TABLE OF CONTENTS

Multiple Ovulation Embryo Transfer (MOET) in Cattle: Procedure, Benefits, and Superovulation Protocol

Multiple ovulation and embryo transfer (MOET) is a technique where a superior female animal is hormonally stimulated to produce multiple eggs, which are fertilized and the resulting embryos are collected and transferred to surrogate females to increase the number of offspring from high-quality animals.

Embryo transfer technology (ETT) is a biotechnological tool by which embryos are collected from a superior donor female and are transferred to recipient females, which serve as surrogate mothers for the remainder of pregnancy.

Multiple ovulation and embryo transfer (MOET), the widely used technology increases the reproduction rate of superior female dairy animals. This is achieved by the stimulation of the ovaries hormonally for the growth of multiple follicles.

Advantages

Embryo transfer propagates superior male and female germplasm.
Transfer of various species across the country in the world is possible through embryo transfer technology.
We can produce identical twins.
It is possible to produce chimeras.
It is used to study ovary embryo uterine relationship.
It is useful to study the maternal recognition of pregnancy.
It is a basic step for transgenic animal production.
It is useful for conservation of endangered species.
It is useful to treat infertility also.

Disadvantages

It requires high technical skills.
Hormones are to be imported. Hence, procedures are costlier.
Every step needs aseptic precautions.

Steps in Embryo Transfer Technology

Selection of Donor
Selection of Recipient
Management of donor and recipient
Synchronization of donor and recipient
Superovulation
Breeding of super ovulated donors
Embryo collection
Evaluation of embryos
Transfer of embryo
Post-flush treatment

1. Selection of Donor

Donor should have superior genetic merit i.e., high yielding animal.
It should be free from genetic diseases, general and systemic diseases.
Its cervix should be easily negotiable.
It should be free from palpable abnormalities of the genital tract.
It should have positive energy balance.
It should be between 3-10 years of age.
It should have good fertility record.
There should not be any infections in the genital tract.
It should have clear oestrual discharge.

2. Selection of Recipient

Recipient should produce sufficient milk.
Its cervix should be easily negotiable.
It should be between 3-10 years of age.
It should have good fertility record.
It should have clear oestrual discharge.

3. Management of Donor and Recipient

Balanced ration should be given.
Proper deworming and vaccination should be done.

4. Synchronization of Donor and Recipient

Synchronize the donor and recipient by any one of the protocol.
For single donor, we have to prepare at least 4-6 recipients.
Both the animals are in oestrus on day-0.

5. Superovulation

Superovulation is defined as “increasing the number of ovulations more than the number that occurs normally by using exogenous gonadotropin.

There are 2 hormones used for superovulation. They are:

FSH
PMSG

PMSG is used as single dose, because, it has half-life of 3-4 days due to sialic acid. FSH has the half-life of 2-2½ hours. Hence, multiple doses of FSH are used (8 doses at 12 hours interval)

Super ovulatory treatment in donor is started from 8^th-12^th day of oestrous cycle. The dose of FSH ranges from 28-40 mg.

Synchronization and Superovulation

Donor cows will be treated with two injections of PGF₂α at 11 days interval for pre- synchronization.
After second dose of PGF₂α, the animals will be observed for estrum (Day 0).
On the 7^th day of reference estrum, CIDR will be inserted intravaginally followed by injection of estradiol benzoate (2 mg I/M) and Hydroxyprogesterone (100 mg I/M).
The recipient animals will be inserted with CIDR on the same day of donor animals.
On day 11, FSH (Folltropin-V) will be diluted with 20 ml of sterile saline aseptically in a laminar airflow.
The FSH diluted will be loaded in syringes separately as per the tapering dosage required.
On day 11, 1.5 ml/ 5.0 ml of FSH (Folltropin-V) will be administered to the donor cows in the morning and evening.
On day 12, 1.5 ml/ 4.0 ml of FSH (Folltropin-V) will be administered to the donor cows in the morning and evening.
On day 12, the recipients will be administered with Inj. PGF₂α in the evening.
On day 13, 1.0 ml/3.5 ml of FSH (Folltropin-V) will be administered to the donor cows in the morning and evening and Inj. PGF₂α will be administered to the donors in the morning and evening.
On day 13, CIDR will be removed from the recipient animals in the evening.
On day 14, CIDR will be removed 12 hours after second PGF₂α injection.
On day 14, 1.0 ml/ 2.5 ml of FSH (Folltropin-V) will be administered to the donor cows in the morning and evening.
On day 15, Inj. GnRH will be administered to the donor cows in the morning (Day 0) (12 hours after last FSH injection).

FSH for native breeds or Bos indicus is half the dose of the exotic breeds. Ex. Exotic breeds require 400 mg of FSH for one schedule, but, native breeds require 200 m of FSH.

6. Breeding of Super Ovulated Donors

Superovulated cows will be inseminated on day 0 evening (12 hours after GnRH injection).
Re-inseminations will be done on day 1 morning and evening (Optional-12 hours apart).
2 straws should be used at every insemination.

7. Embryo Collection

Day-7 post AI, embryo collection is done.
Flushing media: Dulbecco’s phosphate buffer saline with 10% Foetal calf serum / 1% Bovine serum albumin.
Holding media: Dulbecco’s phosphate buffer saline with 20% Foetal calf serum / 4% Bovine serum albumin.
Embryo collection is done on Day 7 post AI.
Trans-cervical recovery of embryos is routinely practiced in cows.
Embryo collection should be done in the early morning.
Feeding should be suspended until embryo collection.
The animals to be flushed should be cleaned and tied near the collection area.
The animal should be restrained properly in a trevis.
Superovulatory response should be checked by per rectum or by ultrasonography.
Two-third of the dung in the rectum should be removed (or) the dung should be removed from the rectum by back raking and complete removal should not be done.
Wash the perineal region of the animal with soap solution and disinfect with povidone iodine.
Epidural anaesthesia should be given using 2% Lignocaine solution at sacro-coccygeal site and checked for tail flaccidity.
Tail should be secured using a cotton bandage roll so that it does not interfere the procedure.
Cervical dilator and stylet should be cleaned with povidone iodine and then wiped with dry cotton and should be kept ready.
Sanitary sheath should be applied over the cervical dilator.
The Foley’s catheter should be checked for the patency of balloon and it should be flushed with flushing medium.
The flushing medium bottle should be hanged from a height and it should be connected to one end of the Y-junction tube.
The person who is going to perform the procedure of embryo flushing should wear full arm gloves and perform per rectal examination and should not remove the hand till the procedure is over.
The person who supports should spread the vulval lips until the cervical dilator or the foley’s catheter is at the desired place.
The hand should not be removed frequently in order to prevent pneumo-rectum.
After holding the cervix, it is dilated using the cervical dilator.
The foley’s catheter should be clamped using artery forceps and cotton.
Foley’s catheter along with the stylet should be passed per vaginally and should be inserted to the right uterine horn and fixed in the anterior 1/3^rd of the horn.
Foley’s catheter should be fixed in the right horn, the forceps should be removed and the balloon should be inflated with 8-10 ml of air.
One end of the Y-junction tube should be connected to the Foley’s catheter and the other end to the embryo filter.
Embryo filter should be covered with aluminium foil to avoid direct sunlight.
The flushing medium should be let to flow through the Y-junction tube from the media bottle to the embryo filter by keeping both the stoppers open to prevent the sticking of embryos to the tube.
Once flushing of the Y-junction tube is done, the right horn should be flushed with 50 ml of flushing medium 8-10 times by simultaneously opening and closing the inlet and outlet stoppers respectively.
The medium collected in the embryo filter should be discarded through the drain tube then and there necessary and the filter should not be allowed to fill with medium.
Then the Foley’s catheter should be removed by deflating the balloon and should be flushed with the flushing medium to remove the mucus attached to the tip of the catheter into the embryo filter.
The same manner, the left horn should also be flushed and then the body flushing.

*Embryo Collection in Multiple Ovulation Embryo Transfer (MOET)*

Embryo Searching

Embryo filter should be transported to the embryo lab carefully by covering it with an aluminium foil.
The filtrate should be washed and drained twice or thrice with Flushing medium to prevent the clouding due to blood cells.
The filter should be placed in an upright position.
The embryos should be searched using 90 mm petridish (graduated).
In case of non-graduated petri dish, graduations should be made using a scale and needle.
The filtrate should be transferred to 90mm petri dish and should be allowed to settle for 10 minutes.
Embryo filter should be kept wet until embryo searching to prevent drying of filter surface.
After 10 minutes, embryos should be searched under stereo zoom microscope at 1-3 X magnification in zig-zag manner.
Each petri dish should be searched atleast twice.
Embryos should be searched under stereo zoom microscope fitted with stage warmer with temperature of 38.5℃.
30 mm petri dish should be placed during searching added with 3 ml holding medium.
Holding medium should be pre-warmed at 38.5℃ prior to transfer of embryos.
After transferring embryos from flushing medium to holding medium, embryos should be washed for 3-5 times in holding medium.
Washing can be done by spreading embryos and transferring individual embryos to the next holding medium.
The procedure should be repeated thrice before freezing or transferring.

8. Evaluation of Embryos

Individual embryos should be graded under inverted or light microscope.
Grading is done based on International Embryo Transfer Society (IETS) standards.
It is graded as:
- Excellen
- Good
- Fair
- Poor

Excellent quality embryo (Code: 1):

Excellent embryo will have spherical zona pellucida.
Spherical blastomeres without vacuoles.
Uniform blastomeres noticed.

Good quality embryo (Code: 2):

Spherical zona pellucida.
Small vacuoles may be present in the blastomeres.

Fair quality embryo (Code: 3):

Zona pellucida distorted.
Vacuoles larger.
Slight pigmentation of blastomeres.

Poor quality embryo (Code: 4):

Distorted zona pellucida.
More pigmentation of blastomeres.
Degenerating blastomeres.

Excellent and good quality embryos are used for transfer.

9. Transfer of Embryo

For fresh transfer, embryos can be loaded in 0.25 ml straws along with holding medium.

Washed individual embryos should be loaded in 0.25 ml straws as follows:

The straw should be fitted with 1 ml insulin syringe containing adapter or embryo loading pipette.
Straws should be flushed with holding medium 5-10 times before loading of embryos without touching the factory seal.

After loading embryos, straws should be sealed with straw plug.

Embryos loaded should be observed under stereo zoom microscope to confirm the presence of embryos.

The transfer of embryo into the uterus of recipient i.e., deposition of embryo in the uterus in Embryo Transfer Technology is called as “Inovulation”.
Usually, the embryo is transferred non-surgically.
The embryo is deposited in the anterior one-third of the uterine horn ipsilateral to the ovary containing corpus luteum.
Ex: If the left ovary contains corpus luteum, the embryo is deposited in the left uterine horn.

10. Post Flush Treatment

PGF₂α should be administered to the donor, in order to kill and flush out the remaining embryos.
Donor will come to heat in 7-10 days.

Note

Surgical embryo collection and transfer is followed in Goats.
It is done on day-3 after breeding.
Fallopian tube is flushed and embryo is collected.