauma results adhesions of genitalia and there is risk of life.

Non-surgical collection (Transcervical method)

⇨ Commonly used in cattle, buffalo and mare.

⇨ This involves two ways or three ways Foley’s catheter which allows flushing fluids to pass into the uterus and at the same time allows fluids to be returned from the uterus to a collecting receptacle. 

⇨ A small balloon near the end of catheter can be inflated just inside the uterine horn to prevent the flushing fluid from escaping through the cervix.

⇨ Bovine embryo descends to the uterus during 4-5 days after estrus and it hatches (shed zona pellucida) on 8-10 days. Therefore, collection of embryo should be made at 6-8 days post-breeding at compact morula or blastocyst stage. 

⇨ The collection of embryo before this time may lead to low recovery of embryos due to their presence in oviduct whereas, late collection may cause presence of hatched blastocyst which is not suitable for transfer.

⇨ In mare, the embryo collection is made at 6-7 days post-ovulation at blastocyst stage.

⇨ The best flushing medium for embryo collection for most of the species is Dulbecco’s Phosphate Buffer Saline (DPBS). Embryos can be kept in this medium for at least 8 hours with no loss of viability and can be cultured for up to 48 hours with acceptable results on transfer.

⇨ During final collection, oxytocin is administered @ 50 i.u. i/v. The donor cows are usually treated with large doses of antibiotics to prevent infection after flushing. Injection of PGF2α is also recommended to speed recoveries of ovaries and to prevent pregnancy, if viable embryos are not dislodged by the flush.

Procedure:

⇨ Secure the donor in specially designed travis to avoid undue movement during flushing of uterus.

⇨ Administer epidural anesthesia (2% Lignocaine HCL – 5 to 7 ml) between last sacro and first coccygeal vertebrae (sacro-coccygeal space) or between first and second coccygeal vertebrae (first inter coccygeal space.

⇨ Clean the perineal region and vulva properly.

⇨ Check the ballon of Foley’s catheter by inflating it.

⇨ Rinse the Foley’s catheter, syringes, flushing tubes, “Y” connector and embryo concentrator using flushing media.

⇨ Stiffen the Foley’s catheter with a metallic stylet.

⇨ Introduce the stiffened Foley’s catheter into the one side of uterine horn through vagina and cervix.

⇨ Fix the catheter 1-2” above the inter cornual junction by inflating the baloon using air with the help of 15 ml air syringe.

⇨ Take out the stylet and introduce 30 ml flushing media into the uterine  horn through the Foley’s catheter using a catheter using a catheter tip syringe.

⇨ Connect the flushing tubes to the Foley’s catheter with the help of “Y” connector.

⇨ The flushing medium (DPBS) is kept in 500 ml bottle hanging on IV drip stand just above the height of the animal.

⇨ The inflow tube is connected to the flushing media container bottle. The outflow tube is connected to embryo concentrator.

⇨ The flushing of uterine horn is done by the gravitation method. The uterine horn is allowed to be filled with flushing medium of its own capacity, thereafter, fluid is drained out and collected through outlet into the embryo concentrator. Care should be taken to recover approximately all the fluid before another filling.

⇨ The uterine horn is filled up and drained out 8 – 10 times using 500 ml flushing medium.

⇨ After completion of the flushing of one horn, the catheter is deflated and removed.

⇨ Similar procedure is followed for the flushing of other horn.

⇨ After flushing of both the horns, the embryo concentrator is taken to the laboratory and contents are poured into the embryo searching dish.  

Advantages

⇨ Method is comparatively easy

⇨ Allows repeated collection of embryo without any damage to the reproductive tract

⇨ Avoids surgery and post – surgical care and complications.

Laparoscopic embryo collection

⇨ In sheep and goat, non-surgical embryo collection and transfer has been hampered by the difficulty of introducing the catheter and inability to perform rectal manipulation of genitalia.

⇨ This has lead to the use of surgical techniques predominately leading to adhesion formation.

⇨ Laparoscopy is considered to results in fewer adhesions than traditional surgery.

7.  Screening and Evaluation of embryo

⇨ Screening and evaluation of embryo is done under the stereozoom microscope.

⇨ Day 7 bovine embryos (compact morula or blastocyst) are about 150-190µm in diameter and are still within the zona pellucida.

Stages of embryo

Stage	Appearance
Preclevage stage	Presence of two polar bodies into the perivitelline space
2 cell, 4 cell, 8 cell, 16 cell stage	Embryo containing 2-16 blastomere cells with the zona pellucida
Early morula	Contains 16-32 blastomere cells
Compact morula	Contains 32-64 blastomere cells Compact arrangement of blastomere cells
Early blastocyst	Embryo containing a small blastoceal cavity
Blastocyst	Embryo  with expanded blastoceal cavity
Expanded blastocyst	Expanded blastoceal cavity and tightly stuck to zona pellucida The perivitelline space is very small Zona pellucida thin The diameter of embryo increases 1/3 of its preliminary size
Hatched blastocyst	Embryo is hatched out of zona pellucida

 

Parameters ascertaining the quality and stage of embryo:

ü  State of zona pellucida

ü  No. of visible blastomere cells

ü  Compactness of the blastomere cells

ü  State of perivitelline space

ü  Size of blastocoele cavity

ü  Colour and appearance of cytoplasm

ü  Regularity of shape

ü  Variation in cell size

ü  Presence of vesicles, extruded cells, cellular debris

 

Morphological description of embryo

 

Grade	Types	Characteristics
I	Excellent	-  Perfectly symmetrical                    - Compact - Well defined distinct outline          - No blastomere extrusion -  Even granulation
II	Good	- Somewhat asymmetric                 - Some blastomere extrusion - Even granulated with distinct outline
III	Fair	- Asymmetric in shape                        - Hazy outline in parts - Some blastomere extrusion and degeneration
IV	Poor	- Uneven granulation                       - Hazy outline - Abnormal shaped              - Much blastomere extrusion and degeneration
V	Unfertilized	- Granular appearance of cytoplasm - No indication of cleavage and formation of blastomere cells

 

8.  Transfer of embryo

⇨ Recipient should be in estrus within 12 hours of the donor so that it should posses good CL at the time of transfer.

⇨ Both surgical and non-surgical methods are used for the transfer of embryos in cattle and buffaloes.

⇨ However, in sheep and goat, the embryos are transferred by surgical method.

⇨ The non-surgical method is easy and avoids surgical interventions but the results of conception is lower than surgical method.

Surgical method:

⇨ Involves laparotomy incision, preferred in sheep, goat and pig. The uterine horn ipsilateral to the ovary with CL is exposed. A small syringe fitted with 21 gauge needle is used to make the transfer.

⇨ When the embryo is placed in the uterus, the needle is carefully inserted through the wall of uterine horn whereas, when embryo is placed in oviduct then the needle is inserted through the infundibulum into the ampulla where the embryo is deposited.

Non-surgical method:

⇨ Used in cattle and mare.

⇨ The embryo is to place in a 0.25 ml plastic straw similar to that used for semen packaging.

⇨ This straw is then placed in the stainless steel embryo transfer gun which is passed through the cervix for deposition in to the uterus.

⇨ The straw gun is carefully passed in to the horn ipsilateral to the ovary with CL.

⇨ Extreme precautions should be taken to prevent injury to the endometrium.

 

9.  Storage and Cryopreservation of embryo

 

⇨ Embryos can be maintained at near body temperature in the media used for flushing during the period between recovery and transfer.

⇨ If embryos are to be held longer than 2 hrs up to 10 hrs, a media containing 20% heat treated serum should be used as a holding medium.

⇨ If embryos are cooled at 5ºC (i.e. refrigerated temperature), they can be maintained for 2-4 days.

⇨ Cryopreservation of embryo is performed for longer period of time

Advantages of Cryopreservation

⇨ Long term storage

⇨ Eliminates estrus synchronization in recipients

⇨ World wide distribution

⇨ Easy export and import

⇨ Embryos must be stored in quiescent stage to retain their viability. Cryoprotactants like glycerol, ethylene glycol and DMSO (Dimethyl sulpher oxide) are always needed for preservation of embryos.

 

A stepwise procedure of freezing

 

⇨ After placing the embryo with holding medium and cryoprotactants in straw, the straw is placed directly into an alcoholic bath freezer (-7 ºC).

⇨ The straw is then seeded by touching metal rod, cooled by immersion in liquid nitrogen, to the straw at allocation away from the embryo.

⇨ After a 5-minutes hold, the straw is cooled at 0.5 ºC per minute to -35 ºC, held for 15 min. and then plunged into liquid nitrogen.

Thawing of straws

⇨ Straws are thawed before transfer of embryo to the recipients.

⇨ If 0.25ml straw – 15 sec. in air and 20 sec in water bath at 37 ºC

⇨ If 0.5 ml straw- 20 sec. in air and 20 sec in water bath at 37 ºC

⇨ Exposure to air reduces damage to the zona pellucida.

Special Thanks to:- 

Dr. C. F. Chaudhari

Associate Professor 

Department of veterinary Gynecology and Obstetrics, SDAU