In recent years, assisted reproduction techniques in sport horses have undergone a turning point that we can define as epochal with the introduction and increasingly extensive use of Ovum Pick Up and ICSI (Intracytoplasmic Sperm Injection).
An Italian researcher, together with his team, is the leader in Europe in the application of this technique, a name now known to anyone involved in breeding, Prof. Cesare Galli.
The OPU-ICSI technique consists of the collection of oocytes from a donor mare, at any time of the year, which are then transferred to an incubator to complete the maturation phase. In a second phase, oocytes are fertilized in vitro through ICSI by direct injection of the sperm. Once fertilized, the oocytes are incubated for about a week. The embryos obtained can be frozen and/or implanted in a recipient mare that will carry the pregnancy to term.
The transvaginal removal of the oocytes under ultrasound control is a method that does not interfere with the normal activity of the donor, even if the mare is involved in sport activity.
OPU-ICSI can be applied with good results, even when limited semen is available or it is of poor fertility, when used for artificial insemination or natural mating. Given the growing interest in this technique and the increasing number of embryos that are produced every year, we interviewed Prof. Galli who deals with this daily at his laboratory, Avantea. Today you can read the first part of the interview which will be followed by the second part in the coming days.
Prof. Galli, can you tell us how Avantea was born?
“The business started in 1991. In those years, my wife and I were in Cambridge in England doing research. The Italian Breeders Association proposed to us to open a laboratory that would use assisted reproduction technologies in the bovine field. So we came back to Cremona where we set up the activity of in vitro embryo production and cloning in cattle, working for the Consorzio Incremento Zootecnico. In fact, in England I was already involved in cloning.
Then at the end of the 90s, there was a strong crisis due to the onset of the Mad Cow Disease. Nobody was interested anymore in investing in this field; there was a lot of fear. If there was only one positive case on a farm, all the animals were slaughtered, and no one slaughtered any more cattle because of fear of the disease.
That was an opportunity to rethink the business and, since I had already started working with horses in England, we started in 2000 with ICSI in horses.
The first foal was born in 2002, then we also cloned the first horse, PROMETEA, in 2003.
The cattle activity, on the other hand, has never recovered to its previous levels, and we have continued with the work on horses.
In 2008, the Italian Breeders Association decided to stop investing in biotechnology, thus deciding to close their activities. At that point, we took over the business unit. This is how Avantea was born, at first we rented the structure, then we acquired it in 2013.
We continued to work mainly following market demands. Today the horse service sector is the most important part.
Following is the part concerning genetically modified pigs. Genetic mutations induced in these pigs are analogous to the mutations that cause genetic diseases in humans. They represent an animal model that can be used to study the mechanisms, prevention, and treatment of various diseases.
We also have pigs with genetic characteristics that make them potential donors of cells and tissues for humans. “
Regarding the horse assisted reproductive technologies, Avantea is today the leader in Europe in the OPU-ICSI technique.
“Yes, we are currently the only laboratory that can apply this technique from start to finish: from egg retrieval to embryo freezing, plus possibly implantation. There is another laboratory that has similar activity to ours but with poor results, as well as a university centre, this too with much lower results than ours.
OPU-ICSI is not a particularly difficult technique, but it takes a lot of manual skill that is acquired with experience, with time, and above all with numbers. It is not an activity that can be done by a single veterinarian who has three or four mares a year from which to collect oocytes.
It needs a team to complete the procedure. Only three people are needed to collect the oocytes. Then there are those in the laboratory who look for the oocytes. Clinics do not always have the opportunity to have this staff and the time to follow this activity.”
Is there also a collaboration with other equine veterinary clinics in Europe?
“The oocytes collection phase can currently be deferred, meaning they can be collected elsewhere and then sent to Avantea. Over the years, we have established collaborations with other equine clinics, the most important being that of the University of Utrecht in the Netherlands. They collect the oocytes that the same day are sent to our laboratory in Cremona. Here we treat the most critical phase: we make them mature, fertilize them through ICSI, grow the embryo for eight days, and finally, freeze it and send it back to Utrecht.
Today we have several clinics in Europe that collaborate with us in this way, collecting oocytes from their clients: three in the Netherlands, two in Belgium, two in Germany, and three in France.
We return the frozen embryo because we do not have enough recipient mares. We have about 250 mares, but last year we produced about 3800 frozen embryos. We can transfer 10% of them; the remaining ones are returned to the clinics which transfer them to their recipients.
We also provide training for veterinarians who want to learn how to transplant frozen embryos. Those interested come to us, we show them how to proceed then they can return and operate, obtaining, in most cases, pregnancy rates similar to ours, around 70%. The advantage of countries like Germany, Holland, and Belgium is that they have a lot of mares available as recipients at much lower costs than ours and are therefore competitive in implanting embryos to carry the pregnancies to term.”
So the mare no longer needs to come to you?
“It depends. Some customers still want me to do the collection of the oocytes. Here, at Avantea, I still do all the OPU. Sometimes in some mares, the oocytes collection is particularly difficult, and therefore, the customer prefers to have the collection done here with us.
The donors of the oocytes, as well as at Avantea, are hosted in two centres: one located on Garda Lake and one in the province of Brescia. Here some veterinarians check them, and when a mare is ready, she can come to us the same day to collect the oocytes, then return to the paddock.”
A close collaboration with the veterinarians of the area.
“Yes, of course, they monitor the donor mares, and when they are ready, they report them to us and send them to us for the collection of the oocytes. In Italy, there is no one else doing this procedure.
The work continues throughout the year, but we work mainly from September to March/April, more than from April to September, we have an inverse seasonality.”
How much staff is employed at Avatea?
“We are eighteen people, but there is also the part concerning the pigs which employs at least five other people.”
How many retrievals of oocytes can be done for a donor mare in a season?
“On average, one per month, about ten retrievals per year. It also depends on the mare and on the interest around this mare. For example, we started with Arabian mares which, due to the high level of inbreeding, are not very fertile. We have mares that have been here for 2-3 years because they don’t produce many embryos. The breeders are very interested, that’s why they moved them here from the Middle East.”
How many embryos can a mare produce?
“From zero to records of 11-12 for each retrieval session, the average is 2 for each retrieval.
The advantage of this technique is that you can use semen from stallions that are either dead or whose semen is available in limited amount or you can use semen which due to its characteristics could not be used in any other way than with the ICSI. For example, for CUMANO or HEARTBREAKER semen, that does not have such a quality that can be used with other artificial fertilization techniques, this is the only option. If you want to use it, you have to use ICSI. ”
Behind the use of this particular semen through ICSI technique, which allows the use of minimal quantities, there is the complaint from the stallion owners when more foals are born than the gestations sold.
“Yes, exactly, the owners of these stallions complain about what is happening with us, but they must also think that they can still sell their semen only because we have the possibility to use it, otherwise they would have finished their business years ago.
On the flip side, they should look at this too. They complain about the use of the semen, but they earn money because they still manage to sell the semen of their stallions that otherwise would not be usable; they don’t remember this at all.
It happens that they ask us for the data of our customers to verify who produces the embryos. But we also have a privacy obligation, we cannot say what we have done for our customers.
The sale of the semen is an agreement between the customer and the seller. We are not responsible. They have to ask the customer what he does with the legitimately purchased seed, not us. The agreements must be done between the stallion owner and the breeder who buys the semen.
This is a problem that existed even before ICSI; it was born when it was possible to use a limited amount of semen for insemination. Now certainly this diatribe has intensified. ”
These embryos usually reach a considerable sum of money both in auctions and in private negotiations. It is a new market for breeders.
“The majority are small breeders but there are also professional breeders. There are also those who are setting it up as a business, producing and selling embryos. It has already happened with cattle, and now it’s happening with horses. The best genetics are moving on to frozen embryos; it’s a natural evolution. The genetic level rises, and these embryos are very competitive on the market because of great commercial value. We have many more offsprings from top sires that will compete than before.
What we are trying to support is opening up to international markets thanks to frozen embryos, so breeders can sell on a large scale, worldwide. An expansion of the market for breeders who produce in this way can be with these important genetics.
We exported the first embryo to the United States, then to Brazil and Hong Kong during the embryo auction of the Asia Horse Week. The Asian market opened with excellent earning prospects for breeders. ”
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