Swine in vitro embryo production: Potential, challenges, and advances.

Pig production, a vital sector of the meat industry, faces demands for improved quality, efficiency, and sustainability. Advancements in breeding, disease control, and artificial insemination have enhanced production, while biotechnologies such as in vitro embryo production (IVP) and genetic engineering offer further progress. In vitro embryo production could facilitate the global exchange of valuable genetic material, accelerate breeding programs, and improve productivity, and it is essential for generating genetically modified (GM) pigs. These GM pigs have two main applications: first, they allow for targeted modifications aimed at improving production traits relevant to pig production in agriculture, such as meat quality and disease resistance. Second, they serve as valuable biomedical models for human disease research, regenerative medicine, and organ transplantation. Yet, despite notable advancements in recent decades, the efficiency of the current IVP systems for porcine embryos remains a challenge. Compared to the in vivo environment, suboptimal culture conditions lead to issues such as elevated polyspermy, poor embryo development, and the production of low-quality blastocysts. This review provides an overview of the key steps and recent advancements in porcine IVP technology. We will emphasize the promising utilization of oocytes from live females of high genetic value through ovum pick-up and the incorporation of extracellular vesicles and cytokines into IVP media. These innovative strategies hold immense potential to significantly enhance embryo development and overall success rates in porcine IVP, and could open the door for significant progress in both agriculture and biomedicine applications.

Vitrification of pig embryos dysregulates the microRNA transcriptome profile.

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4-6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139-3p, miR-214 and miR-885-3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-ß signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-ß (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.

Cryotop vitrification of large batches of pig embryos simultaneously provides excellent postwarming survival rates and minimal interference with gene expression.

The most commonly used technique to vitrify pig embryos is the super open pulled straw (SOPS), where a maximum of 6 embryos can be vitrified simultaneously per device without compromising the minimum volume necessary for optimal preservation. Since optimal embryo transfer (ET) demands a transfer of 20-40 embryos per recipient, the customary use of SOPS complicates embryo warming and ET in field conditions. Such complications could be avoided when using the Cryotop® (OC) system, which has been proven to be an effective option for vitrifying at least 20 porcine embryos simultaneously. This study aimed to investigate the changes in the transcriptome of blastocysts caused by vitrification using both systems. In vivo-derived blastocysts were OC- (n = 60; 20 embryos/device) and SOPS- (n = 60; 4-6 embryos/device) vitrified and cultured for 24 h after warming. Nonvitrified blastocysts (n = 60) cultured for 24 h postcollection acted as controls. At the end of culture, 48 viable embryos from each group (6 pools of 8 embryos) were selected for microarray (GeneChip® Porcine Genome Array, P/N 900624, Affymetrix) analysis of differentially expressed genes (DEGs). The survival rate of embryos vitrified with the OC and SOPS systems (>97%) was similar to that of the control embryos (100%). Microarray analysis of each vitrification system compared to the control group showed 245 DEGs (89 downregulated and 156 upregulated) for the OC system and 210 (44 downregulated and 166 upregulated) for the SOPS system. Two pathways were enriched for the DEGs specifically altered in each vitrification system compared to the control (glycolysis/gluconeogenesis and carbon metabolism pathways for the OC system and amino sugar and nucleotide sugar metabolism and lysosome pathways in the SOPS group). The OC group showed 31 downregulated and 24 upregulated genes and two enriched pathways (mineral absorption and amino sugar and nucleotide sugar metabolism pathways) when compared to the SOPS group. In summary, vitrification with the OC system altered fewer genes related to apoptosis and activated genes related to cell proliferation. We conclude that vitrification with either the OC or SOPS system has a moderate to low effect on the transcriptome of in vivo-derived porcine blastocysts. Further investigation is needed to elucidate how the differences in the transcriptome of embryos vitrified with these systems affect their subsequent developmental ability after ET.