Brazilian company perfects in vitro production of bovine embryos

Located in inland São Paulo State, In Vitro Brasil, an animal reproduction biotechnology company, holds more than 45% of the world’s market share.

By Elton Alisson

Agência FAPESP – In recent years, herds of dairy cattle all over Brazil have enjoyed the addition of a dairy cow considered to be high-producing like Holsteins, while at the same time resistant to disease, climate changes and scarcity of water and food like Gir cattle.

The result of genetic crossing between these two breeds, this type of hybrid cattle has been increasing its presence on dairy farms in states such as Pará and Alagoas thanks to a technique known as in vitro embryo production (IVEP), which has been perfected by the company In Vitro Brasil.

With headquarters in Mogi Mirim, in inland São Paulo, the Brazilian animal reproduction biotechnology company has had research studies funded by the FAPESP Innovative Research in Small Business Program (PIPE) since 2007. With the ever-growing demand for more efficient embryos, the company has begun to direct resources toward strengthening its department of research, development and innovation (RD&I).

“We have more than 45% of the world market for in vitro production of bovine embryos,” Andrea Cristina Basso, director of research at In Vitro Brasil told Agência FAPESP. “In 2013, we produced nearly 266,000 bovine embryos, and we estimate that our share has increased even more in this market, which Brazil leads.”

The company was established in 2002 at a point when IVEP began to emerge as a business in Brazil, the first country to use the technology commercially.

In the early stages, the technique presented some limitations, such as time restrictions for the collection and laboratory submission of the oocytes (sex cells produced in the animal ovaries), in addition to the fact that at that time, it was not possible to freeze and store the embryos. Furthermore, the production of embryos was low, the gestation rates were unsatisfactory, and there were many issues at birth.

For these reasons, the embryos produced using this technique were very expensive for the cattle farmers and were therefore used, for the most part, only in animals with high genetic value, known as “elite cattle,” particularly in Nelore beef cattle, said Basso.

“In vitro production of bovine embryos was not efficient enough to be made into a business, nor did it present significant outcomes because it was still underdeveloped. It needed to be made commercially applicable,” the researcher said.

Investment in research

To improve the technology, increase productivity and make it feasible to use the embryos not only in “elite animals” but in the entire herd to reduce the interval between generations and to accelerate the process of genetic improvement, the company decided to turn its efforts towards research to change the concepts that were then behind the IVEP technique.

With an undergraduate degree in veterinary medicine from the Jaboticabal campus of São Paulo State University (Unesp), and with masters and doctoral degrees from the University of São Paulo (USP) obtained through a FAPESP scholarship, Basso was hired in 2007 when the company began its first research project.

Conducted with funding from the FAPESP Innovative Research in Small Business Program (PIPE), the project made it possible for the company to certify the validity of its culture media – the liquid media used in the production of in vitro embryos.

“To ensure that the culture media presented suitable conditions for the embryos, we would prepare them every day before the technical staff left for the farms to collect the oocytes from the donor cows,” Basso said.

“As we attracted more clients in different parts of Brazil and increased the number of laboratories, this daily process became unfeasible,” the researcher said.

Using analyses conducted with resources from a project at the Thomson Laboratory of Mass Spectrometry of the Institute of Chemistry at the University of Campinas (Unicamp), the researchers proved that the embryonic culture media produced by the company could remain stable for up to 90 days.

Thus, the company began to produce and store large quantities of culture media, which became its principal product and gave the company its market advantage.

“Today, we have a lot of flexibility in handling the embryonic culture media. We send the media to our laboratories and are assured of being able to work with them for quite a long time because they have a long shelf-life and are sent only after they have passed through a rigorous quality-control process,” Basso said.

With the second project, conducted through a postdoctoral fellowship from FAPESP in collaboration with Colorado State University, in the United States, the company began to use an ultra-rapid freezing method for the embryos.

Honed with tools developed during completion of the project, the method proved to be so efficient that it soon became another commercial product for the company. Today, nearly 20% of the embryos produced by the company are frozen and stored, Basso said.

“This technique has changed the notion of in vitro bovine fertilization in Brazil and has allowed greater flexibility in managing the embryos, shipping them to other parts of Brazil and even exporting them,” she added.

Obstacles to exporting embryos

According to Basso, one of the main obstacles to exporting embryos produced in vitro is the absence of a health protocol between countries.

There is a health protocol that is valid only for embryos produced in vivo (i.e., in the uterus of the cow) that have undergone some type of treatment after extraction to eliminate any pathogens present from the semen of the donor bull or from the donor cow itself.

In the case of embryos produced in vitro, studies have shown that procedures utilized with in vivo embryos are not effective because pathogens have been found in these embryos, even after treatment, the researcher explained.

“There is enormous demand for us to export frozen embryos from cows that adapt well to the climate of other countries, but we are not in a position to do so because there is no health protocol for this type of embryo,” Basso said.

The company is now planning to develop a protocol with health monitoring agencies in Brazil and abroad to certify that the in vitro embryos are free of pathogens.

“We need to make not only our embryos but also the entirety of Brazilian livestock genetics available to the rest of the world,” Basso said. “We are planning to develop a methodology that proves that our system for producing embryos is free of viruses and protozoa and that can be recognized by the Ministry of Agriculture, Livestock and Supply in Brazil and by responsible entities in countries that may buy our products in the future,” she said.

The Brazilian company already has a presence in South Africa, Russia, Colombia, Argentina, Australia, Paraguay, Panama, Uruguay, Venezuela and, most recently, in the United States, through joint ventures with animal reproduction biotechnology companies.

In Brazil, in addition to the main office in Mogi Mirim, the company has a reference center for animal reproduction in Uberaba, in the state of Minas Gerais, and a laboratory in Xinguara, in the state of Pará.

Since 2010, the company has begun to offer in vitro embryo production technology to several affiliated laboratories located throughout Brazil, Basso explained.

“These affiliated laboratories are companies that had in vitro production technology already in place or that had experienced unsatisfactory results,” Basso stated.

“They purchase our culture media, and we provide full assistance to them in setting up their laboratories and training technical personnel in the methodology for freezing embryos,” she said.

According to Basso, the company has been growing between 20% and 25% per year because it began to invest in research and development and to modify its business plan to add value to the embryos it produces and sells, thus increasing its profit margin.

“We began to really move forward when we started to conduct research through programs such as FAPESP’s PIPE to perfect our products,” said the researcher, who has become a partner in the company.

“Today we have one doctorate, one master’s degree and two undergraduate degree holders, and we are expanding our department of research, development and innovation through the use of fellows,” said Basso.

The company is currently working on two projects with PIPE funding from FAPESP. One of them, in partnership with the Unesp Araçatuba campus, is in its final phase and is seeking to generate single nucleotide polymorphism (SNP) genetic markers in high- and low-oocyte-producing Gir and Girolando cattle.

Through another project in partnership with USP that began recently, the company plans to develop tools for the in vitro production of embryos in 3-5-month-old Nelore and Holstein calves and in pre-pubescent heifers.

“This would contribute to an even more significant reduction of the interval between generations and would drastically accelerate genetic improvement of the herds,” said Basso.