Supplementing diets with rumen-protected lysine is a common strategy to meet the nutritional needs of high-producing dairy cows. This work addressed two separate but crucial issues: the lysine protection degree across the entire digestive tract as well as the production scalability of the proposed delivery systems. This was achieved by evaluating, in vitro or ex vivo, previously developed rumen-resistant lipid nanoparticles regarding their stability in the digestive tract and in the bloodstream of the dairy cow as well as how their production could be scaled-up. Results showed that the developed nanoparticles were able to resist digestion along the digestive tract but were degraded in the blood over 24 h. Thus, releasing their content to be used by the animal. In vitro viability assays were also performed, with the nanoparticles being found not to be inherently toxic when using nanoparticle concentrations up to 1 mg/mL. Results showed that neither the purity of the used lipids nor the production method significantly altered the nanoparticles' properties or their ruminal resistance. Furthermore, the shelf-life of these nanoparticles was assessed, and they were found to retain their properties and remain usable after at least 1 month of storage. Moreover, a pilot-scale production allowed the production of nanoparticles with similar properties to the previous ones made using standard methods. To summarize, the proposed rumen-resistant nanoparticles presented potential as orally ingested lysine delivery systems for dairy cattle supplementation, being capable of a large-scale production using cheaper components while maintaining their properties and without any efficiency loss. It should however be noted that these results were obtained mainly in vitro and further in vivo bioavailability and production experiments are needed before this technology can be confirmed as a viable way of delivering lysine to dairy cows.
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