The dairy industry needs a gold-standard method that is accurate, precise, and simple to replicate.
By Dr. Rafael Caputo Oliveira Key points: - Balancing diets through supplementation of specific amino acids helps to improve milk yield and efficiency of protein utilization, increasing economic return to the dairy. - The lack of a standard method to estimate bioavailability of commercial AA products makes it difficult for nutritionists to choose the most cost-effective product. - In summary, nutritionists should only rely on bioavailability values of commercial products if they have been evaluated by in vivo experiments and published in a peer-reviewed journal. Protein is the most expensive nutrient in a dairy cow ration. Moreover, dairy cows are very inefficient in converting dietary protein into tissues or milk (e.g., 20 to 35% efficiency). Balancing diets through supplementation of specific amino acids (AA) helps to improve milk yield and efficiency of protein utilization, potentially increasing economic return. The most important parameter when choosing a specific rumen-protected AA (rpAA) is the cost per unit of bioavailable AA supplied. However, because there are multiple methods to estimate supply of AA from products, the main concern is that a lack of method standardization in the industry makes it difficult for end-users to properly decide on the most cost-effective product. This lack of a standardized method also has implications for researchers trying to understand dose-response mechanisms from AAs fed to dairy cows. Therefore, the dairy industry needs a gold-standard method that is precise, simple to replicate in different research facilities, and most importantly, its bioavailability outputs are applicable for cows under commercial dairy conditions. This concern was addressed in a symposium at the 2021 ADSA annual meeting. The topic called “Bioavailability of amino acids: Methods and lessons learned” was discussed by Dr. Zanton (researcher at the USDA Dairy Forage), Dr. Whitehouse (professor at University of New Hampshire), Dr. Hanigan (Professor at Virginia Tech), and moderated by Dr. McFadden (professor at Cornell University). The main goal was to discuss the limitations and inference from in vitro, in situ, and in vivo methods to evaluate bioavailability of rpAA products so the dairy industry can move forward. In vivo methods Two in vivo methods were discussed by Dr. Whitehouse and Dr. Hanigan as options to standardize the industry. To be a valid technique, the inference from these methods should be for cows under commercial dairy conditions. Therefore, it is important that all treatment doses are within physiological range to avoid changes in the normal metabolism of these cows. Feed abrasion, chewing, and rumination can all potentially break the coating protecting rpAA products, thus it is very important that products are incorporated in the TMR and fed to cows to mimic conditions at commercial dairies. 1) Plasma AA dose response approach: This method measures bioavailability of rpAA products by comparing the increase in plasma AA concentrations after feeding groups of cows with physiological doses of rpAA products relative to a positive control (Figure 1). The positive control group of cows is supplemented by continuous infusions (throughout the day and for 7d) of physiological doses of unprotected AA into the abomasum, therefore, intestinal absorption is assumed to be 100%. Each group of cows are fed a TMR containing different doses of rpAA 3 times per day for 7d. M
easurements only start after blood AA concentrations are no longer varying within day or among days, meaning that plasma AA entry and exit are in equilibrium. Because it is simple to replicate, it has been adopted in multiple dairy research facilities such as University of New Hampshire, The William H. Miner Agricultural Research Institute, University of Delaware, and The Autonomous University of Barcelona in Spain. Dozens of commercial rumen-protected methionine, lysine, and histidine products have been evaluated by this technique in modern lactating dairy cows. In conclusion, this relatively simple technique has high precision and accuracy. If linearity of responses is confirmed, this methodology has no apparent limitations. 2) Stable isotope-based approach: Stable isotopes are types of atoms that have the same atomic number but with different atomic mass (e.g., 13C or 15N). In summary, groups of animals are fed with rpAA products while being infused with unprotected AA(s) containing stable isotopes used as tracers. This method assesses bioavailability of rpAA products through the relative difference in dilution of stable isotopes-containing AA in blood from animals fed rpAA. This technique requires advanced mathematical modeling experience to fit the data, increasing the level of expertise necessary to perform it. In conclusion, this technique has high precision and as long as it is performed using modern lactating dairy cows it can be applied to the dairy industry. 3) Milk selenium dilution-based approach Although not discussed in that symposium, the milk selenium dilution method can also be a viable solution for evaluating specific commercial methionine (Met) products. This method estimates the relative supply of metabolizable Met based on changes in the concentration of selenomethionine (SeMet) relative to Met in milk. Because SeMet and Met are interchangeably used by cells as building blocks to make proteins, SeMet can be used as a tracer of Met. In this method if intake of SeMet is constant and supply of metabolizable Met increases, the ratio between SeMet and Met in milk is decreased. For simplicity, the authors proposed to measure concentrations of Se and N in milk as a proxy for concentrations of SeMet and Met in milk. If multiple doses for each treatment are tested and linearity of response occurs, this simpler method could be a simple solution to evaluate bioavailability of commercial Met products.
In vitro and in situ methods In vitro methods simulate ruminal escape and intestinal digestion of AA through techniques carried out entirely in laboratories. Because there is no animal and dietary influence on the outcome, the results are an inaccurate estimation of true AA bioavailability in dairy cows under farm conditions. Similarly, in situ methods also artificially simulate ruminal escape and intestinal digestion of AA. In summary, rpAA products are placed in nylon bags, introduced in the rumen, and retrieved after some time to estimate the product’s ruminal protection. Because this in situ bags can prevent bacteria from reaching the product, this method overestimates the product’s rumen protection. Following ruminal incubation, mobile bags are introduced into the intestine of animals through duodenal cannulas and retrieved in the manure to estimate intestinal AA digestibility. This method fails to consider that products are subjected to hindgut digestion by bacteria, overestimating the product’s intestinal absorption. Additionally, important factors that can impact bioavailability of rpAA products such as feed abrasion, chewing, and rumination are not accounted for in this technique. In conclusion, in vitro and in situ techniques overestimate the bioavailability of rpAA. Nutritionists should only rely on bioavailability values of rpAA products if they have been evaluated by in vivo experiments and published in a peer-reviewed journal. As longs as assumptions are met and tests are performed in cows under commercial dairy conditions, all in vivo methods mentioned previously can potentially become the standard technique to estimate AA supply from rpAA products. Cost and simplicity will determine what in vivo technique will be adopted by the dairy industry as a gold-standard method and so far, the plasma AA dose response method is ahead in this race.
