The main challenge of the swine industry is to optimize pork meat production while ensuring sustainability and an efficient cost. The improvement of emerging reproductive technologies is one of the most important vectors to reach this challenge and our research program will move through this vector to implement the scientific results in real innovative programmes in farms.

Our research program also opens its objectives to interspecies applications such as the development of animal models for research in human regenerative medicine. For the next 4 years, we propose several research activities sorted in four areas:

1. Area 1: Development and implementation of a practical procedure for non-surgical deep uterine transfer with fresh preserved porcine embryos.
2. Area 2: Generation of pigs with specific genetic modifications via intracytoplasmic injection of in vivo-derived zygotes with the CRISPR/Cas9 system.
3. Area 3: Response to the pending challenge of the swine industry to find boar fertility biomarkers.
4. Area 4: Development of an iPSC-specific humanized pancreas in a pancreatic pig through interspecies blastocyst complementation (IBC).

Porcine embryo transfer (ET) technology has been in demand for decades due to its potential to provide important sanitary, economic and animal welfare benefits. However, the commercial use of ET is practically non-existent today. Our program will evaluate the use of fresh in vivo-derived morulae and blastocysts stored in liquid stage up to 72 h, the ovarian superovulatory response in different breeds of pig, and the usefulness of whole seminal plasma (SP)- or SP-specific components, as cytokine TGF-β1, to improve ET efficiency. The results of this research may provide means to enhance outcomes of ET technology, which would represent a fundamental advance in the widespread use of ET by the pig industry.

The purpose in Area 2 is to generate pigs with specific genetic modifications via intracytoplasmic injection of in vivo-derived zygotes with the CRISPR/Cas9 system. The specific goal is to disrupt the porcine CD163 gene, which appears to be required for productive infection by porcine reproductive and respiratory syndrome virus (PRRSV). The technology based on the CRISPR/Cas9 system is completely revolutionizing genome engineering and is in place to generate these animals now. Because PRRSV is endemic in most pig producing countries worldwide and causes enormous economic losses to the swine industry, there is a huge need to demonstrate the feasibility of generating genetically modified pigs that are resistant to that disease.

Artificial insemination (AI) is extensively used in swine industry and the current AI-programs require few boars, increasing the relevance of the boar on farm fertility outcomes. In this context, subfertile boars have an important negative economic impact for farmers. To achieve this goal, we will evaluate the effectiveness of the measurement of sperm nuclear DNA damage and, if effective, define a threshold able to difference between fertile and subfertile boars. We will also intend to build the SP-proteome in order to identify qualitative and/or quantitative protein differences as fertility biomarkers. Finally, inquire whether one or more of the many measurable cytokines present in boar-SP could also be used as fertility biomarkers.

Our on-going effort intends to develop an iPSC-specific humanized pancreas in a pancreatic pig through interspecies blastocyst complementation (IBC). Pig closely resembles human in many aspects including anatomy, physiology and organ size. The technologies to be used in this project attempt to overcome the current limitations associated with safety and efficiency of standard iPSC differentiation protocols. If successful, IBC technology would revolutionize stem cell biology and regenerative medicine research, bringing extraordinary opportunities in the effective application of iPSCs for research and clinical translation.