Embryo transfer technique-ETT

What is ETT It involves removal of embryo from a female of superior genetics(donar) and placement of embryo into reproductive tract of female of average genetics(recipient),where embryo complete its development. Traditionally cows produce 1 calf per year, ET allows the production of many off spring many offspring within 1 year from single cow.

What is goal of ETT To obtain maximum no. of genetically superior embryos in minimum amount of time

Advantages It can increase genetic potential of herd in relatively short period of time. Increase no. of calves of genetically superior cows. ET can increase milk production in dairy herds. It can increase weaning weight in beef and dairy herd. To take superior genetics, because frozen embryos can be shipped almost every where. Preserves the superior genetics for future generation due to embryo freezing.

Advantages. Continued Increase the marking opportunities of off springs and embryos. Ease of import and export Embryos can be stored for indefinitely time period

Disadvantages Increased the expenses Oestrous detection required Synchronization for recipient and donor. Specialized equipment and trained personnel. Time consuming

Selection of Donor and recipient cow Free of reproductive abnormalities Outstanding reproductive capacities High milking ability High growth rate Bull should have superior genetics For recipient cow, average genetics holding, recipient cows serve as surrogate mothers to calf, but contribute no genetic information. R cows must maintain her pregnancy to term and produce adequate milk supply for calf

Synchronizing oestrous cycle Once the donor and recipient cows selected, they must be synchronized , they are on same phase of oestrous cycle. Reproductive environment of donor and recipient must be identical in order for embryo to survive. The oestrous cycle is controlled by production and secretion of hormone at proper time during cycle. PGF-2ₐ is a hormone used to synchronize the oestrous cycle of donar and recipient cows

Superovulation Donor cow is superovulated with series of injection of FSH Ovulation is a process of releasing eggs Superovulation causes ovary to produce many follicles Follicles are small blister like structure develop on ovary containing 1 egg each. When follicles ovulate, egg are released Superovulation ensures many eggs will be released because there are many follicle are present.

Superovulation of donor cows Day 0 to 4….FSH 20ml injection 2 times daily Day 0 starts 10 days following oestrous cycle Day-1 4ml Day-2 3ml Day 3 2ml Day 4 1ml Day 3 0r 4rth Prostaglandin injection given and repeated after 12hr causes CL regression to bring animal in oestrous Day 5 onset of oestrous

Management of recipient cow Observation of natural oestrous based on based on synchronization protocol. PGF-2 ₐ injection given 12 hr before the 1st treatment of PGF-2ₐ of donor cows Recipient cows ovulating same day as donor cows is preferred. It is expected 75-90% cows will respond to superovulation treatment 20-30% cows of flushed cows do not produce embryo of transferable quality. 0-20 per flush embryo 5-7 embryo considered good

AI of superovulated donor AI when in oestrous usually 5 days after superovulation Usually 12hrs, 24hrs, 36hrs, after onset of oestrous Preferably more than 1 straw of high semen quality to be used.

Embryo collection At day 7, when uterine stage embryos are expected to be collected. Donor cow is palpated Flushing of embryos from uterus Epidural anaesthesia with 5-7ml of idocaine. The perineum is washed French Foley type catheter placed Once inside the uterus body , catheter is advanced in uterine body and cuff is inflated with 15-20ml of air

Foley catheter

Embryo collecting sieve

Each uterine horn is then flushed with commercially available, complete, and ready-to-use flushing media-these embryo flushing solutions also contain antibiotics and bovine serum albumin as a source of protein. Some commercial preparations of complete flush media also contain surfactants to minimize the formation of foam and bubbles in the embryo search dish. 5) The uterine horn may be lavaged by repeated small volume (25–50 mL) infusions of flush media that are allowed to drain into an embryo filter.

Alternatively, the uterine horn may be flushed continuously; 1–2 L of flush media is used to flush a cow uterus. After the flush is completed, the cuff is deflated and the contents of the catheter are carefully allowed to flow into the embryo filter located at the end of the outflow tubing. The embryo filter is then taken to the laboratory, and its contents are dispensed into a search dish with grid and visualized using a stereoscope (dissecting microscope) at 10× magnification.

All embryos are transferred to a clean dish (with wells) containing holding media similar in composition to the flush media except for a higher concentration of bovine fetal/calf serum (10%–20%) or bovine serum albumin (0.4%). As with flush media, complete holding media are also available commercially. Embryos are then visualized at a higher magnification (40–60×) and classified according to their morphology, stage of development (unfertilized oocytes, early morula, tight morula, blastocyst, expanded blastocyst, hatched blastocyst, etc) quality (excellent, good, fair, poor, degenerate).

The quality score is based on morphologic assessment of the physical integrity of embryos and morphologic characteristics according to the stage of embryonic development, compaction status areas of cellular degeneration,

Only embryos classified as fair, good, or excellent should be transferred. Embryos are kept in holding media after being “washed” at least three times. Embryo washing is performed by transferring embryos into different, clean wells containing holding media. This procedure, recommended by the International Embryo Transfer Society, aids in removing cellular debris and potential pathogens adhered to the zona pellucida. Embryos are kept in holding media at room temperature until they are transferred to recipients or prepared for freezing.

Alternatively, embryos can be refrigerated in transfer medium for up to 24 hr with no appreciable loss of viability. Embryo transfer gun