Production of rumen- and gastrointestinal-resistant nanoparticles to deliver lysine to dairy cows.

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.

Validation of a Simple HPLC-Based Method for Lysine Quantification for Ruminant Nutrition.

Robust and selective quantification methods are required to better analyze feed supplementation effectiveness with specific amino acids. In this work, a reversed-phase high-performance liquid chromatography method with fluorescence detection is proposed and validated for lysine quantification, one of the most limiting amino acids in ruminant nutrition and essential towards milk production. To assess and widen method applicability, different matrices were considered: namely Li2CO3 buffer (the chosen standard reaction buffer), phosphate buffer solution (to mimic media in cellular studies), and rumen inoculum. The method was validated for all three matrices and found to be selective, accurate (92% ± 2%), and precise at both the inter- and intra-day levels in concentrations up to 225 µM, with detection and quantification limits lower than 1.24 and 4.14 µM, respectively. Sample stability was evaluated when stored at room temperature, 4 °C, and -20 °C, showing consistency for up to 48 h regardless of the matrix. Finally, the developed method was applied in the quantification of lysine on real samples. The results presented indicate that the proposed method can be applied towards free lysine quantification in ruminant feeding studies and potentially be of great benefit to dairy cow nutrition supplementation and optimization.

Applying nanotechnology to increase the rumen protection of amino acids in dairy cows.

The amino acid requirements of high-production dairy cows represent a challenge to ensuring that their diet is supplied with available dietary resources, and thus supplementation with protected amino acids is necessary to increase their post-ruminal supply. Lysine is often the most limiting amino acid in corn-based diets. The present study proposes the use of lipid nanoparticles as novel rumen-bypass systems and assesses their capability to carry lysine. Solid lipid nanoparticles, nanostructured lipid carriers and multiple lipid nanoparticles were considered and their resistance in a rumen inoculum collected from fistulated cows was assessed. All nanoparticles presented diameters between 200-500 nm and surface charges lower than -30 mV. Lysine encapsulation was achieved in all nanoparticles, and its efficiency ranged from 40 to 90%. Solid lipid nanoparticles composed of arachidic or stearic acids and Tween 60 resisted ruminal digestion for up to 24 h. The nanoparticles were also proven to protect their lysine content from the ruminal microbiota. Based on our findings, the proposed nanoparticles represent promising candidates for rumen-bypass approaches and should be studied further to help improve the current technologies and overcome their limitations.