The techniques used in embryo transfer (ET ) are very similar to those used in human IVF. These days ET is a widespread practice in veterinary medicine that is regarded as safe and effective. At the same time, to protect the welfare and well-being of the actual sheep involved, the procedure includes measures to avoid pain or discomfort to the animals. ET creates new life and accelerates genetic advancement, breeding only from superior animals, for the good of the breed. At the completion of the protocol, donor ewes are returned to their flock, resume normal cycling and subsequently lamb normally.
At Coolibah Stud, for 5 of the 6 years from 2014 to 2020, we annually selected the best 6 ewes and did ET. This was time consuming and expensive but incredibly rewarding. In this way, we were able to make a very rapid genetic advance. One of our good imported ewes “144” had 24 lambs in 15 months during 2019 and 2020 (from 2 flushes), of which 19 were daughters. This is twice the lifetime production of a good ewe -- all just in 15 months. She and 10 other original imports are still at Coolibah and are now 8 years old (having been implanted themselves as imported frozen embryos in 2017). I still find it amazing when I look down the microscope at an early embryo, just a small group of cells and think to myself that I will see, and indeed meet, that individual again in just 5 months when it is a living air- breathing lamb chasing after its foster mum across the grass.
So how is embryo transfer actually done? I have described below an actual protocol. For some breeders, this information may be overly detailed and perhaps more information than they actually need. Hopefully, however, this information will assist breeders, who are considering doing ET, in making their decision about whether or not to proceed. For those looking for a simpler description of the technique, I have provided a summary at the end
Donor ewes
Each donor is given a prostaglandin injection, e.g. 1 ml intramuscular of Ovuprost, Estrumate, Estromil or Estroplan, to ensure that they are not pregnant. Prostaglandins break down a corpora lutea on the ovary if present. Corpora lutea produce the hormone, progesterone that maintains a pregnancy. Some clinicians also recommend a course of antibiotics, e.g. Allamycin LA, Engemycin, Trisoprim, at this time. The same treatment can be given to recipients. It is regarded as good practice to start with clean, non-pregnant recipients.
Recipient ewes
Insert CIDR into vagina of recipient ewes. CIDRs are progesterone-impregnated suppositories. They block ovulation. When removed, ewes predictably start cycling after about 40 hours. In this way, oestrus cycles are synchronized.
Donor ewes: Insert CIDR into vagina of donor ewes
Both donor and recipient ewes commence a high-protein , high-energy supplement, e.g. full feed pellets, oaten or lucerne hay, so that they are in an ascending plane of nutrition and condition so that the donor ewes are more likely to multi-ovulate and the harvested embryos are more likely to implant in the recipients.
Some clinicians recommend further antibiotics for the donors during this time.
Donor ewes
7am - a second CIDR is inserted into the vagina of donor ewes. If the vagina won’t accommodate this, replace the first CIDR with the second.
7pm - a second prostaglandin injection is given (the same injection as on day 1).
Some clinicians recommend further antibiotics be given. It is important the CIDR insertion occurs hygienically
Donor ewes
7am -- Donor ewes are injected in the muscle with 2.5 ml Folltropin V.
7pm -- Donor ewes are injected in the muscle with 2.5 ml Folltropin V.
For the best response, the two Folltrophin injections are injected 12 hours apart. Folltropin contains FSH (Follicle Stimulating Hormone), which stimulates the development of follicles on the ovaries.
Donor ewes
7am -- Donor ewes are injected in the muscle with 2.0 ml Folltropin V.
7pm -- Donor ewes are injected in the muscle with 1.5 ml Folltropin V.
Donor ewes
7 am -- Donor ewes are injected in the muscle with 1.0 ml Folltropin V.
Recipient ewes
8 am – remove CIDRs and inject 2.5ml of Pregnecol into the muscle.
Pregnecol contains ECG (Equine Chorionic Gonadotrophin), which stimulates ovulation.
Donor ewes
4 to 6 pm – remove both CIDRs and inject with 1.0 ml Folltropin V and 1ml Pregnecol.
Donor ewes
7 am -- Donor ewes are injected in the muscle with 1.0 ml Folltropin V.
6 pm – if the donor ewe is going to be inseminated by laparoscopic AI (which requires an anaesthetic), it is safest if they do not eat or drink for 12 hours before the procedure. Some clinicians recommend drenching with 50ml of Ketol. Ketol is an energy supplement for sheep with added minerals. It is used as an aid in the treatment and prevention of ketosis (acetonaemia) in sheep. It elevates the blood glucose and depresses the blood ketone levels.
Recipient ewes
Teaser rams can be used to detect heat in recipients every 12 hours. Those on heat have their tags recorded or are marked with a coloured dot.
8am – it is now approximately 40 hours since the donor CIDRs have been removed. Removal of the CIDRs combined with the Folltropin injections will now lead to the ewes ovulating. As the optimal time for insemination is 4 to 14 hours before ovulation, then insemination should occur 42 to 46 hours after CIDR removal. If there are a number of donors to inseminate, it can be a busy few hours. Semen is collected from the ram and each donor ewe is anaesthetised and inseminated. Semen is not placed in the vagina. Normal mating or vaginal AI may only fertilise approximately 60% or less of the available ova. Obviously, as many embryos as possible are wanted. Rams are artificially ejaculated. Collected semen is placed in an extender fluid. Each donor ewe is anaesthetised and the semen deposited via endoscope directly into the fallopian tubes where fertilisation occurs. This ensures that more eggs are fertilized. To do this, two small incisions are placed in the ewe’s abdomen near the navel. Through one incision, an endoscope is passed (basically a fine torch) while through the second a fine syringe containing the semen is passed. While looking through the endoscope, the veterinarian can guide the syringe into the fallopian tube and deposit the semen there.
Some clinicians recommend inseminating the donors again later in the day after 4 pm, I,e, 45 to 50 hours after CIDR removal. After insemination, the ewes are given pain relief (usually meloxicam), sometimes antibiotics and are returned to feed. Usually hay is offered initially and later concentrates.
Through the day the recipients continue to be monitored for the onset of heat and their details recorded.
At any time during the morning, a CIDR is inserted into the donor ewes. Some clinicians recommend that antibiotics be given. The progesterone in the CIDR creates a uterine environment conducive to survival of the new embryos.
In the morning, the donor ewes are removed from feed and several hours later given a Ketol drench. At noon, the recipients are removed from food and water.
Today is the day that the embryos are transferred from the donor to the recipient ewes. At 9am, the donor ewes are anaesthetised. An incision approximately 3 cm long is made near the navel. A section of the uterus is exteriorised and sterile saline used to flush any embryos present into a petri dish. After embryo collection, several stitches are placed to close the incision. The procedure takes about 5 minutes. Some clinicians recommend giving antibiotics and a prostaglandin injection (to prevent pregnancy in the donor) after surgery. The embryos are examined microscopically to make sure they are healthy. Later in the day, the recipients are anaesthetized and, similar to the procedure with the donors, an incision is made near the navel and part of the uterus exteriorised. Usually 2 embryos are placed into each recipient. After surgery, both the donors and recipients are intially fed cereal hay. Of course, the recipient ewe ovum (or ova) are not fertilised and therefore fail to implant.
Some clinicians recommend injecting the donors with antibiotics.
Some clinicians recommend injecting the donors with Prostaglandin.
The donors have now fully healed and are joined back with a ram.
This might all sound a bit boggling and complicated at first but it is actually quite straightforward. Basically both the recipient and donor ewes need to be in good condition and ideally on a rising plane of nutrition. Progesterone-impregnated vaginal pessaries called CIDRs are inserted. These stop the ewes reproductive cycling and block the onset of heat (i.e. oestrus when ovulation occurs and the ewe is capable of conceiving). When the CIDRs are removed, all of the ewes start cycling together. Most do this at a predictable time, which means that all of the donor and recipient ewes’ oestrus (heat) cycles have been synchronised. A drug called Follicle Stimulating Hormone (Folltopin V) is then given to the donors only to stimulate the development of multiple follicles on the ovaries. A further drug, Equine Serum Gonadotrophin (Pregnacol) is then given at the correct time to the donors and recipients, which stimulates ovulation (i.e. each follicle to rupture and release its egg). The donor ewes are then inseminated and several days later their uterine horns are flushed to collect the newly formed embryos. Because the recipient ewes’ reproductive cycles have been synchronised, this means that when the embryos from the donors are inserted into them, the uterine environment is receptive to the embryo so that most implant and a pregnancy begins. Five months later, the recipient ewes give birth to the donor ewes’ lambs.
Further information can be found on the Australian Persian Sheep Association (APSA) website, under Increasing the number of quality purebred Persians in Australia.