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Dietary replacement of soybean meal with heat-treated soybean meal or high-protein corn distillers grains on nutrient digestibility and milk composition in mid-lactation cows

Open AccessPublished:November 01, 2022DOI:https://doi.org/10.3168/jds.2022-21904

      ABSTRACT

      Lactation diets dependent on rumen undegradable protein (RUP) sources derived from soybean meal (SBM) products are generally high in Lys and poor in Met. We conducted an experiment to evaluate the effects of increasing dietary RUP and altering digestible AA supply by inclusion of heat-treated soybean meal (HTSBM) or high-protein corn dried distillers grains with soluble (DDGS) on performance in mid-lactation dairy cows. Twenty-four Holstein cows (200 ± 40 d in milk and 30.0 ± 3.92 kg/d of milk yield) blocked according to parity, milk yield, and days in milk were used in a 3 × 3 Latin square design experiment with 21-d periods. Treatments were (1) control (CON), a diet with 6.0% RUP containing 15.9% SBM as the main protein source; (2) HTSBM, a diet with 6.7% RUP containing 4.4% HTSBM partially replacing SBM; and (3) high-protein DDGS (FP; FlexyPro, SJC Bioenergia), a diet with 6.9% RUP containing 5.34% FP partially replacing SBM and ground corn. Diets had similar crude protein (16.9%) and net energy of lactation. Data were submitted to ANOVA using the mixed procedure of SAS software (SAS Institute Inc.). Treatment differences were evaluated using orthogonal contrasts: (1) increasing RUP (SBM vs. HTSBM + FP) and (2) altering digestible AA supply (HTSBM vs. FP). Cows fed HTSBM and FP had greater intake (values in parentheses represent treatment means of CON, HTSBM, and FP, respectively) of neutral detergent fiber (7.14, 7.35, and 7.69 kg/d), crude protein (4.27, 4.37, and 4.51 kg/d), and ether extract (0.942, 0.968, and 1.04 kg/d) compared with cows fed CON. Feeding FP resulted in greater intake of neutral detergent fiber and ether extract compared with HTSBM. Cows fed HTSBM and FP had lower sorting index for feed particles <4 mm than cows fed CON (1.029, 1.008, and 1.022). Feeding FP resulted in greater intake of feed particles <4 mm compared with HTSBM. Treatments containing HTSBM or FP tended to decrease organic matter digestibility (72.4, 71.2, and 71.1%), but no other effects were detected in digestibility of neutral detergent fiber, crude protein, or ether extract. No evidence for differences among treatments was detected in excretion of purine derivatives in milk and urine. Milk yield was greater in cows fed HTSBM or FP than in cows fed CON (28.0, 28.9, and 28.8 kg/d, respectively). Cows fed HTSBM or FP tended to have greater energy-corrected milk and protein yield compared with those fed CON. Milk protein concentration was greater in DDGS cows than those in the HTSBM group (3.45 and 3.40%, respectively). No differences were detected in milk fat yield and concentration, milk urea nitrogen, feed efficiency, or serum concentrations of urea and glucose. Overall, increasing dietary RUP by feeding HTSBM or FP improved intake of nutrients and milk yield without affecting feed efficiency. Altering digestible AA supply while maintaining similar dietary RUP had negligible effects on performance of cows.

      Key words

      INTRODUCTION

      In Brazil, soybean meal (SBM) products (heat treated or chemical treated) are the main ingredients providing RUP to the diets because corn gluten meal is scarce in certain regions and rumen-protected AA are relatively expensive. Furthermore, Brazilian federal regulations do not allow the inclusion of animal-derived products in diets of cattle to avoid any possible case of bovine spongiform encephalopathy (“mad cow disease”); thus, blood meal or fishmeal cannot be added to diets of ruminants (. Because a large portion of RUP in Brazilian diets is derived from SBM products, lactation diets are generally high in Lys and poor in Met. Dried distillers grains with soluble (DDGS) is a by-product from the corn ethanol industry that is rich in CP (especially RUP) and Met (
      • Belyea R.L.
      • Rausch K.D.
      • Tumbleson M.E.
      Composition of corn and distillers dried grains with solubles from dry grind ethanol processing.
      ;
      • Liu K.
      Chemical composition of distillers grains, a review.
      ). A commercially available high-protein (46% CP, of which 81.9% is RUP) corn DDGS containing protein derived from Saccharomyces spp. and from gluten (FP; FlexyPro, SJC Bioenergia) may be an alternative to commonly fed protein sources and other RUP supplements such as SBM and heat-treated soybean meal (HTSBM), respectively, in dairy cow diets.
      Responses to increased RUP by lactating cows have varied in the literature, but positive effects in performance are observed, especially when treated SBM is used as the main RUP source in the diet (
      • Santos F.A.P.
      • Santos J.E.P.
      • Theurer C.B.
      • Huber J.T.
      Effects of rumen-undegradable protein on dairy cow performance: A 12-year literature review.
      ;
      • Bateman II, H.G.
      Response to increased rumen undegradable protein intake by lactating dairy cows.
      ). Improvements in performance when supplementing RUP depend on intestinal digestibility and AA profile of RUP sources, as well as whether the increased flow of feed protein at the small intestine is offset, or not, by decreased microbial protein flow (
      • Bateman II, H.G.
      Response to increased rumen undegradable protein intake by lactating dairy cows.
      ). Feeding more RUP (+0.6% of diet DM) as expeller SBM resulted in improved feed efficiency and greater FCM and fat yield (
      • Broderick G.A.
      • Reynal S.M.
      Effect of source of rumen-degraded protein on production and ruminal metabolism in lactating dairy cows.
      ). Similarly,
      • Zanton G.I.
      • Heinrichs A.J.
      • Jones C.M.
      Short communication: Effects of level of rumen-degradable protein and corn distillers grains in corn silage-based diets on milk production and ruminal fermentation in lactating dairy cows.
      reported marginal increases in milk yield (+1.37 kg/d) and FCM (+1.48 kg/d) with similar DMI when increasing dietary RUP from 7.1 to 7.9%. Recently,
      • Brown W.E.
      • Bradford B.J.
      Effects of a high-protein corn product compared with soy and canola protein sources on nutrient digestibility and production responses in mid-lactation dairy cows.
      reported positive effects on milk and protein yield and feed efficiency in dairy cows fed diets with RUP sources (soy bypass protein combined with SBM or canola meal) replacing SBM.
      Corn DDGS have relatively low Lys (1.87% of RUP) and high Met (2.40% of RUP) compared with typical protein sources such as SBM and HTSBM (
      • Anderson J.L.
      • Schingoethe D.J.
      • Kalscheur K.F.
      • Hippen A.R.
      Evaluation of dried and wet distillers grains included at two concentrations in the diets of lactating dairy cows.
      ). According to estimated duodenal Lys supply based on
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      , partially replacing SBM (∼30% of total SBM in diet) with FP would increase digestible Met supply from 44 to 50 g/d and digestible Lys supply from 190 to 194 g/d. Thus, using FP as a RUP source compared with HTSBM may improve the AA profile (i.e., a Lys-to-Met ratio closer to 3:1) and potentially increase productivity of dairy cows. However, although adding DDGS may improve digestible AA profile, DDGS in a diet may cause sorting by cows.
      • Brown W.E.
      • Bradford B.J.
      Effects of a high-protein corn product compared with soy and canola protein sources on nutrient digestibility and production responses in mid-lactation dairy cows.
      fed a high-protein corn product in place of SBM in diets of dairy cows and reported greater orts CP concentration and lower CP intake as a percentage of total DMI compared with the other treatments, suggesting that cows sorted against the high-protein corn product. The authors speculated that this product imparted a flavor or odor that led cows to select against the high-protein corn diet (
      • Brown W.E.
      • Bradford B.J.
      Effects of a high-protein corn product compared with soy and canola protein sources on nutrient digestibility and production responses in mid-lactation dairy cows.
      ).
      We hypothesized that higher dietary RUP would improve animal performance by increasing digestible AA supply and that cows fed FP would have greater milk protein yield compared with those fed HTSBM because of differences in AA profile. The objectives of this study were to evaluate the effect of increased RUP on nutrient intake, feed sorting index, nutrient total-tract apparent digestibility, excretion of purine derivatives (PD) in milk and urine, milk yield and composition, and serum concentrations of urea-N and glucose by including FP in diets of lactating cows and to determine whether the responses observed were due to digestible AA profile.

      MATERIALS AND METHODS

      This study was carried out in the Laboratório de Pesquisa em Bovinos de Leite (LPBL; Laboratory on Dairy Cattle Research, Pirassununga, Brazil) under the approval of the Ethics Committee on Animal Use from the School of Veterinary Medicine and Animal Sciences, University of São Paulo (protocol #5986020620).

      Treatments and Experimental Design

      Twenty-four Holstein cows (6 primiparous and 18 multiparous; 200 ± 40 DIM, 599 ± 78 kg of BW, and 30.0 ± 3.92 kg/d of milk yield at the start of the experiment) blocked according to parity, milk yield, and DIM were enrolled in a replicated 3 × 3 Latin square design experiment. Experimental periods lasted 21 d, with the first 14 d for treatment adaptation and 7 d for data collection. Treatments were (1) control (CON), a diet with 6.0% RUP containing 15.9% SBM as the main protein source; (2) HTSBM, a diet with 6.7% RUP containing 4.4% HTSBM (Soypass, Cargill Animal Health and Animal Nutrition) partially replacing SBM; and (3) high-protein DDGS, a diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn. Diets were formulated to achieve similar CP and NEL. According to the manufacturer's information, FP is a type of DDGS, a by-product of corn ethanol production containing protein derived from Saccharomyces spp. and from gluten at a 20:80 ratio. According to the manufacturer's information, FP contains 46.0% CP, of which 81.9% is considered RUP, 3.5% Lys (% of CP), and 1.9% Met (% of CP). The RUP content of FP reported by the company was analyzed by a commercial laboratory using the method described in
      • Krishnamoorthy U.
      • Sniffen C.J.
      • Stern M.D.
      • Van Soest P.J.
      Evaluation of a mathematical model of rumen digestion and an in vitro simulation of rumen proteolysis to estimate the rumen-undegraded nitrogen content of feedstuffs.
      . Wet chemistry analysis demonstrated that FP had 88.6% DM, 49.8% NDF, 28.2% ADF, 44.9% CP, 9.13% ether extract (EE), 4.61% Lys (% of CP), and 1.93 Met (% of CP). An in situ procedure was used to estimate RUP of HTSBM and FP (
      • Chumpawadee S.
      • Sommart K.
      • Vongralub T.
      • Pattarajinda V.
      Estimation of rumen undegradable protein with in situ nylon bag and in vitro enzymatic technique in tropical concentrate feedstuffs.
      ). Briefly, nylon bags (R1020, Ankom Technology Corp.) of 10 × 20 cm were filled with 5 g of the protein sources (25 mg/cm2) and incubated in the rumen of 1 cannulated lactating dairy cow fed a corn silage-based diet. Bags were inserted into the rumen in reverse order for 48, 24, 16, 12, 8, 4, and 2 h, and all bags were removed simultaneously. Bag incubation residues were dried at 65°C for 72 h, weighed, and the residues were analyzed for CP. Bag residue CP was not corrected for microbial protein contamination. The RUP content in feeds was calculated according to
      • Chumpawadee S.
      • Sommart K.
      • Vongralub T.
      • Pattarajinda V.
      Estimation of rumen undegradable protein with in situ nylon bag and in vitro enzymatic technique in tropical concentrate feedstuffs.
      considering a 5% passage rate (kp). Average RUP values for HTSBM and FP were 73.4 and 81.8% CP, respectively. Soybean meal and HTSBM were chosen as treatments for comparison because they are the typical protein and bypass protein sources, respectively, fed on Brazilian farms and can be used as standard benchmarks for comparison with FP.
      Animals were individually fed twice daily (0700 and 1300 h) in equal amounts, and the feeding rate was adjusted to allow refusals between 5 and 10% on an as-fed basis. Diets (Table 1) were formulated according to the
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      to meet or exceed nutrient estimate requirements of cows and were designed to have similar CP contents. Both HTSBM and DDGS diets were formulated to yield 10.0% (diet DM) RDP content. Cows were housed in a barn with individual pens (17 m2 of area), sanded beds, fans, and free access to water. Samples of feed ingredients (Table 2) were collected during the last 7 d of each experimental period and analyzed for contents of DM (method 930.15;
      • AOAC International
      Official Methods of Analysis.
      ), ash (method 942.05;
      • AOAC International
      Official Methods of Analysis.
      ), CP (N × 6.25; Kjeldahl method 984.13;
      • AOAC International
      Official Methods of Analysis.
      ), and EE (method 920.39;
      • AOAC International
      Official Methods of Analysis.
      ). Neutral detergent fiber (
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      ) was analyzed using α-amylase (TE-149 fiber analyzer; Tecnal Equipamentos para Laboratório Inc.), and ADF and lignin (method 973.18) were analyzed according to
      • AOAC International
      Official Methods of Analysis.
      . Nonfiber carbohydrate was calculated according to
      • Hall M.B.
      Calculation of non-structural carbohydrate content of feeds that contain non-protein nitrogen. Bulletin 339.
      . Feed ingredients were analyzed for contents of starch using an enzymatic degradation method (amyloglucosidase, Novozymes Latin America Ltda.) and absorbances measured by spectrophotometer (SBA-200, Celm) according to
      • Hendrix D.L.
      Rapid extraction and analysis of nonstructural carbohydrates in plant tissues.
      . Feed ingredient samples were pooled per period and shipped to a commercial laboratory (Laboratório CBO) for AA profiling (Table 3), except for tryptophan. Samples were submitted to HCl (6 N) hydrolysis and further derivatization with phenylisothiocyanate; AA were analyzed using HLPC and UV detection (
      • White J.A.
      • Hart R.J.
      • Fry J.C.
      An evaluation of the Waters Pico-Tag system for the amino-acid analysis of food materials.
      ;
      • Cohen S.A.
      • Strydom D.J.
      Amino acid analysis utilizing phenylisothiocyanate derivatives.
      ). Digestibility of RUP (%) in protein sources was considered 93.0 (
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      ), 98.0 (
      • Schumacher E.
      • Erickson G.E.
      • Wilson H.
      • MacDonald J.
      • Watson A.
      • Klopfenstein T.
      Comparison of Rumen Undegradable Protein Content of Conventional and Organic Feeds.
      ), and 80.0 (
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      ) for SBM, HTSBM, and FP, respectively.
      Table 1Ingredient and chemical composition (% of DM, unless otherwise noted), balance of digestible Met and Lys, and particle size distribution of diets
      ItemTreatment
      Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass, Cargill Animal Health and Animal Nutrition) partially replacing SBM; and high protein DDGS (FP): diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      CONHTSBMFP
      Ingredient
       Corn silage
      Chemical (% of DM): 26.4% DM (as-fed), 95.9% OM, 40.8% NFC, 44.1% NDF, 28.6% ADF, 8.84% CP, 2.28% ether extract, and 26.4% starch.
      48.048.048.0
       Ground corn18.318.617.4
       Citrus pulp8.208.208.19
       Soybean meal, 46% CP solvent15.911.211.6
       Whole raw soybean6.476.476.47
       Heat-treated soybean meal
      Soypass (Cargill Animal Health and Animal Nutrition).
      4.40
       Dried distillers grains with solubles
      FlexyPro (SJC Bioenergia). Chemical (% of DM): 88.6% DM (as-fed), 97.0% OM, 25.0% NFC, 49.8% NDF, 28.2% ADF, 44.9% CP, 9.13% ether extract, and 5.70% starch.
      5.34
       Sodium bicarbonate0.820.820.82
       Mineral mixture
      Contained per kg: 215 g Ca, 60 g P, 20 g S, 20 g Mg, 35 g K, 70 g Na, 15 mg Co, 700 mg Cu, 600 mg F, 10 mg Cr, 700 mg Fe, 40 mg I, 1,600 mg Mn, 20 mg Se, 2,500 mg Zn, 200,000 IU vitamin A, 50,000 IU vitamin D3, and 1,500 IU vitamin E.
      1.291.291.29
       Limestone0.730.730.73
       Salt0.260.260.26
      Chemical composition
       DM41.542.442.6
       OM92.492.592.4
       Starch29.329.528.6
       CP16.916.817.0
       RDP
      Estimated according to NRC (2001).
      10.910.110.2
       RUP
      Estimated according to NRC (2001).
      6.006.706.90
       Ether extract3.703.734.12
       NDF28.028.229.3
       Forage NDF26.526.526.5
       NFC
      Calculated according to Hall (2000).
      42.539.435.1
       NEL,
      Estimated according to NRC (2001).
      Mcal/kg of DM
      1.591.591.58
       Ca0.950.940.93
       P0.410.400.40
       Mg0.220.220.22
       Cl0.430.420.43
       K1.261.261.19
       Na0.430.430.43
       S0.200.200.22
      Protein balance,
      Calculated according to NRC (2001) using as inputs the outcomes of the current study (BW, milk yield and composition, and DMI).
      g/d
       RDP290104124
       RUP526662855
       MP449537712
      MP requirements,
      Calculated according to NRC (2001) using as inputs the outcomes of the current study (BW, milk yield and composition, and DMI).
      g/d
      2,3002,3512,293
      AA balance, g/d
       dLys
      Requirements
      Requirements of digestible Met (dMet) and digestible Lys (dLys) were calculated as 2.2 and 6.6%, respectively, of MP requirements (NRC, 2001; Schwab et al., 2005; Lee et al., 2012).
      152155151
      Supply
      Calculated according to NRC (2001) using as inputs the outcomes of the current study (BW, milk yield and composition, and DMI).
      190196197
      Balance384146
       dMet
      Requirements
      Requirements of digestible Met (dMet) and digestible Lys (dLys) were calculated as 2.2 and 6.6%, respectively, of MP requirements (NRC, 2001; Schwab et al., 2005; Lee et al., 2012).
      50.651.750.4
      Supply
      Calculated according to NRC (2001) using as inputs the outcomes of the current study (BW, milk yield and composition, and DMI).
      444650
      Balance−6.6−5.7−0.4
      Particle size distribution, % as-fed
       >19 mm12.210.411.4
       19–8 mm36.938.835.2
       8–4 mm18.516.921.0
       <4 mm32.333.832.7
      1 Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass, Cargill Animal Health and Animal Nutrition) partially replacing SBM; and high protein DDGS (FP): diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      2 Chemical (% of DM): 26.4% DM (as-fed), 95.9% OM, 40.8% NFC, 44.1% NDF, 28.6% ADF, 8.84% CP, 2.28% ether extract, and 26.4% starch.
      3 Soypass (Cargill Animal Health and Animal Nutrition).
      4 FlexyPro (SJC Bioenergia). Chemical (% of DM): 88.6% DM (as-fed), 97.0% OM, 25.0% NFC, 49.8% NDF, 28.2% ADF, 44.9% CP, 9.13% ether extract, and 5.70% starch.
      5 Contained per kg: 215 g Ca, 60 g P, 20 g S, 20 g Mg, 35 g K, 70 g Na, 15 mg Co, 700 mg Cu, 600 mg F, 10 mg Cr, 700 mg Fe, 40 mg I, 1,600 mg Mn, 20 mg Se, 2,500 mg Zn, 200,000 IU vitamin A, 50,000 IU vitamin D3, and 1,500 IU vitamin E.
      6 Estimated according to
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      .
      7 Calculated according to
      • Hall M.B.
      Calculation of non-structural carbohydrate content of feeds that contain non-protein nitrogen. Bulletin 339.
      .
      8 Calculated according to
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      using as inputs the outcomes of the current study (BW, milk yield and composition, and DMI).
      9 Requirements of digestible Met (dMet) and digestible Lys (dLys) were calculated as 2.2 and 6.6%, respectively, of MP requirements (
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      ;
      • Schwab C.G.
      • Huhtanen P.
      • Hunt C.W.
      • Hvelplund T.
      ;
      • Lee C.
      • Hristov A.N.
      • Cassidy T.W.
      • Heyler K.S.
      • Lapierre H.
      • Varga G.A.
      • de Veth M.J.
      • Patton R.A.
      • Parys C.
      Rumen-protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.
      ).
      Table 2Chemical composition of feed ingredients (% of diet DM, unless otherwise stated)
      ItemSoybean mealHeat-treated soybean mealDDGS
      Dried distillers grains with solubles (FP; FlexyPro, SJC Bioenergia).
      Corn silageGround cornWhole raw soybeansCitrus pulp
      DM, % as-fed87.086.188.626.484.388.084.6
      OM93.696.097.096.098.395.094.2
      NFC22.919.42.5040.869.89.1666.1
      NDF20.024.949.844.133.631.517.8
      ADF8.9712.828.228.612.215.913.2
      CP48.650.344.98.8410.136.77.96
      NDICP
      Neutral detergent insoluble CP.
      6.3510.822.31.692.1311.02.08
      ADICP
      Acid detergent insoluble CP.
      2.594.7514.91.150.943.771.62
      Ether extract1.842.129.132.284.5017.32.08
      Lignin0.680.550.555.200.540.610.53
      Starch5.453.545.7025.078.74.469.36
      Indigestible NDF1.422.052.4619.12.241.293.29
      1 Dried distillers grains with solubles (FP; FlexyPro, SJC Bioenergia).
      2 Neutral detergent insoluble CP.
      3 Acid detergent insoluble CP.
      Table 3Amino acid profile of feed ingredients and experimental diets
      AA, % of DMCorn silage
      Chemical (% of DM): 26.4% DM (as-fed), 95.9% OM, 40.8% NFC, 44.1% NDF, 28.6% ADF, 8.84% CP, 2.28% ether extract, and 26.4% starch.
      Ground cornCitrus pulpSoybean mealWhole raw soybeanHTSBM
      Heat-treated soybean meal (HTSBM; Soypass, Cargill Animal Health and Animal Nutrition).
      FPTreatment
      Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      CONHTSBMFP
      Ala0.7510.6900.3242.231.672.263.240.9760.9731.047
      Arg0.6970.5930.6054.703.464.752.781.4641.4531.405
      Asp0.3700.7110.6696.284.656.323.451.6621.6471.570
      Cys0.0540.1400.0320.6640.5350.6710.7710.1950.1930.206
      Glu0.8491.780.5078.976.689.0797.5402.6332.6162.634
      Gly0.3160.3770.3022.131.642.161.830.6900.6860.692
      His0.1310.4100.1941.991.471.971.970.5650.5600.581
      Hyp0.0330.0220.1300.0740.0730.0750.0320.0470.0470.045
      Ile0.2500.3230.1732.261.752.261.700.6650.6600.656
      Leu0.6751.110.3353.852.943.925.081.3561.3511.452
      Lys0.2290.3660.2383.913.063.832.071.0161.0020.955
      Met0.0650.1620.0000.5480.4090.5640.8660.1750.1740.196
      Phe0.2720.4420.2162.511.922.552.120.7520.7480.754
      Pro0.4900.8620.9722.551.942.593.791.0041.0001.089
      Ser0.2070.4420.2382.601.882.662.310.7350.7310.743
      Thr0.2500.3020.1511.991.482.031.840.6000.5970.610
      Tyr0.1740.3130.1402.011.492.002.040.5690.5630.588
      Val0.3380.4530.2272.311.832.312.330.7480.7430.770
      1 Chemical (% of DM): 26.4% DM (as-fed), 95.9% OM, 40.8% NFC, 44.1% NDF, 28.6% ADF, 8.84% CP, 2.28% ether extract, and 26.4% starch.
      2 Heat-treated soybean meal (HTSBM; Soypass, Cargill Animal Health and Animal Nutrition).
      3 Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.

      Nutrient Intake, Sorting Index, and Apparent Digestibility

      Feed offered and refusals were recorded daily to determine feed intake. Refusals were sampled during the last 7 d of each experimental period, pooled by cow per period, and frozen for further chemical analysis according to the methods described earlier. Samples of TMR and refusals were collected for 2 consecutive days during the collection period for determination of particle size distribution (
      • Maulfair D.D.
      • Heinrichs A.J.
      Review: Methods to measure forage and diet particle size in the dairy cow.
      ) and sorting index (
      • Silveira C.
      • Oba M.
      • Yang W.Z.
      • Beauchemin K.A.
      Selection of barley grain affects ruminal fermentation, starch digestibility, and productivity of lactating dairy cows.
      ). Feed particles were stratified using the Penn State particle size separator to the following fractions: long (>19 mm), medium (19 to 8 mm), short (8 to 4 mm), and fine (<4 mm) particles. The sorting index was calculated using the following equations:
      Expected intake (kg/d) = intake (kg as-fed)/d × PTMR (kg/kg),


      Observed intake (kg/d) = [offered (kg/d) × PTMR (kg/kg)] − [refusals (kg/d) × PRefusals (kg/kg)],


      Sorting index = observed intake (kg/d)/expected intake (kg/d).


      The intake corresponding to each sieve was expressed as the percentage of the total estimated intake, where PTMR is the TMR particle size and Prefusals is the particle size distribution of refusals. A sorting index of 1 indicates no sorting, a sorting index <1 indicates sorting against, and a sorting index >1 indicates sorting for particles on the particular screen.
      Indigestible NDF (iNDF) contents in feeds, refusals, and feces were used to estimate fecal excretion of DM. Fecal samples (n = 8) were collected directly from the rectum of cows every 9 h during 3 consecutive days (d 15: 0600, 1500, and 0000 h; d 16: 0900 and 1800 h; d 17: 0300, 1200, and 2100 h) and pooled for further analyses. For the iNDF analysis, ground samples (2-mm) of feeds, refusals, and feces were placed in nonwoven fabric bags (12 µm pore size, 5 × 5 cm at 20 mg DM/cm2) and incubated in the rumen of 2 cannulated dry cows for 288 h (
      • Huhtanen P.
      • Kaustell K.
      • Jaakkola S.
      The use of internal markers to predict total digestibility and duodenal flow of nutrients in cattle given six different diets.
      ;
      • Casali A.O.
      • Detmann E.
      • Valadares Filho S.D.C.
      • Pereira J.C.
      • Henriques L.T.
      • De Freitas S.G.
      • Paulino M.F.
      Influência do tempo de incubação e do tamanho de partículas sobre os teores de compostos indigestíveis em alimentos e fezes bovinas obtidos por procedimentos in situ.
      ). After removal from the rumen, bags were washed in running tap water and dried, and the NDF content was determined. Digestibility of DM and nutrients were calculated using following equations:
      DMdigestibility(%)=100-[100×(%iNDFintake%iNDFinfeces)],


      Nutrientdigestibility(%)=100-[100×(%iNDFintake%iNDFinfeces)×(%nutrientinfeces%nutrientintake)].


      Excretion of Purine Derivatives

      Urine samples (20 mL) were collected at the same time points of feces. Urine samples were diluted (1:4 ratio) into a sulfuric acid solution at 0.036 N to preserve PD (
      • Chen X.B.
      • Gomes M.J.
      Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivatives—An overview of the technical details. Occasional publication.
      ). Samples were stored frozen for total N, allantoin, uric acid, and creatinine analyses. Daily urine volume was estimated considering a daily creatinine excretion of 29 mg/kg of BW (
      • Valadares R.F.D.
      • Broderick G.A.
      • Valadares Filho S.C.
      • Clayton M.K.
      Effect of replacing alfalfa silage with high moisture corn on ruminal protein synthesis estimated from excretion of total purine derivatives.
      ). Body weights were measured (on 2 consecutive days) after the morning milking at the beginning of experiment and during the last 2 d of each experimental period. Urine creatinine concentration was assessed using commercial kits (kinetic creatinine catalog no. K-067, Bioclin) and absorbances measured on spectrophotometer (SBA 200, Celm). Allantoin concentrations in urine and milk were assessed by a colorimetric method according to
      • Chen X.B.
      • Gomes M.J.
      Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivatives—An overview of the technical details. Occasional publication.
      , with absorbances measured on a microplate reader (Biochrom Asys UVM 340). Uric acid concentration in urine was analyzed using a commercial kit (uric acid stable liquid: catalog no. K-052; Bioclin). Total PD was calculated as the sum of allantoin excreted in milk and allantoin and uric acid excreted in urine (
      • Orellana Boero P.
      • Balcells J.
      • Martín-Orúe S.M.
      • Liang J.B.
      • Guada J.A.
      Excretion of purine derivatives in cows: Endogenous contribution and recovery of exogenous purine bases.
      ).

      Milk Yield and Composition and Serum Metabolites

      Cows were milked twice daily (0600 and 1700 h), and production was recorded electronically (Alpro, DeLaval). Data from the last 7 d of milk yield were averaged and used for statistical analysis. Milk samples (300 mL) were collected for 3 consecutive days during each experimental period to assess concentrations of protein, fat, and lactose using a mid-infrared method (Lactoscan, Entelbra). Fat-corrected milk was calculated according to
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      , where 4.0% FCM = 0.4 × milk (kg/d) + 15 × fat (kg/d). Milk samples were deproteinized with trichloroacetic acid solution (25%; 2:1 vol/vol;
      • Shahani K.M.
      • Sommer H.H.
      The protein and non-protein nitrogen fractions in milk. I. Methods of analysis.
      ) and stored at −20°C for allantoin and MUN analyses; MUN was determined using commercial kits (catalog no. K-056; Bioclin). Blood samples were collected from the tail veins on d 16 of each period, 4 h after the morning feeding. After clotting, blood samples were centrifuged (15 min, 2,000 × g at room temperature), and the serum was harvested and stored at −20°C for further glucose and urea analyses. Blood urea N was calculated by multiplying the urea concentration by 0.4667. Blood glucose (catalog no. K082) and urea (catalog no K056) were measured using colorimetric commercial kits (Bioclin) and absorbances were measured on a semi-automatic biochemistry analyzer.

      Statistical Analysis

      Data were submitted to ANOVA using the PROC MIXED of SAS 9.4 (SAS Institute Inc.) according to the following model:
      Yijkl=μ+Ai+Pj+Sk+ASik+αkl+εijkl,


      where αklN(0,σc2) and εklN(0,σe2), where N = Gaussian distribution; σc2 = estimated variance associated with cows; and σe2 = estimated residual variance, Yijkl = observation on animal l, given treatment i, at period j, in square k; k = overall mean, Ai = fixed effect of the ith treatment (i = 1 to 3); Pj = fixed effect of the jth period (j = 1 to 3); Sk = fixed effect of the kth Latin square (k = 1 to 8); ASik = interaction fixed effect between treatment and Latin square; αkl = random effect of animal within square (l = 1 to 24); and εijkl = random error associated with each observation. Means were adjusted by LSMEANS, and degrees of freedom were calculated using the
      • Kenward M.G.
      • Roger J.H.
      Small sample inference for fixed effects from restricted maximum likelihood.
      method. Treatment differences were evaluated by orthogonal contrasts (C), as follows: C1 = CON versus high-RUP sources (HTSBM + FP) and C2 = different digestible AA profile (HTSBM vs. FP). The significance level was set at P ≤ 0.05 and tendencies were considered when 0.05 < P < 0.10.

      RESULTS

      Increasing dietary RUP by using HTSBM or FP tended to increase (P ≤ 0.07) intake (kg/d) of DM and OM and increased (P ≤ 0.03) the intake of NDF, CP, and EE (Table 4). Dry matter intake was similar when comparing treatments with different digestible AA profile (HTSBM vs. FP). Cows fed FP tended to have greater (P = 0.09) NDF intake and had greater (P < 0.01) EE intake than cows in the HTSBM group. In terms of intake as a percent of BW, greater (P ≤ 0.01) DM and NDF intakes were observed in cows fed high-RUP treatments, whereas NDF intake was greater in FP group compared with cows fed HTSBM. Sorting index for feeds with small particle size (between 8 and 4 mm and <4 mm) was lower (P ≤ 0.03) in high-RUP treatments, whereas the FP group had greater (P ≤ 0.05) sorting index for feeds with small particle size than the HTSBM group. No differences were detected for sorting index for feeds with particle size >19 mm and 19 to 8 mm. Organic matter digestibility tended to be lower (P = 0.09) in high-RUP treatments than in CON. No other treatment differences were detected for digestibility coefficients of DM, NDF, CP, and EE among groups.
      Table 4Nutrient intake, sorting index, and total-tract apparent digestibility of mid-lactation cows fed different RUP sources partially replacing soybean meal in the diet
      ItemTreatment
      Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      SEMP-value
      Orthogonal contrasts: C1 = CON vs. high-RUP sources (HTSBM + FP); C2 = different digestible AA profile (HTSBM vs. FP).
      CONHTSBMFPC1C2
      Intake, kg/d
       DM25.325.826.20.830.060.37
       OM23.223.624.10.810.070.22
       NFC12.012.011.80.400.670.34
       NDF7.147.357.690.237<0.010.09
       CP4.274.374.510.1430.030.13
       Ether extract0.9420.9681.040.0331<0.01<0.01
      Intake, % of BW
       DM3.843.963.980.1460.010.66
       NDF1.101.131.170.041<0.010.03
      Sorting index
      No sorting = 1, values <1 indicates sorting against, and values >1 indicates sorting for particles on the particular particle size range. Sorting index was calculated according to Silveira et al. (2007).
       >19 mm0.9200.9380.9350.01650.210.85
       19–8 mm0.9770.9410.9680.01890.150.15
       8–4 mm1.0050.9780.9980.00660.020.02
       <4 mm1.0291.0081.0220.00690.030.05
      Digestibility coefficient, %
       DM70.270.068.70.750.180.11
       OM72.471.271.10.910.090.96
       NDF36.037.336.60.950.360.61
       CP69.969.668.70.970.440.42
       Ether extract80.479.379.71.540.450.76
      1 Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      2 Orthogonal contrasts: C1 = CON vs. high-RUP sources (HTSBM + FP); C2 = different digestible AA profile (HTSBM vs. FP).
      3 No sorting = 1, values <1 indicates sorting against, and values >1 indicates sorting for particles on the particular particle size range. Sorting index was calculated according to
      • Silveira C.
      • Oba M.
      • Yang W.Z.
      • Beauchemin K.A.
      Selection of barley grain affects ruminal fermentation, starch digestibility, and productivity of lactating dairy cows.
      .
      Body weight tended to be greater (P = 0.10) in cows under high-RUP treatments than in CON, whereas FP cows had greater (P = 0.04) BW than those in HTSBM group (Table 5). Estimated urine output was similar between CON and treatments with high RUP, but urine output tended to be greater (P = 0.10) in cows fed FP compared with HTSBM. Similar PD excretion (allantoin in milk and urine, and uric acid in urine) was observed among treatment groups.
      Table 5Purine derivative excretion of mid-lactation cows fed different RUP sources partially replacing soybean meal in the diet
      ItemTreatment
      Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      SEMP-value
      Orthogonal contrasts: C1 = CON vs. high-RUP sources (HTSBM + FP); C2 = different digestible AA profile (HTSBM vs. FP).
      CONHTSBMFPC1C2
      BW, kg65065165613.20.100.04
      Estimated urine, L/d19.618.821.01.280.800.10
      Purine derivatives, mmol/d
       Milk allantoin23.820.326.53.830.830.14
       Urine allantoin24627225923.90.510.69
       Urine uric acid1181261279.60.350.93
       Total38841844433.10.280.57
      1 Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      2 Orthogonal contrasts: C1 = CON vs. high-RUP sources (HTSBM + FP); C2 = different digestible AA profile (HTSBM vs. FP).
      Yields of actual milk and lactose were greater in high-RUP treatments than in CON (Table 6). Energy-corrected milk and protein yield tended to increase (P ≤ 0.10) when feeding high RUP treatments. No differences in milk and solids yield were observed between HTSBM and FP groups. Increasing dietary RUP had no effect on milk concentration of fat, protein, or lactose, but milk protein concentration was greater (P = 0.01) in cows fed FP than in cows fed HTSBM. Milk urea nitrogen concentration, feed efficiency, and serum concentrations of urea-N and glucose were not affected by treatments.
      Table 6Milk yield and composition, feed efficiency, and blood metabolites of mid-lactation cows fed different RUP sources partially replacing soybean meal in the diet
      ItemTreatment
      Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      SEMP-value
      Orthogonal contrasts: C1 = CON vs. high-RUP sources (HTSBM + FP); C2 = different digestible AA profile (HTSBM vs. FP).
      CONHTSBMFPC1C2
      Yield, kg/d
       Milk28.028.928.80.570.050.82
       4% FCM
      FCM was calculated according to NRC (2001), where 4.0% FCM = 0.4 × milk (kg/d) + 15 × fat (kg/d).
      29.730.730.50.600.120.70
       ECM
      ECM was calculated according to NRC (2001), where ECM (kg) = [0.327 × milk (kg)] + [12.95 × fat (kg)] + [7.65 × protein (kg)].
      32.533.533.40.590.100.87
       Fat1.241.281.260.0300.220.69
       Protein0.960.980.990.0190.070.44
       Lactose1.441.491.480.0280.040.80
      Composition, %
       Fat4.454.474.430.1060.950.64
       Protein3.433.403.450.0190.760.01
       Lactose5.145.175.150.0310.290.47
       MUN, mg/dL9.809.249.190.4060.230.90
      Efficiency
       Milk yield ÷ DMI1.121.141.120.0400.460.17
       FCM ÷ DMI1.291.321.280.0380.670.20
       ECM ÷ DMI1.301.321.300.0390.640.26
      Blood metabolites, mg/dL
       Urea-N18.718.818.30.470.710.38
       Glucose57.156.357.70.990.900.24
      1 Control (CON): diet with 6.0% RUP containing 15.9% soybean meal (SBM) as the main protein source; heat-treated soybean meal (HTSBM): diet with 6.7% RUP containing 4.4% HTSBM (Soypass) partially replacing SBM; and high-protein DDGS (FP), diet with 6.9% RUP containing 5.34% FP (FlexyPro, SJC Bioenergia) partially replacing SBM and ground corn.
      2 Orthogonal contrasts: C1 = CON vs. high-RUP sources (HTSBM + FP); C2 = different digestible AA profile (HTSBM vs. FP).
      3 FCM was calculated according to
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      , where 4.0% FCM = 0.4 × milk (kg/d) + 15 × fat (kg/d).
      4 ECM was calculated according to
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      , where ECM (kg) = [0.327 × milk (kg)] + [12.95 × fat (kg)] + [7.65 × protein (kg)].

      DISCUSSION

      We hypothesized that increasing dietary RUP by using either HTSBM or FP would improve performance, and altering digestible AA profile (i.e., Lys-to-Met ratio closer to 3:1) would result in enhanced milk protein yield when comparing HTSBM and FP treatments. It is important to highlight that the model for estimating protein degradability in the rumen used by
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      contains inaccuracies, as pointed out in
      • Tedeschi L.O.
      • Fox D.G.
      • Fonseca M.A.
      • Cavalcanti L.F.L.
      Models of protein and amino acid requirements for cattle.
      and in
      • NASEM (National Academies of Sciences, Engineering, and Medicine)
      Nutrient Requirements of Dairy Cattle.
      . For instance,
      • Broderick G.A.
      • Huhtanen P.
      • Ahvenjärvi S.
      • Reynal S.M.
      • Shingfield K.J.
      Quantifying ruminal nitrogen metabolism using the omasal sampling technique in cattle—A meta-analysis.
      meta-analyzed data of 32 studies and reported that
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      underpredicted RDP (i.e., overpredicted RUP) by 22% compared with observed omasal values. Inaccuracies in RDP and RUP estimates by
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      support positive responses in milk yield and DMI observed in literature when feeding more RUP to cows. Although in the current experiment, RDP and RUP amounts were fed in excess according to
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      estimates, feeding protein sources rich in protein fractions not degraded in the rumen increased milk yield without affecting total-tract digestibility of CP and excretion of PD. Improvements in performance when feeding more RUP may be observed if the increased flow of feed protein at the small intestine is not offset by decreased microbial protein flow (
      • Bateman II, H.G.
      Response to increased rumen undegradable protein intake by lactating dairy cows.
      ). Data of different studies, collectively, suggest that a minimum of 10% RDP is required to not impair DMI and microbial protein supply in dairy cows (
      • Reynal S.M.
      • Broderick G.A.
      Effect of dietary level of rumen-degraded protein on production and nitrogen metabolism in lactating dairy cows.
      ;
      • Broderick G.A.
      • Reynal S.M.
      Effect of source of rumen-degraded protein on production and ruminal metabolism in lactating dairy cows.
      ). Note that the current
      • NASEM (National Academies of Sciences, Engineering, and Medicine)
      Nutrient Requirements of Dairy Cattle.
      recommends a minimum diet RDP of 10%, which was used as the RDP threshold level in diet formulations for the current study. Improved performance of cows fed HTSBM and FP is associated with greater intake of DM, NDF, and CP, and supported by no differences in feed efficiency. The greater sorting for shorter feed particles observed in HTSBM and FP over CON might be a potential factor for differences in milk yield. An increase in sorting index for shorter feed particles when feeding FP contrasts with speculations by
      • Brown W.E.
      • Bradford B.J.
      Effects of a high-protein corn product compared with soy and canola protein sources on nutrient digestibility and production responses in mid-lactation dairy cows.
      regarding flavor or odor imparted during the production of a type of DDGS.
      Increased dietary RUP promoted feed intake of cows but tended to reduce total-tract apparent digestibility of OM. Greater DMI results in an increase in feed passage rate and less time available for degradation in the rumen and digestion throughout the gut. The reasons for the marginal increase in DMI when feeding RUP sources are not clear, based on the data reported in this study. Aligning with the results observed in this study, increasing dietary RUP by adding HTSBM or DDGS has been shown to increase DMI and milk yield (
      • Janicek B.N.
      • Kononoff P.J.
      • Gehman A.M.
      • Doane P.H.
      The effect of feeding dried distillers grains plus solubles on milk production and excretion of urinary purine derivatives.
      ;
      • Benchaar C.
      • Hassanat F.
      • Gervais R.
      • Chouinard P.Y.
      • Julien C.
      • Petit H.V.
      • Massé D.I.
      Effects of increasing amounts of corn dried distillers grains with solubles in dairy cow diets on methane production, ruminal fermentation, digestion, N balance, and milk production.
      ;
      • Martins C.M.M.R.
      • Fonseca D.C.M.
      • Alves B.G.
      • Arcari M.A.
      • Ferreira G.C.
      • Welter K.C.
      • Oliveira C.A.F.
      • Rennó F.P.
      • Santos M.V.
      Effect of dietary crude protein degradability and corn processing on lactation performance and milk protein composition and stability.
      ). The replacement of urea (0.78% of diet DM) and SBM (10.3% of diet DM) with HTSBM (at 4.47% of diet DM) in corn silage- and ground corn-based diets increased DMI and milk yield of mid-lactation cows (
      • Martins C.M.M.R.
      • Fonseca D.C.M.
      • Alves B.G.
      • Arcari M.A.
      • Ferreira G.C.
      • Welter K.C.
      • Oliveira C.A.F.
      • Rennó F.P.
      • Santos M.V.
      Effect of dietary crude protein degradability and corn processing on lactation performance and milk protein composition and stability.
      ). Replacing SBM (13.2% of diet DM) with different doses of DDGS (10 to 30% of diet DM) has linearly increased DMI and milk yield of cows fed diets with varied CP contents (
      • Benchaar C.
      • Hassanat F.
      • Gervais R.
      • Chouinard P.Y.
      • Julien C.
      • Petit H.V.
      • Massé D.I.
      Effects of increasing amounts of corn dried distillers grains with solubles in dairy cow diets on methane production, ruminal fermentation, digestion, N balance, and milk production.
      ). Feeding corn DDGS (at either 10 or 20% of diet DM) replacing SBM and ground corn improved milk (+1.9 kg/d) and protein yields and feed efficiency in dairy cows (
      • Anderson J.L.
      • Schingoethe D.J.
      • Kalscheur K.F.
      • Hippen A.R.
      Evaluation of dried and wet distillers grains included at two concentrations in the diets of lactating dairy cows.
      ). Other studies have reported either no effects on milk yield and DMI (
      • de Lima J.A.
      • Gavioli I.L.C.
      • Barbosa C.M.P.
      • Berndt A.
      • Gimenes F.M.A.
      • Paz C.C.P.
      Silagem de soja e silagem de ponta de cana no desempenho de cordeiros.
      ;
      • Maxin G.
      • Ouellet D.R.
      • Lapierre H.
      Effect of substitution of soybean meal by canola meal or distillers grains in dairy rations on amino acid and glucose availability.
      ;
      • Castillo-Lopez E.
      • Ramirez Ramirez H.A.
      • Klopfenstein T.J.
      • Hostetler D.
      • Karges K.
      • Fernando S.C.
      • Kononoff P.J.
      Ration formulations containing reduced-fat dried distillers grains with solubles and their effect on lactation performance, rumen fermentation, and intestinal flow of microbial nitrogen in Holstein cows.
      ) or tendencies for decreased DMI (
      • Anderson J.L.
      • Schingoethe D.J.
      • Kalscheur K.F.
      • Hippen A.R.
      Evaluation of dried and wet distillers grains included at two concentrations in the diets of lactating dairy cows.
      ;
      • Morris D.L.
      • Kim S.H.
      • Kononoff P.J.
      • Lee C.
      Continuous 11-week feeding of reduced-fat distillers grains with and without monensin reduces lactation performance of dairy cows.
      ) when replacing SBM with DDGS. Earlier studies have not shown evidence for effects of HTSBM on DMI and milk yield (
      • Ahrar M.
      • Schingoethe D.J.
      Heat-treated soybean meal as a protein supplement for lactating cows.
      ;
      • Driver L.S.
      • Grummer R.R.
      • Schultz L.H.
      Effects of feeding heat-treated soybeans and niacin to high producing cows in early lactation.
      ). We can ask whether changing RUP dietary levels from 6.0% to 6.70 to 6.90% is sufficient to alter milk production; we did show that altering RUP levels from 5.15 to 6.30% increased milk production of cows (
      • Martins C.M.M.R.
      • Fonseca D.C.M.
      • Alves B.G.
      • Arcari M.A.
      • Ferreira G.C.
      • Welter K.C.
      • Oliveira C.A.F.
      • Rennó F.P.
      • Santos M.V.
      Effect of dietary crude protein degradability and corn processing on lactation performance and milk protein composition and stability.
      ).
      • Janicek B.N.
      • Kononoff P.J.
      • Gehman A.M.
      • Doane P.H.
      The effect of feeding dried distillers grains plus solubles on milk production and excretion of urinary purine derivatives.
      altered dietary RUP from 6.87 to 7.49%, leading cows to produce 3.2 kg/d more milk. These studies demonstrated that relatively small differences in RUP levels can promote differences in milk yield even larger than those observed in the current experiment. Those studies, however, did not report values or balances of estimated MP supply to determine whether diets were sufficient in RUP. It is worth mentioning that the cows used in the current study are less efficient in terms of milk yield for every kilogram of DM consumed compared with the cows of studies cited above (
      • Janicek B.N.
      • Kononoff P.J.
      • Gehman A.M.
      • Doane P.H.
      The effect of feeding dried distillers grains plus solubles on milk production and excretion of urinary purine derivatives.
      ;
      • Benchaar C.
      • Hassanat F.
      • Gervais R.
      • Chouinard P.Y.
      • Julien C.
      • Petit H.V.
      • Massé D.I.
      Effects of increasing amounts of corn dried distillers grains with solubles in dairy cow diets on methane production, ruminal fermentation, digestion, N balance, and milk production.
      ).
      Most studies evaluating dietary inclusion of DDGS in diets have reported either an increase or no effect on DMI and milk yield (
      • Paz H.A.
      • de Veth M.J.
      • Ordway R.S.
      • Kononoff P.J.
      Evaluation of rumen-protected lysine supplementation to lactating dairy cows consuming increasing amounts of distillers dried grains with solubles.
      ;
      • Castillo-Lopez E.
      • Ramirez Ramirez H.A.
      • Klopfenstein T.J.
      • Hostetler D.
      • Karges K.
      • Fernando S.C.
      • Kononoff P.J.
      Ration formulations containing reduced-fat dried distillers grains with solubles and their effect on lactation performance, rumen fermentation, and intestinal flow of microbial nitrogen in Holstein cows.
      ;
      • Foth A.J.
      • Brown-Brandl T.
      • Hanford K.J.
      • Miller P.S.
      • Garcia Gomez G.
      • Kononoff P.J.
      Energy content of reduced-fat dried distillers grains with solubles for lactating dairy cows.
      ;
      • Ranathunga S.D.
      • Kalscheur K.F.
      • Anderson J.L.
      • Herrick K.J.
      Production of dairy cows fed distillers dried grains with solubles in low- and high-forage diets.
      ;
      • Testroet E.D.
      • Beitz D.C.
      • O'Neil M.R.
      • Mueller A.L.
      • Ramirez-Ramirez H.A.
      • Clark S.
      Feeding reduced-fat dried distillers grains with solubles to lactating Holstein dairy cows does not alter milk composition or cause late blowing in cheese.
      ), whereas few studies have reported negative effects of DDGS on performance of dairy cows (
      • Morris D.L.
      • Kim S.H.
      • Kononoff P.J.
      • Lee C.
      Continuous 11-week feeding of reduced-fat distillers grains with and without monensin reduces lactation performance of dairy cows.
      ). The reasons why
      • Morris D.L.
      • Kim S.H.
      • Kononoff P.J.
      • Lee C.
      Continuous 11-week feeding of reduced-fat distillers grains with and without monensin reduces lactation performance of dairy cows.
      found lowered performance in cows fed DGGS are not clear but might be related to the high inclusion rate of DDGS and consequently large amounts of PUFA in the diet, dietary RDP level (not reported), or AA balancing. For example, the control diet had a Lys:Met ratio of 4.00, whereas the DDGS diet had a Lys:Met ratio of 2.11. In addition, cows fed DDGS had a lower Lys plasma concentration than CON cows (
      • Morris D.L.
      • Kim S.H.
      • Kononoff P.J.
      • Lee C.
      Continuous 11-week feeding of reduced-fat distillers grains with and without monensin reduces lactation performance of dairy cows.
      ). The studies cited above used higher DDGS inclusion rates (≥9.7% of diet DM) than those in the present study, suggesting that even small changes in dietary protein profile (i.e., degradability and AA content) may improve productivity of cows. For instance, increasing the dietary content of RUP by less than 1% and Met by 0.021% resulted in 0.80 kg/d more milk produced by cows fed FP compared with CON. Some studies feeding more RUP by changing protein sources or supplementing rumen-protected AA, however, have reported no effects in performance of dairy cows (
      • Castillo-Lopez E.
      • Ramirez Ramirez H.A.
      • Klopfenstein T.J.
      • Hostetler D.
      • Karges K.
      • Fernando S.C.
      • Kononoff P.J.
      Ration formulations containing reduced-fat dried distillers grains with solubles and their effect on lactation performance, rumen fermentation, and intestinal flow of microbial nitrogen in Holstein cows.
      ;
      • Lee C.
      • Giallongo F.
      • Hristov A.N.
      • Lapierre H.
      • Cassidy T.W.
      • Heyler K.S.
      • Varga G.A.
      • Parys C.
      Effect of dietary protein level and rumen-protected amino acid supplementation on amino acid utilization for milk protein in lactating dairy cows.
      ;
      • Ranathunga S.D.
      • Kalscheur K.F.
      • Anderson J.L.
      • Herrick K.J.
      Production of dairy cows fed distillers dried grains with solubles in low- and high-forage diets.
      ). The reasons for the lack of responses could be related to inaccuracies in MP estimation (
      • Broderick G.A.
      • Huhtanen P.
      • Ahvenjärvi S.
      • Reynal S.M.
      • Shingfield K.J.
      Quantifying ruminal nitrogen metabolism using the omasal sampling technique in cattle—A meta-analysis.
      ;
      • NASEM (National Academies of Sciences, Engineering, and Medicine)
      Nutrient Requirements of Dairy Cattle.
      ) and imbalances in MP and digestible AA, which are often not reported in studies with protein and AA nutrition.
      Because one objective of this study was to evaluate the effects of altering the digestible AA profile of diets with similar RUP, the inclusion level of FP was based on dietary RUP of HTSBM treatment. Low inclusion rates of FP may also avoid potential negative effects of DDGS in milk fat when fed at levels >10% of diet DM as reported in some studies (
      • Zanton G.I.
      • Heinrichs A.J.
      • Jones C.M.
      Short communication: Effects of level of rumen-degradable protein and corn distillers grains in corn silage-based diets on milk production and ruminal fermentation in lactating dairy cows.
      ;
      • Morris D.L.
      • Kim S.H.
      • Kononoff P.J.
      • Lee C.
      Continuous 11-week feeding of reduced-fat distillers grains with and without monensin reduces lactation performance of dairy cows.
      ). High inclusions of DDGS may increase the dietary concentration of 18:2 fatty acid (as % of long-chain fatty acids), resulting in greater milk trans-10,cis-12 18:2 fatty acid concentration (
      • Morris D.L.
      • Kim S.H.
      • Kononoff P.J.
      • Lee C.
      Continuous 11-week feeding of reduced-fat distillers grains with and without monensin reduces lactation performance of dairy cows.
      ). It is well known that trans-10,cis-12 18:2 downregulates milk fat synthesis (
      • Harvatine K.J.
      • Boisclair Y.R.
      • Bauman D.E.
      Time-dependent effect of trans-10,cis-12 conjugated linoleic acid on gene expression of lipogenic enzymes and regulators in mammary tissue of dairy cows.
      ). We speculate that the relatively low inclusion of DDGS in the current study did not promote an increase in blood trans-10,cis-12 concentration large enough to impair milk fat synthesis.
      • Castillo-Lopez E.
      • Ramirez Ramirez H.A.
      • Klopfenstein T.J.
      • Hostetler D.
      • Karges K.
      • Fernando S.C.
      • Kononoff P.J.
      Ration formulations containing reduced-fat dried distillers grains with solubles and their effect on lactation performance, rumen fermentation, and intestinal flow of microbial nitrogen in Holstein cows.
      fed DDGS at 9.7% of diet DM and did not find differences in milk fat concentration and production (
      • Castillo-Lopez E.
      • Ramirez Ramirez H.A.
      • Klopfenstein T.J.
      • Hostetler D.
      • Karges K.
      • Fernando S.C.
      • Kononoff P.J.
      Ration formulations containing reduced-fat dried distillers grains with solubles and their effect on lactation performance, rumen fermentation, and intestinal flow of microbial nitrogen in Holstein cows.
      ). It is important to highlight that different responses of altering protein nutrition in performance (i.e., milk yield and feed efficiency) of cows might be observed in short-term (3- to 4-wk periods, Latin square) or in long-term (randomized complete block design) study designs; however, interaction effects between experimental design (changeover vs. continuous design) and treatments were not observed for yields of milk fat and protein (
      • Zanton G.I.
      Analysis of production responses to changing crude protein levels in lactating dairy cow diets when evaluated in continuous or change-over experimental designs.
      ).
      It is important to note that even though HTSBM or DDGS increased CP intake without altering CP digestibility, treatments did not affect MUN or BUN, supporting the trend for greater milk protein yield over the CON group. In contrast to most studies demonstrating that feeding DDGS at ≤20% has no effect on milk protein percentage (
      • Anderson J.L.
      • Schingoethe D.J.
      • Kalscheur K.F.
      • Hippen A.R.
      Evaluation of dried and wet distillers grains included at two concentrations in the diets of lactating dairy cows.
      ;
      • Kleinschmit D.H.
      • Schingoethe D.J.
      • Hippen A.R.
      • Kalscheur K.F.
      Dried distillers grains plus solubles with corn silage or alfalfa hay as the primary forage source in dairy cow diets.
      ;
      • Schingoethe D.J.
      • Kalscheur K.F.
      • Hippen A.R.
      • Garcia A.D.
      Invited review: The use of distillers products in dairy cattle diets.
      ), feeding DDGS resulted in greater milk protein concentration compared with HTSBM treatment in this study. Replacing soybean products with DDGS has also increased milk protein concentration and yield without affecting DMI in early lactation cows (
      • Mjoun K.
      • Kalscheur K.F.
      • Hippen A.R.
      • Schingoethe D.J.
      • Little D.E.
      Lactation performance and amino acid utilization of cows fed increasing amounts of reduced-fat dried distillers grains with solubles.
      ). Differences in milk protein concentration between cows fed HTSBM and FP are negligible because no differences were detected in milk protein yield when comparing both treatments. Studies that attempted to replace bypass protein sources with DDGS are scarce in the literature (
      • Mjoun K.
      • Kalscheur K.F.
      • Hippen A.R.
      • Schingoethe D.J.
      • Little D.E.
      Lactation performance and amino acid utilization of cows fed increasing amounts of reduced-fat dried distillers grains with solubles.
      ;
      • Brown W.E.
      • Bradford B.J.
      Effects of a high-protein corn product compared with soy and canola protein sources on nutrient digestibility and production responses in mid-lactation dairy cows.
      ). Feeding a novel high-protein corn product (56.1% CP; co-product from ethanol production) in place of SBM and expeller SBM, authors reported lower ECM, fat, and protein yield, and DM digestibility (
      • Brown W.E.
      • Bradford B.J.
      Effects of a high-protein corn product compared with soy and canola protein sources on nutrient digestibility and production responses in mid-lactation dairy cows.
      ). These authors related the negative effects of the high-protein corn product to formation of Maillard products during drying (
      • Brown W.E.
      • Bradford B.J.
      Effects of a high-protein corn product compared with soy and canola protein sources on nutrient digestibility and production responses in mid-lactation dairy cows.
      ).
      As we expected, all diets were apparently excessive for Lys and poor in Met. Note that
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      considers the RUP fraction of feeds to have the same AA profile as the feed CP, and we did not observe treatment differences in PD excretion (a proxy of rumen microbial protein synthesis). Earlier studies have proposed an approximately 3:1 ratio of Lys to Met in MP to improve milk protein synthesis (
      • Armentano L.E.
      • Bertics S.J.
      • Ducharme G.A.
      Response of lactating cows to methionine or methionine plus lysine added to high protein diets based on alfalfa and heated soybeans.
      ;
      • Rulquin H.
      • Graulet B.
      • Delaby L.
      • Robert J.C.
      Effect of different forms of methionine on lactational performance of dairy cows.
      ;
      • Wang C.
      • Liu H.Y.
      • Wang Y.M.
      • Yang Z.Q.
      • Liu J.X.
      • Wu Y.M.
      • Yan T.
      • Ye H.W.
      Effects of dietary supplementation of methionine and lysine on milk production and nitrogen utilization in dairy cows.
      ;
      • Osorio J.S.
      • Ji P.
      • Drackley J.K.
      • Luchini D.
      • Loor J.J.
      Supplemental Smartamine M or MetaSmart during the transition period benefits postpartal cow performance and blood neutrophil function.
      ). In the current study, the FP treatment had the lowest Lys-to-Met ratio (3.98), whereas other treatments had values around 4.3. Furthermore, the balance of digestible Met (supply – requirements) was greater in FP than in other treatments. Differences in Lys-to-Met ratio between treatments were expected to be greater than we observed; the Lys content in DDGS reported by the manufacturer (3.5% of CP) was lower than that observed (4.61% of CP) in the current study. In addition, the Met content in diet might have been underestimated because some sulfur AA can be lost during the HCl hydrolysis used for AA profile determination. Cysteine and methionine are unstable AA and can be degraded during acid hydrolysis because of the reaction of sulfur with residual oxygen by the formation of methionine sulfoxide or sulfone and cysteine sulfenic, sulfenic, or sulfonic acid (
      • Lamp A.
      • Kaltschmitt M.
      • Lüdtke O.
      Improved HPLC-method for estimation and correction of amino acid losses during hydrolysis of unknown samples.
      ). One caveat is that we did not measure the intestinal digestibility of RUP fraction of HTSBM and FP to accurately estimate MP supply. According to
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      calculations, treatments containing sources rich in RUP (HTSBM and FP) would provide greater MP balance than CON, which could be associated with increases in milk yield. According to PD excretion, FP dietary inclusion did not alter microbial protein supply in the current study, which is in line with previous studies in the literature (
      • Janicek B.N.
      • Kononoff P.J.
      • Gehman A.M.
      • Doane P.H.
      The effect of feeding dried distillers grains plus solubles on milk production and excretion of urinary purine derivatives.
      ;
      • Maxin G.
      • Ouellet D.R.
      • Lapierre H.
      Effect of substitution of soybean meal by canola meal or distillers grains in dairy rations on amino acid and glucose availability.
      ).
      In summary, we demonstrated that our hypothesis—that increasing dietary RUP using either HTSBM or FP would improve performance of dairy cows—was confirmed by increased milk yield, which is supported by increased DMI and higher balances of digestible AA. The expected improvement in protein yield when altering digestible AA profile using FP to replace soybean products was not evident in this study. Feeding DDGS in place of soybean products had minor effects on nutrient intake and performance of cows, demonstrating that DDGS can be a valuable substitute for HTSBM, depending on market price fluctuations and product availability. Furthermore, replacing corn starch and SBM products with DDGS has been economically beneficial to dairy producers when included up to 20% of diet DM (
      • Ranathunga S.D.
      • Kalscheur K.F.
      • Hippen A.R.
      • Schingoethe D.J.
      Replacement of starch from corn with nonforage fiber from distillers grains and soyhulls in diets of lactating dairy cows.
      ,
      • Ranathunga S.D.
      • Kalscheur K.F.
      • Anderson J.L.
      • Herrick K.J.
      Production of dairy cows fed distillers dried grains with solubles in low- and high-forage diets.
      ). In the current study, FP (at 5.34% FP) replaced 0.90% ground corn and 4.3% (of diet DM) SBM in the CON diet. In other words, for every 1,000 kg of diet DM, around 1 kg of ground corn and 4 kg of SBM were replaced by 5 kg of DDGS. According to
      • ERS (Economic Research Service)
      Feed Outlook: April 2022, FDS-22d.
      , in 2020–2021, the average cost of corn was $178/t, that of SBM was $230/t, and that of distillers dried grains was $198/t. Thus, if the performance is maintained, replacing SBM with DDGS in diets of dairy cows seems economically favorable.

      CONCLUSIONS

      Partially replacing SBM with either HTSBM (4.40% of diet DM) or a high-protein corn DDGS (5.34% of diet DM) to increase dietary RUP improved feed intake and milk yield without affecting DM digestibility or PD excretion in mid-lactation cows. Compared with HTSBM, feeding the high-protein corn DDGS had negligible effects on milk protein concentration as no differences in milk yield and milk protein concentration were observed. This study demonstrated that the high-protein corn DDGS (at 5.34% of diet DM) can be a suitable substitute for HTSBM.

      ACKNOWLEDGMENTS

      This study received no external funding. The authors have not stated any conflicts of interest.

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