Optimal amino acid system for early embryo development in sows based on response surface methodology and high-throughput screening cell models

Abstract

Early embryo development plays a pivotal role in determining pregnancy outcomes, postnatal development, and lifelong health. Therefore, the strategic selection of functional nutrients to enhance embryo development is of paramount importance. In this study, we established a stable porcine trophectoderm cell line expressing dual fluorescent reporter genes driven by the CDX2 and TEAD4 gene promoter segments using lentiviral transfection. Three amino acid metabolites—kynurenic acid, taurine, and tryptamine—met the minimum z-score criteria of 2.0 for both luciferase and Renilla luciferase activities and were initially identified as potential metabolites for embryo development, with their beneficial effects validated by qPCR. Given that the identified metabolites are closely related to methionine, arginine, and tryptophan, we selected these three amino acids, using lysine as a standard, and employed response surface methodology combined with our high-throughput screening cell model to efficiently screen and optimize amino acid combination conducive to early embryo development. The optimized candidate amino acid system included lysine (1.87 mmol/L), methionine (0.82 mmol/L), tryptophan (0.23 mmol/L), and arginine (3 mmol/L), with the ratio of 1:0.43:0.12:1.60. In vitro experiments confirmed that this amino acid system enhances the expression of key genes involved in early embryonic development and improves in vitro embryo adhesion. Transcriptomic analysis of blastocysts suggested that candidate amino acid system enhances early embryo development by regulating early embryonic cell cycle and differentiation, as well as improving nutrient absorption. Furthermore, based on response surface methodology, 400 sows were used to verify this amino acid system, substituting arginine with the more cost-effective N-carbamoyl glutamate (NCG), a precursor of arginine. The optimal dietary amino acid requirement was predicted to be 0.71% lysine, 0.32% methionine, 0.22% tryptophan, and 0.10% NCG for sows during early gestation. The optimized amino acid system ratio of the feed, derived from the peripheral release of essential amino acids, was found to be 1:0.45:0.13, which is largely consistent with the results obtained from the cell model optimization. Subsequently, we furtherly verified that this optimal dietary amino acid system significantly increased total litter size, live litter size and litter weight in sows. In summary, we successfully established a dual-fluorescent high-throughput screening cell model for the efficient identification of potential nutrients that would promote embryo development and implantation. This innovative approach overcomes the limitations of traditional amino acid nutrition studies in sows, providing a more effective model for enhancing reproductive outcomes.