| Summary: | Determining the AA availability and metabolism in ruminant is a big challenge due to the rumen fermentation and complicated post absorption utilization. Current techniques used for direct determination of AA absorption and metabolism are laborious and expensive with large variation. The objectives of this project were to investigate AA availability of rumen undegradable protein, develop a stable isotope technique for determination of microbial protein and to evaluate the metabolism of amino acids in mammary glands of dairy cattle using stable isotope-based approaches. In the first experiment, seven heifers (258 ± 28 kg BW) were randomly chosen and assigned to 8 treatment sequences in a 7 x 8, incomplete, Latin square design. Treatments were a basal diet (BD), and 10% (DM basis) of BD replaced by corn silage (CS), grass hay (GH), alfalfa hay (AH), dried distillers grain (DDGS), soybean hulls (SH), wet brewers grain (BG), or corn grain (CG). Individual essential AA availabilities for corn silage, grass hay, alfalfa hay, dried distillers grain, soyhulls, brewers grain and corn grain were 33.4, 29.9, 34.1, 40.6, 28.8, 41.2, and 36.5% of the essential AA in each of the respective ingredients when a loss of 8.27% to splanchnic utilization during first pass was assumed; however, availability varied across individual essential AA. In the second experiment, twelve cows were blocked into 3 groups according to days in milk and randomly assigned to 4 treatments in a repeated 4 x 4 Latin square design with 2 factors to evaluate the essential AA availability from microbial protein and rumen undegradable protein under different rumen fermentation conditions. The 4 treatments were high rumen undegradable protein and high starch (HPHS), low rumen undegradable protein and high starch (LPHS), high rumen undegradable protein and low starch (HPLS) and low rumen undegradable protein and low starch (LPLS). Microbial protein synthesis calculated from purine derivatives was positively associated with rumen degradable protein, which was consistent with total microbial AA entry derived from the isotope dilution model indicating that the isotope based approach was representative. The individual essential AA availability from microbial protein was determined by isotope technique, whereas the PD method was just total PD absorption reflecting CP absorption. The metabolizable AA estimates from NDS nutritional model was similar to results from isotope dilution models, but with smaller difference among treatments. The microbial protein estimated from White's model showed the same trend among treatments compared to isotope dilution model, which may imply it represents the rumen fermentation better. The average essential AA digestibility for microbial AA was 82%, which varied across individual AA and treatments. In the third experiment, four cows (78 ± 10 DIM) were used to study the effects of jugular infusion of 2 groups of AA on essential AA uptake and metabolism by mammary glands in a 4 x 4 Latin square design. Treatments were jugular infusion of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL (MKH+IL). The MKH increased milk protein yield in high producing dairy cows. The IL infusion increased milk and milk lactose yields. The production response was associated with a change in mammary plasma flow together with changes in AA uptake and metabolism in mammary gland. Mammary uptake of essential AA was 135 % of milk protein output. Efflux of EAA from the mammary to blood was 13-61% of influx, which was high for BCAA but low for Met and Lys. Changes in influx and efflux resulted in net uptake difference of infused essential AA that were responsive to varying supplies resulting in maintenance of homeostasis. The proportion of AA catabolized and used for milk protein was affected by EAA infusion, which demonstrated plasticity of mammary gland in AA metabolism. Overall, results suggested essential AA availability from rumen undegraded protein and microbial protein varied across individual AA and diets and can be affected by rumen fermentation. After absorption, EAA transport into mammary tissue was bi-directional and their metabolism was affected by AA supply and energy. Using a single coefficient to represent all AA digestibility in MCP or feed ingredient and an integrated efficiency of MP-AA converted into milk protein is inaccurate. === Doctor of Philosophy === Studies in monogastric animals have showed that balancing AA supply with animal requirements can improve the efficiency of N utilization. In order to build a model for AA balanced diet formulation, the composition of feed ingredients, the profile and digestibility of EAA for the rumen undegradable protein and microbial protein, the partition and efficiency of EAA utilization in mammary glands must be determined accurately. However, current AA degradation, digestibility and metabolism data used in nutritional models are from in vitro and in situ studies, which have not been fully validated against in vivo observations. This research used an in vivo stable isotope-based approach to determine amino acid availability for commonly used feed ingredients in dairy industry. The microbial protein AA and rumen undegradable protein AA availability was determined by adapting this isotope technique and introducing another isotope into rumen to label microbes. In addition, by coupling stable isotope tracers with arterio-venous difference technique and compartmental modelling, essential AA metabolism in mammary glands of dairy cows were qualified. Total essential AA availabilities for corn silage, grass hay, alfalfa hay, dried distillers grain, soyhulls, brewers grain and corn grain were similar to values from meta-analysis of mobile bag results, but the availabilities of individual AA were more variable compared to in vitro and in situ results. The model derived microbial AA availability was consistent with the microbial protein calculated from NDS and Felming's model. However, our model predicted a lower proportion of metabolizable AA from microbial protein under diets including low rumen degradable protein, which might imply the NDS nutritional model overestimates microbial protein under low protein diets. The microbial protein estimated from White's model showed the same trend among treatments compared to isotope dilution model, which may imply it represents the rumen fermentation better. The averaged essential AA digestibility form microbial protein was 82%, which varied across individual AA and treatments. After absorption, mammary uptake of essential AA was 135 % of milk protein output. Cellular efflux represented 13 to 61% of essential AA uptake. The proportion of AA catabolized and used for milk protein was affected by essential AA infusion, which demonstrated the plasticity of mammary glands in AA metabolism. In conclusion, the results from isotope technique quantified the essential AA availability from rumen undegradable protein for various feed ingredients and from microbial protein under different feeding conditions. The essential AA transport and metabolism in mammary glands were regulated by multi factors and essential AA supply.
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