|Abstract:||High starch diets, because of their increased energy density, are known to enhance animal performance. However, HS diets can have negative impacts on the animal when fed for an extended period, thus reducing animal productivity and performance. The addition of autolyzed yeast (AY) has been shown to have beneficial effects while feeding HS diets, resulting in positive effects on animal performance. The objective of the following study were to examine the effects of a commercially-available autolyzed yeast product (Levabon® Rumen E, BIOMIN Holding GmbH, Austria) in HS diets on lactation performance, rumen environment parameters, inflammatory markers, opportunity to improve N utilization, nutrient bioavailability, apparent digestibility, and in situ degradability, gene expression, and the rumen microbiome in lactating Holstein cows. Fifteen multiparous rumen-cannulated Holstein cows were assigned to 1 of 5 treatments in a replicated 5 × 5 Latin square design balanced to measure carryover effects. Treatments were: low starch diet without AY (LS0; control), high starch diet without AY (HS0), high starch diet with either 15 g (HS15), 30 g (HS30), or 45 g (HS45) of AY supplementation. Each period (21 d) was divided into an adaptation phase (d 1 to 14) and a measurement phase (d 15 to 21). Cows were milked 3 times per d, and samples were collected at each milking on d 15 and 20 of each period. Rumen fluid was sampled on d 15 and 16 starting at time point 0, which was related to feeding at 1400 h. Blood was sampled on d 15, 18, and 21. During the measurement phase, total mixed ration samples were collected on d 18 through 20 and orts on d 18 through 21 and analyzed for dry matter (DM), neutral detergent fiber (NDF), crude protein, starch, organic matter (OM), total N, and undegradable NDF at 120 h (uNDF 120 h). Urine samples were collected by manually stimulating urination at 0800, and 2030 h on d 20 of each period. Fecal samples were collected on d 18 through 20, every 8 h for a total of 8 collections per period. The fecal samples, TMR, and orts were used to calculate total-tract digestibility. In situ degradability determination of a standard alfalfa hay, corn silage (CS), and soybean meal was performed on d 19 through 21 of each period. Rumen papillae and liver biopsies were conducted on d 21 of each period. One single degree-of-freedom contrast LS0 compared to HS0 was used, and linear, quadratic, and cubic treatment effects of HS0, HS15, HS30, and HS45 for statistical inferences. Cows in HS0 had increased dry matter intake (DMI; 24.9 kg/d), milk yield (34.1 kg/d), and energy corrected milk (ECM; 34.4 kg/d) compared to LS0 (19.9 kg/d, 30.9 kg/d, and 31.3 kg/d, respectively). Cows in HS0 had greater protein (1.1 kg/d), casein (0.43 kg/d), and lactose yield (1.6 kg/d) when compared to LS0 (0.94 kg/d, 0.32 kg/d, and 1.4 kg/d, respectively). Cubic treatment effects were present for milk components, AY HS30 tended to increase milk yield, 3.5% fat corrected milk (FCM), lactose yield, and increased ECM, protein and casein percentage, while improving MUN. The cows in LS0 had greater FCM/DMI (1.6), ECM/DMI (1.6), and milk/DMI (1.6) efficiencies when compared to HS0 (1.3, 1.3, and 1.4, respectively). Cows in HS45 had increased DMI (25.6 kg/d) compared to cows in HS0 (24.9 kg/d). Cows in HS15 tended to improve 3.5% FCM/DMI (1.4), and ECM/DMI (1.4) efficiencies than HS0 (1.3, and 1.3, respectively). Cows in HS0 had lower ruminal pH (6.1), nadir pH (5.5), and fecal pH (6.7) when compared to LS0 (6.4, 5.7, and 6.9, respectively). Cows in HS0 had greater propionate and decreased acetate concentrations when compared to LS0, and tended to have a cubic treatment effect for acetate concentration. Cows in HS0 had greater N intake (719.6 vs. 583.7 g/d), milk protein N (166.0 vs. 140.3 g/d), and fecal N (268.4 vs. 219.8 g/d) with a tendency for greater microbial N production when compared to LS0. Supplementing AY decreased fecal N excretion, lowest at HS15. Cows in HS0 had greater nutrient intakes for OM (22.6 vs. 18.6 kg/d), crude protein (4.5 vs. 3.7 kg/d), and starch (6.6 vs. 4.7 kg/d) when compared to cows in LS0. Cows in LS0 had a tendency for greater digestibility for starch (95.0 vs. 93.7 %) and NDF (52.0 vs. 45.7 %), when compared to HS0, AY increased starch and NDF digestibility. Cows in LS0 had a greater degradable fraction of OM (0.35 vs. 0.28), NDF (0.75 vs. 0.37), and acid detergent fiber (0.66 vs. 0.43) for CS when compared to HS0. For CS NDF kd and acid detergent fiber degradable fraction tended to increase with AY, while undegradable starch decreased. There were no differences for LS0 compared to HS0 for liver and rumen papillae gene expression. Yeast supplementation increased expression of NFKB1 and TLR4 with increased AY dosage (greatest expression at 0.99 and 1.05, respectively). There were no differences for genes associated with acute phase protein or metabolism associated genes in hepatic tissue. Additionally, for rumen papillae there was a quadratic treatment effect for CD14, expressed greatest at HS15 (1.03). There was a cubic treatment effect for TLR2, expressed greatest at HS15 (1.00). Tight junction gene CLDN1 tended to linearly increase expression with increased AY dosage. Cows in HS45 (0.96) had the greatest CLDN1 expression compared other HS treatments. There were no differences for genes associated with metabolism in rumen papillae. High starch diets increased DMI, milk production, milk protein, casein, and lactose yields, lowered production efficiencies, increased VFA propionate concentration, and produced and excreted more N compared to LS0. Supplementing AY increased DMI, rumen pH, nadir pH, improved production efficiencies, milk components, increased VFA propionate concentration with greater AY dosage, reduced fecal N excretion, improved rate of degradation, and starch digestibility for CS, had greater CD 14, TLR2 and CLDN1 rumen papillae and NFKB1 and TLR4 in hepatic tissue gene expression when compared to HS0. In conclusion, adding AY to a HS diet had many benefits when fed to dairy cattle. This could be an important supplement to be considered by nutritionists that wish to formulate HS diets for dairy cows.