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After parturition, dairy cows mobilize AA from skeletal muscle to meet metabolizable protein (MP) requirements. High mobilization may compromise cow health and longer-term milk production. Postpartum diets with higher MP concentrations, improved AA profiles, or MP increased at the expense of forages rather than nonforage fiber sources may attenuate muscle catabolism; however, the molecular mechanisms responsible need investigation. We evaluated mRNA expression in the longissimus dorsi of cows fed postpartum diets differing in MP concentration, AA profile, and fiber source. From 0 to 25 d after parturition, 40 multiparous cows received the following diets: (1) 13% deficient in MP (D-MP), (2) adequate in MP using primarily soy protein (A-MP), (3) adequate in MP using blends of proteins and individual AA to improve the AA profile (Blend), or (4) similar to Blend except additional protein replaced forage (Blend-fNDF). Biopsies were taken approximately -5, 7, and 25 d relative to parturition. Greater dietary MP concentration (D-MP vs. A-MP and Blend) decreased expression of genes related to protein synthesis (MTOR, RPS6KB1) and degradation (FOXO1), inflammation (IFNG, TLR4), and endoplasmic reticulum (ER) stress (HSPA5, DDIT) and increased genes associated with lipogenesis (PPARG) and glucose oxidation (LDH, MB). In Blend versus A-MP (i.e., effect of AA profile), expression related to apoptosis (CASP8) and inflammation (TNFA) decreased and genes associated with cell cycle progression (E2F1) and fast-twitch glycolytic muscle fiber type (MYH4) increased. Less forage (Blend-fNDF vs. Blend) decreased genes associated with lipogenesis (PPARG, ACACA) and ER stress (BCL2, DDIT3, EIF2AK3, PPP1R15A) and increased genes associated with inflammation (TNF), inhibition of myogenesis (MSTN), and autophagy (PEBP1). In summary and based on mRNA expression, increasing MP supply may attenuate muscle turnover and ER stress. However, an unbalanced AA supply reduced cell cycle progression and protein synthesis. Lower energy supplies may reduce cell growth and cause autophagy., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Increasing the supply of metabolizable protein (MP) and improving its AA profile may attenuate body protein mobilization in fresh cows and lead to increased milk production. Increasing the concentration of rumen-undegradable protein (RUP) to increase MP supply and replacing RUP sources from forages instead of nonforage fiber sources may further decrease tissue mobilization if it improves dry matter intake (DMI). Our objective was to determine whether increasing MP concentrations and improving the AA profile at the expense of either nonforage or forage fiber (fNDF) would affect MP balance and empty body (EB) composition (measured using the urea dilution method) in early postpartum dairy cows of different parities. In a randomized block design, 40 primigravid [77 ± 1.5 kg of EB crude protein (CP) at 8 ± 0.6 d before calving] and 40 multigravid (92 ± 1.6 kg of EB CP at 5 ± 0.6 d before calving) Holsteins were blocked by calving date and fed a common prepartum diet (11.5% CP). After calving to 25 d in milk (DIM), cows were fed 1 of 4 diets: (1) a diet deficient in MP meeting 87% of MP requirements (DMP, 17% CP, 24% fNDF), (2) 104% of MP requirements using primarily soy protein to make MP adequate (AMP, 20% CP, 24% fNDF), (3) 110% of MP requirements using a blend of proteins and rumen-protected (RP) AA to make MP adequate (Blend, 20% CP, 24% fNDF), or (4) a diet similar to Blend but substituting added RUP for fNDF rather than nonforage NDF (Blend-fNDF, 20% CP, 19% fNDF). Blend was formulated to have a RUP supply with a similar AA profile to that of casein. Cows were fed a common diet (16.3% CP) from 26 to 50 DIM. Calculated MP balance (supply - requirements) was less than zero for DMP and Blend-fNDF from 1 to 4 wk of lactation (WOL), whereas that for AMP was positive from 1 to 4 WOL and that for Blend was close to zero from 3 to 4 WOL. Daily MP balance was greater from 5 to 7 WOL for DMP compared with AMP and Blend (100 vs. 22 g/d). From -7 to 7 d relative to calving, losses of EB CP were greater for DMP than for AMP and Blend (-121 vs. average of 11 g/d). From 7 to 25 DIM, cows fed AMP (-139 g/d) and Blend-fNDF (-147 g/d) lost EB CP but cows fed Blend (-8 g/d) maintained EB CP. Increased DMI for Blend versus AMP led to reduced losses of EB lipid in primiparous cows from 7 to 25 d relative to calving (-1.0 vs. -1.3 kg/d of EB lipid), whereas lipid mobilization was similar in multiparous cows (average -1.1 kg of EB lipid/d). By 50 DIM, EB lipid and CP were similar across treatments and parities (average 60.2 kg of EB lipid and 81.6 kg of EB CP). Overall, feeding fresh cows a high MP diet with a balanced AA profile improved DMI and attenuated EB CP mobilization, which could partly explain positive carryover effects on milk production for multiparous cows and reduced lipid mobilization for primiparous cows., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Because of low feed intake during the first weeks of lactation, dietary concentration of metabolizable protein (MP) must be elevated. We evaluated effects of providing additional rumen-undegradable protein (RUP) from a single source or a blend of protein and AA sources during the first 3 wk of lactation. We also evaluated whether replacing forage fiber (fNDF) or nonforage fiber with the blend affected responses. In a randomized block design, at approximately 2 wk prepartum, 40 primigravid (664 ± 44 kg of body weight) and 40 multigravid (797 ± 81 kg of body weight) Holsteins were blocked by calving date and fed a common diet (11.5% crude protein, CP). After calving to 25 d in milk (DIM), cows were fed 1 of 4 diets formulated to be (1) 20% deficient in metabolizable protein (MP) based on predicted milk production (17% CP, 24% fNDF), (2) adequate in MP using primarily RUP from soy to increase MP concentration (AMP; 20% CP, 24% fNDF), (3) adequate in MP using a blend of RUP and rumen-protected AA sources to increase MP concentration (Blend; 20% CP, 24% fNDF), or (4) similar to Blend but substituting fNDF with added RUP rather than nonforage neutral detergent fiber (Blend-fNDF; 20% CP, 19% fNDF). The blend was formulated to have a RUP supply with an AA profile similar to that of casein. A common diet (17% CP) was fed from 26 to 92 DIM, and milk production and composition were measured from 26 to 92 DIM, but individual dry matter intake (DMI) was measured only until 50 DIM. During the treatment period for both parities, AMP and Blend increased energy-corrected milk (ECM) yields compared with the diet deficient in MP based on predicted milk production (40.7 vs. 37.8 kg/d) and reduced concentrations of plasma 3-methyl-His (4.1 vs. 5.3 µmol/L) and growth hormone (9.0 vs. 11.9 ng/mL). Blend had greater DMI than AMP (17.4 vs. 16.1 kg/d), but ECM yields were similar. Blend had greater plasma Met (42.0 vs. 26.4 µmol/L) and altered metabolites associated with antioxidant production and methyl donation compared with AMP. Conversely, the concentration of total essential AA in plasma was less in Blend versus AMP (837 vs. 935 µmol/L). In multiparous cows, Blend-fNDF decreased DMI and ECM yield compared with Blend (19.2 vs. 20.1 kg/d of DMI, 45.3 vs. 51.1 kg/d of ECM), whereas primiparous cows showed the opposite response (15.3 vs. 14.6 kg/d of DMI, 32.9 vs. 31.4 kg/d of ECM). Greater DMI for multiparous cows fed Blend carried over from 26 to 50 DIM and was greater compared with AMP (23.1 vs. 21.2 kg /d) and Blend-fNDF (21.3 kg/d). Blend also increased ECM yield compared with AMP (49.2 vs. 43.5 kg/d) and Blend-fNDF (45.4 kg/d) from 26 to 92 DIM. Few carryover effects of fresh cow treatments on production were found in primiparous cows. Overall, feeding blends of RUP and AA may improve the balance of AA for fresh cows fed high MP diets and improve concurrent and longer-term milk production in multiparous cows. However, with high MP diets, multiparous fresh cows require greater concentrations of fNDF than primiparous cows., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

We hypothesized that dairy cows fed oscillating metabolizable protein (MP) and crude protein (CP) concentrations on a 24-h frequency for a diet formulated to be below MP requirements would use N more efficiently (i.e., increased milk protein yields and less manure N) without increasing mobilization of body protein stores than would cows fed the same deficient MP diet continuously, although both treatments would on average have equal MP concentrations. In a randomized block design, 30 Holstein cows (119 ± 21 d in milk; 667 ± 69 kg of body weight) were blocked according to milk yield within a parity (3 primiparous and 7 multiparous blocks) and fed 1 of 3 treatments: (1) diet with 16.2% CP (109% of MP requirements) fed continuously (109MP), (2) diet with 14.1% CP (95% of MP requirements) fed continuously (95MP), or (3) diets oscillating on a 24-h cycle from the 109MP diet and a diet with 11.9% CP (∼78% of MP requirements) such that average CP and MP concentration would be the same as 95MP (OSC). Dry matter intake was similar between 109MP and 95MP (22.9 vs. 23.2 kg/d) but tended to be lower for OSC (22.2 kg/d) compared with 95MP. Milk yield was greater for 109MP compared with 95MP (36.6 vs. 35.1 kg/d) and similar between 95MP and OSC (35.3 kg/d). Milk protein and energy-corrected milk yields were similar among treatments. Milk urea N (MUN) concentration was higher for 109MP compared with 95MP (12.8 vs. 10.2 mg/dL), and tended to be higher for OSC (10.9 mg/dL) compared with 95MP. Higher MUN concentration for OSC occurred despite lower N intake (474 vs. 512 g of N/d) and similar milk N outputs compared with 95MP (164 vs. 179 g/d). On days when cows on OSC were fed high versus low MP diets, yields of milk (34.8 vs. 36.3 kg/d) and milk protein (1.0 vs. 1.1 kg/d) and MUN concentration (9.3 vs. 12.5 mg/dL) followed the oscillation pattern but lagged the change in diet CP by 1 d, whereas dry matter intake, yields of milk fat, plasma energy metabolites, AA, and 3-methyl-His were similar between days. Nutrient digestibility was similar for major nutrients across treatments except for CP, which was greater for 109MP (65.2%) and OSC (65.3%) compared with 95MP (61.7%). Compared with 95MP, OSC did not increase milk N relative to N intake (averaged 0.35 g of milk N/g of N intake) or N balance; however, urinary N output was increased for OSC versus 95MP (0.32 vs. 0.24 g of urine N/g of N intake). Body composition estimated using urea dilution was similar across treatments, and all cows accreted lipid and energy during the trial. Empty body CP did not change over the 50-d treatment period. Overall, greater CP digestion, urinary N excretion, and MUN concentrations with lesser N intake and similar milk N outputs for OSC compared with 95MP suggests that the lower energy intake by OSC cows may have limited potential responses to altered N metabolism., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Nitrogen concentrations in feeds, feces, milk, and urine samples were measured using 2 analytical methods following different drying procedures. Ten samples of corn silage, alfalfa silage, and concentrates collected from 2017 to 2018 at Krauss Dairy Research Center, The Ohio State University (Wooster), were used. A 4-d total collection digestion trial provided fecal samples from 10 cows (1 sample/cow), and another 10 cows were used to collect milk samples (1 sample/cow) and spot urine samples (1 sample/cow). Spot urine samples were acidified immediately to pH <3.0 when collected. Feed samples were oven dried (55°C) or lyophilized and analyzed using the Kjeldahl (KJ; copper sulfate as a catalyst) method and a combustion method (elemental analyzer; EA). Feces, urine, and milk samples were analyzed for N using the following methods: (1) fresh samples by KJ (referred to as wet KJ), (2) lyophilization (urine and milk for 8 h; feces for 120 h) followed by EA (LYO-EA), and (3) oven drying (milk and urine for 1 h; feces for 72 h at 55°C) followed by EA (OD-EA). Additionally, changes in N content of acidified urine at -20° over 180 d of storage were examined. Nitrogen concentrations in corn silage, alfalfa silage, and concentrates were greater for EA by 6.1, 4.8, and 8.3%, respectively, compared with KJ. Analysis of dried samples via EA compared with wet KJ resulted in lower fecal N content (27.8 vs. 29.3 g/kg of DM). Nitrogen concentration in fecal samples via KJ after lyophilization was lower by 5% compared with wet KJ but did not differ from LYO-EA, suggesting that N losses occurred during drying. Nitrogen determination with EA after drying of samples resulted in greater milk N (5.70 vs. 5.50 g/kg) and urinary N (9.16 vs. 9.06 g/kg) content compared with wet KJ. However, drying method (i.e., lyophilization vs. oven drying) did not affect N content of milk, urine, or feces. The use of EA resulted in lower percentage deviation of N content from duplicate sample assays for most samples (no difference was found for concentrate and fecal N), suggesting that EA was more precise than KJ. In conclusion, drying of feces caused N losses regardless of drying methods. For urine and milk samples, if drying is necessary (i.e., EA), oven drying at 55°C can be used rather than lyophilization. The N content was greater in feeds, milk, and urine when determined with EA versus KJ. In addition, N content in acidified and undiluted urine at -20° changed and should be analyzed within 90 d of storage. The results in the current study, however, did not account for laboratory-to-laboratory variation., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The California Net Energy System (CNES) used a combination of measured and tabular metabolizable energy (ME) values and changes in body composition gain to determine net energy requirements for maintenance and gain and their corresponding dietary concentrations. The accuracy of the CNES depends on the accuracy of the feed ME values. Feed or diet ME values can be measured directly but are expensive and require specialized facilities; therefore, most ME values are estimated from digestible energy (DE) values, which are often estimated from the concentration of total digestible nutrients (TDN). Both DE and TDN values are often from tables and not based on actual nutrient analysis. The use of tabular values eliminates important within-feed variation in composition and digestibility. Furthermore, the use of TDN to estimate DE does not account for important variation in the gross energy value of feeds. A better approach would be to estimate DE concentration directly from nutrient composition or in vitro (or in situ) digestibility measurements. This approach incorporates within-feed variation into the energy system and eliminates the issues of using TDN. A widely used summative equation based on the commonly measured feed fractions (ash, crude protein, neutral detergent fiber, and fat) has been shown to accurately estimate DE concentrations of many diets for cattle; however, deficiencies in that equation have been identified and include an overestimation of DE provided by fat and an exaggerated negative effect of intake on digestibility. Replacing the nonfiber carbohydrate term (which included everything that was not measured) in the equation with measured starch concentration and residual organic matter (i.e., nonfiber carbohydrate minus starch) should improve accuracy by accounting for more variation in starch digestibility. More accurate estimates of DE will improve the accuracy of ME values, which will ultimately lead to more accurate NE values., (© The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Spray-dried plasma protein (SDP) compared with blood meal (BM) may contain various functional and active components that may benefit animal health. The objective of this experiment was to investigate the effects of feeding SDP or BM on production and blood profile in dairy cows during the transition and early-lactation periods. Seventy-two Holstein cows at 14 d before calving were used in a randomized block design. During the prepartum period, cows were fed a typical late-gestation diet containing BM (100 g/cow per day; 100BM, n = 24) or SDP (100 g/cow per day; 100SDP; n = 48). After calving, cows that were fed BM prepartum were fed a typical lactation diet formulated to provide 100 g/d of BM (100BM). Half the cows that were fed 100SDP prepartum were fed a lactation diet formulated to provide 100 g/d of SDP (100SDP; n = 24), and half were fed a diet formulated to provide 400 g/d of SDP (400SDP; n = 24) on a dry matter basis where SDP replaced BM (100SDP) or BM and soybean products (400SDP). All diets were balanced for crude protein concentration and metabolizable protein supply assuming BM and SDP were equal in rumen-degradable protein and rumen-undegradable protein. All data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) as a randomized block design where contrasts were made for 100BM versus 100SDP for prepartum variables and 100BM versus 100SDP and 100SDP versus 400SDP for postpartum variables. Prepartum supplementation of SDP had no effect on plasma fatty acids and β-hydroxybutyrate (2 d before calving). Plasma fatty acids (255 ± 29 µEq/mL) and β-hydroxybutyrate (675 ± 70 µmol/L) at 8 and 14 d of lactation were not affected by SDP in the diet. Feeding SDP at 100 g/d compared with 100BM increased or tended to increase milk fat, protein, and lactose contents for 16 wk after parturition. Providing SDP at 400 g/d in the diet increased milk yield (42 vs. 39 kg/d), energy-corrected milk (44 vs. 41 kg/d), energy-corrected milk per kilogram of dry matter intake, and yields of milk fat (1.60 vs. 1.48 kg/d), protein (1.21 vs. 1.16 kg/d), and lactose compared with 100SDP. Body weight losses tended to be lower for 100SDP compared with 100BM without a difference between 100SDP and 400SDP. Plasma histidine concentration (d 14 of lactation) was lower for SDP compared with 100BM. In addition, plasma 1-methyl-l-histidine tended to be lower as inclusion rate of SDP increased. In conclusion, SDP at 400 g/d increased milk and milk component yields without an increase in feed intake. Studies evaluating effects of functional and active compounds in SDP on gut microbiome, gut health, and immune functions may be needed to determine mode of action., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Nutrient balance studies require measuring urine volume, and urinary excretion can be used to assess Mg bioavailability. A less laborious method than total collection of urine could make balance studies more feasible and expand the utility of using urinary Mg as an index of bioavailability, but the method needs to be accurate and sensitive. Sampling interval can affect accuracy because excretion must be at steady state. Two experiments were conducted to (1) determine whether urinary creatinine could be used to accurately estimate urinary output of nutrients markedly excreted via urine (N, K, Na, S, and Mg; experiment 1) and (2) determine the appropriate sampling schedule to evaluate Mg excretion after abrupt diet changes (experiment 2). Experiment 1 was originally designed to evaluate the interaction of monensin [0 vs. 14 mg of monensin/kg of dry matter (DM)] and Mg source (MgO vs. MgSO 4 ; total diet Mg: 0.36% of DM) under antagonism from increased dietary K (2.11% of DM) on urinary Mg excretion. Experiment 2 evaluated the interaction of Mg concentration (basal vs. supplemental MgO; total diet Mg: 0.20 vs. 0.42% of DM) and K (basal vs. supplemental K 2 CO 3 ; total diet K: 1.60 vs. 2.57% of DM) on urinary Mg excretion over time. Using 4-d composite samples from total collection of urine (n = 34 cow-periods), the average daily excretion of creatinine was similar to previous estimates (29.0 ± 1.16 mg of creatinine/kg of body weight) but was variable among cows (root mean squared error = 2,980 mg/d; 14% of mean). Treatment-average estimated excretion of urine and urinary N, K, Na, S, and Mg were similar to actual values; however, differences between actual and estimated values could be substantial for individual cows. Using the mean creatinine excretion per kilogram of body weight for all cows to estimate urine eliminates the lack of fit variance resulting in artificially low within-treatment variation for estimated urine volume. The standard error of the mean for estimated urine volume was 23% less (1.93 vs. 2.51) than that for actual urine production. This inflated the type I error rate, and, consequently, statistical inferences on N and K excretion differed when urine output was estimated rather than measured. The standard error of the mean for excretion of Mg calculated with actual or estimated urine production were almost identical (0.92 vs. 0.97); however, similar standard error of the mean was likely caused by differences in the covariance of urinary Mg concentration with estimated or actual urine output. Based on spot sampling (experiment 2), urinary Mg reached steady state by 2 d following an increase in dietary K regardless of Mg level, whereas excretion of urinary Mg following an increase in dietary Mg continued to increase through 7 d. Estimating nutrient excretion with urinary creatinine and body weight on average is accurate, but variance is likely underestimated. Knowing the time course of urinary Mg excretion will improve the value of using urinary Mg concentration to assess diet adequacy or Mg bioavailability., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The interaction of monensin and 2 supplemental Mg sources (MgO and MgSO 4 ) on total-tract digestion of minerals and organic nutrients and milk production was evaluated in lactating dairy cattle. Eighteen multiparous Holstein cows (139 ± 35 DIM) were used in a split-plot experiment with 0 or 14 mg/kg diet DM of monensin as the whole-plot treatments and Mg source as split-plot treatments. During the entire experiment (42 d), cows remained on the same monensin treatment but received a different Mg source in each period (21 d) of the Latin square. Diets were formulated to contain 0.35% Mg with about 40% of that provided by MgO or MgSO 4 . Diets were formulated to have similar concentrations of major nutrients and K concentrations were elevated (2.1% of DM) with K 2 CO 3 to create antagonism to Mg absorption. Apparent digestibility was measured by total collection of urine and feces. Supplemental MgSO 4 decreased DMI (26.9 vs. 25.7 kg/d) and tended to decrease milk yield (40.2 vs. 39.3 kg/d), but increased the digestibility of OM (68.3 vs. 69.2%) and starch (91.9 vs. 94.4%) compared with MgO. Feeding MgSO 4 with monensin decreased NDF digestibility compared with other treatments (46.7 vs. 50.2%). That diet also had decreased apparent absorption of Mg compared with diets without monensin (15.6 vs. 19.2%), whereas MgO with monensin had greater apparent absorption of Mg (23.0%) than other treatments. Cows consuming MgSO 4 had increased apparent Ca absorption (32.2 vs. 28.1%) and balance. A diet with MgSO 4 without monensin increased the concentration of long-chain fatty acids in milk, suggesting increased mobilization of body fat or decreased de novo fatty acid synthesis in the mammary gland. Overall, when dietary Mg was similar, MgO was the superior Mg source for lactating dairy cattle, but inclusion of monensin in diets should be considered when evaluating Mg sources., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Many nutrition models rely on summative equations to estimate feed and diet energy concentrations. These models partition feed into nutrient fractions and multiply the fractions by their estimated true digestibility, and the digestible mass provided by each fraction is then summed and converted to an energy value. Nonfiber carbohydrate (NFC) is used in many models. Although it behaves as a nutritionally uniform fraction, it is a heterogeneous mixture of components. To reduce the heterogeneity, we partitioned NFC into starch and residual organic matter (ROM), which is calculated as 100 - CP - LCFA - ash - starch - NDF, where crude protein (CP), long-chain fatty acids (LCFA), ash, starch, and neutral detergent fiber (NDF) are a percentage of DM. However, the true digestibility of ROM is unknown, and because NDF is contaminated with both ash and CP, those components are subtracted twice. The effect of ash and CP contamination of NDF on in vivo digestibility of NDF and ROM was evaluated using data from 2 total-collection digestibility experiments using lactating dairy cows. Digestibility of NDF was greater when it was corrected for ash and CP than without correction. Conversely, ROM apparent digestibility decreased when NDF was corrected for contamination. Although correcting for contamination statistically increased NDF digestibility, the effect was small; the average increase was 3.4%. The decrease in ROM digestibility was 7.4%. True digestibility of ROM is needed to incorporate ROM into summative equations. Data from multiple digestibility experiments (38 diets) using dairy cows were collated, and ROM concentrations were regressed on concentration of digestible ROM (ROM was calculated without adjusting for ash and CP contamination). The estimated true digestibility coefficient of ROM was 0.96 (SE = 0.021), and metabolic fecal ROM was 3.43 g/100 g of dry matter intake (SE = 0.30). Using a smaller data set (7 diets), estimated true digestibility of ROM when calculated using NDF corrected for ash and CP contamination was 0.87 (SE = 0.025), and metabolic fecal ROM was 3.76 g/100 g (SE = 0.60). Regardless of NDF method, ROM exhibited nutritional uniformity. The ROM fraction also had lower errors associated with the estimated true digestibility and its metabolic fecal fraction than did NFC. Therefore, ROM may result in more accurate estimates of available energy if integrated into models., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)