If you liked the first part of the interview with Cesare Galli ( HERE IS THE FIRST PART ), you certainly can’t miss this second part!
We also reported here the data of the work carried out at Avantea in the last 10 years, which you can read at the end of the interview, just published in the “Journal of Equine Veterinary Science” … good reading!

 Regarding the health of foals produced through ICSI, are there any data about problems at birth?

“We come from the experience with cattle where a small percentage of calves born with this technique are overweight, while in the human field the children are more delicate and underweight. Fortunately for us, there are no differences in the horse. What is reported in some cases are placentas that deviate slightly from the norm, but without any reflection on the health or well-being of the foal. In general, mares don’t give birth here except for the first foals that were born here because the technique was still experimental. For this reason, usually, we don’t see the births of the foals, but no one has ever told us about any problem; we would certainly have known.

In the case of horses born from cloning, things are different: we know that there may be late abortion, weakness or fragility at birth. It is a well-known fact. But for horses born through ICSI there is nothing relevant reported.”

 You are also involved in cloning. Can you tell us something about it?

“Yes, we are the only ones in Europe that do it on horses now, as well as in the United States. Also in Argentina and Brazil they work with cloning, but in Europe and the Middle East, we are the only ones.
However, it is an occasional job; there are few requests, and we work with few horses per year. Surely, costs are a limiting factor. In America, they make more clonings because there is more money. Some people go to South America because it costs less, but I don’t know anything about the results.”

Clone di Pieraz – Ph: ©Avantea

This year has there been a downturn due to the Covid-19 pandemic?

“Fortunately, there were no restrictions on veterinary activities. We had some difficulties with some foreign clients based on the news that the press released about the infection progress in our country, which certainly was not reassuring. Someone took a mare home out of fear.

In some cases, donor owners have had trouble travelling to bring them here. For example, mares coming from southern Germany or Austria, since they are a few hours away by car, usually arrived here for the ovum pick up and come back home in the same day. But at that time they couldn’t cross the border, so they had some difficulties.

Mares have declined a bit, but overall the numbers are similar to last year. The University of Utrecht, which is our main customer that ships oocytes, is a public facility and closed with the lockdown at the end of March, until then we worked with them too. Instead, another private clinic did not stop and even these days has sent oocytes.

In general, however, the activity in the summer is quite calm, regardless of the virus, and in the autumn it should start again.
Today the problem is: how much economic activities have been damaged by the pandemic? Many breeders do it for passion or as a second job; consequently, if they don’t sell horses due to the economic crisis, then they will not make the embryos. This is the problem for the future, the economic aspect.”

With the OPU-ICSI technique, the most established bloodlines are almost always used to obtain the embryos and often old stallions are used.

“Yes, for example we have produced many CHACCO BLUE embryos in 2019. They will become inflated, but this happens when there is a famous stallion. Then some take risks and go on new lines. Some people have understood that they must reproduce with the young stallion to shorten the generation interval. We therefore also work on young mares, even if they produce fewer embryos than older mares that have been breeding for years. The young mares are also more difficult to manage, but it is still all doable.

As for the choice of the stallion, it is something we want to stay away from completely. The same thing for what concerns the marketing of the semen. It is a very slippery matter. The semen must be regular. It is delivered to us and we use it. We do not own the semen. The breeders manage their semen; we do not want to get involved in the diatribes which are involved with who bought the semen for one pregnancy and then also use it for ICSI.”

At Avantea you also screened embryos produced by in vitro technologies, what can you test?

“We screened embryos for sex determination before embryo transfer or embryo freezing. Once sexed, we declare the sex on the production certificate, even if the owner might have wanted the opposite sex.
It may happen that those who want the female and instead have the male (or vice versa depending on the sex they are interested in) would like to sell it without declaring the sex, but if the embryo is sexed we always declare the sex.

We only do the screening on the embryo because the sexed semen in the horse does not give good results. You can do something but with poor results. With the embryo, however, it is not possible to predetermine the sex, we know it only afterwards.

We have also set up the Warmblood Fragile Foal Syndrome (WFFS) test as a preimplantation diagnosis. Any genetic test that can be done on a born foal, we can do also for the embryo.
There are a lot of genetic diseases that could be diagnosed in the embryo before implanting it, as it happens in humans. This is because the embryo that grows in vitro is easily accessible and cells can be removed without damaging it too much. This is more difficult to do on flushing embryos (taken for embryo transfer) because they are removed at a later stage and suffer more from manipulation.”

 Can you tell us about other projects for biomedical research?

“We use animal models to study genetic diseases in humans by reproducing them in pigs in collaboration with several partners in European projects.
We developed the animal model for Amyotrophic Lateral Sclerosis that has been going on for ten years, works very well and is being used to study this disease.

Moreover, we collaborate with a Belgian University producing pigs to transplant pancreatic islets as a therapy in type I diabetes. Clinical trials will begin there in a year or two. The pigs produced for this purpose have been transferred to Belgium because they must remain in a sterile environment as those pancreatic islets will have to be transplanted into human patients.

We have also recently started working with genetically modified pigs to produce antibodies against Covid-19. Our pigs have been transferred to Belgium where they are immunized with virus proteins to produce serum antibodies against Covid-19.
The extraction, purification, and evaluation of the serum produced by pigs are carried out by Xenothera, a company that we co-founded in 2014, which will take care of bringing the antisera produced against the virus to clinical trials. Initial feedback is very positive.

In the case of pigs breed to produce serum against Covid-19, there is no need for the sterile environment because once collected it is purified and any pathogens are eliminated.

We are also trying to expand our facilities to have more possibilities for research, at the moment we are limited by space. Genetically modified pigs cannot be placed elsewhere, by law they can only go to authorized centres for experimentation.
The expansion of the existing structure will also concern the horse facilities.”

At Avantea you work also to counteract the decline of species in danger of extinction. Recently the news of the embryos produced by the Northern White Rhino, a species now almost extinct, has had a significant echo, can you tell us something about it?

“Yes, we produced three Northern White Rhino embryos from the last two females left on earth. We developed the technique on the Southern White Rhinos, which are the most widespread, and then we moved it to the Northern Rhinos that are disappearing. We went to Kenya, and on the first try, we were successful.

The next step will be the pregnancy through embryo transfer on Southern Rhinos, we are working on it with our German partners.
At the moment we have not been able to have pregnancies for the first 7-8 implanted embryos.

Currently, the most important thing is to be able to freeze embryos of Northern White Rhino before these last two females become too old. Then we’ll implant them on Southern Rhino females. But we will do that when we’ll manage to have pregnancies with embryos of Southern White Rhino implanted on Southern females.

This is an opportunity to safeguard endangered species, perhaps it should be put in place before reaching only two live females, even the genetic variability at this point is very low.”

– The numbers of the OPU-ICSI in Avantea in the last 10 years –

In a recent publication, whose data should have been presented in Pisa during a Congress that could not be held due to Covid-19, the Avantea team presented a retrospective analysis of in vitro embryo production of equine embryos after OPU-ICSI over the last ten years.

From the published data it is evident the growth of the number of embryos produced is thanks to the advancement of technologies related to OPU-ICSI.
These numbers show us clearly Avantea’s leadership in assisted reproduction techniques related to OPU-ICSI.

Overall, from 2010 to 2019, 4264 oocyte retrievals were performed in Avantea and 2466 in other clinics in Europe which were sent to Avantea for ICSI and embryo production, reaching a total of 6730 ICSI carried out in the last ten years.

The overall number of donor mares, adding those in Avantea and those whose oocytes were collected in other clinics, is 2459while the number of stallions used is 619.

The number of embryos produced via ICSI and subsequently frozen or transferred in a recipient mare in the last 10 years is 8408. 

The number of ICSI following OPU performed at Avantea in 2016 and 2017 remained substantially unchanged (965 and 934). In 2018, it increased considerably to 1472. In 2019, it increased again to 2045, thanks to oocytes retrieval performed in clinics outside Avantea.

The optimization of the technique allowed movement from one embryo for each OPU-ICSI in 2017 to two embryos for each OPU in 2019. Moreover, in 2019 over 30% of successful OPU-ICSI delivered three

or more freezable embryos versus 15% in 2017.

If we distinguish the embryos produced on Arabian mares from those produced by mares of other breeds for equestrian sports, in the last 3 years (2017-2019) the number of embryos produced in Avantea by non-Arab mares was 3379.

The pregnancies rate following the 1008 embryo transfers (of which 248 on Arabian mares) in Avantea was averaging 70% after 17 days of gestation for the 3-year period from 2017 to 2019 and 59% after 50 days.

 

Lazzari, G., Colleoni, S., Crotti, G., Turini, P., Fiorini, G., Barandalla, M.,… & Galli, C. (2020). Laboratory production of equine embryos. Journal of Equine Veterinary Science, Vol 89. 103097.

 

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