# Feed intake, milk production, and nutrient use efficiency with early lactating Norwegian Red dairy cows fed concentrates with varying levels of local ingredients

K.S. Eikanger, M Eknæs, J.K. Sommerseth, I.J. Karlengen, I. Schei, M.

Silberberg, A. Kidane

► To cite this version:

K.S. Eikanger, M Eknæs, J.K. Sommerseth, I.J. Karlengen, I. Schei, et al. Feed intake, milk production, and nutrient use efficiency with early lactating Norwegian Red dairy cows fed concentrates with varying levels of local ingredients. Livestock Science, 2025, 300, pp.105790. 10.1016/j.livsci.2025.105790. hal-05242533

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# Feed intake, milk production, and nutrient use efficiency with early lactating Norwegian Red dairy cows fed concentrates with varying levels of local ingredients

K.S. Eikanger<sup>a,∗</sup> ID, M. Eknæs<sup>a</sup>, J.K. Sommerseth<sup>b ID</sup>, I.J. Karlengen<sup>c</sup>, I. Schei<sup>b</sup>, M. Silberberg<sup>d</sup>, A. Kidane<sup>a ID</sup>

## HIGHLIGHTS

* Alkalised barley is a valid non-protein nitrogen alternative to soya protein.

* Alkalised barley improves feed use efficiency compared with urea-treated barley.

* Ingredient pre-pelleting structure affects starch digestibility of concentrates.

## ARTICLE INFO

**Keywords:**

Dairy cow

Alternative protein

Nutrient use efficiency

Serum metabolites

## ABSTRACT

We evaluated the effects of replacing imported soya protein and carbohydrate ingredients in concentrate feeds with alkalised barley in dairy cow diets with grass silage as a basal diet. Eight multiparous early lactation Norwegian Red dairy (NRF) cows with an initial daily milk yield (MY±SD) of 30.5 ± 5.94 kg were used in a 4 × 4 replicated Latin square design experiment of four 35-day experimental periods. Four roughly isonitrogenous and isoenergetic diets were included: a positive control with a higher share of imported ingredients, including soya as a protein source (Soya-F), two concentrates including alkalised barley (Alka-diets), differing in pre-pelleting particle size (i.e., Alka fine = Alka-F, and Alka coarse = Alka-C), and a negative control with the same basal ingredients as the Alka-diets but with untreated barley and added feed-grade urea replacing the alkalised barley (Urea-F). Grass silage was offered ad libitum and concentrate requirement for individual cows was calculated based on Soya-F. The Soya-F was then quantitatively replaced by the other three concentrate feeds. Feed intake, nutrient digestibility, milk production, nutrient use efficiency, and serum metabolites were measured. Replacing Soya-F with Alka-diets did not affect feed intake, digestibility, and milk production. However, Alka-diets supported a greater milk yield than Urea-F, which in turn improved feed utilisation efficiency for Alka-diets without altered N efficiency. A coarser pre-pelleting structure of Alka-C resulted in a lower starch digestibility compared to Alka-F. In conclusion, alkaline-treated barley has the potential to replace soya in concentrates for NRF dairy cows without compromising production efficiency.

## 1. Introduction

In recent years, there has been an enhanced interest in revising and adapting the use of local resources to strengthen national self-sufficiency. This is also evident in Norway, a country where only 3.5

% of the land is arable (Statistics Norway, 2023). As of 2023, a mere 10 % of all protein ingredients included in concentrates sold for farm animals in Norway were locally produced (Landbruksdirektoratet, 2024). With ruminants consuming approximately 49 % of the total concentrate sold (Landbruksdirektoratet, 2024), this situation results in a

<footnote><sup>a</sup> Faculty of Biosciences, Norwegian University of Life Sciences, P.O. Box 5003, NO-1432 Ås Norway</footnote>

<footnote><sup>b</sup> TINE SA, BTB - NMBU P.O. Box 5003, NO-1432 ÅS, Norway</footnote>

<footnote><sup>c</sup> Norgesfor AS, Akershusstranda 27, 0150 Oslo, Norway</footnote>

<footnote><sup>d</sup> Université Clermont Auvergne, INRAE, VetAgro Sup, UMR 1213 Herbivores, F-63122 Saint-Genès-Champanelle, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: katrinee@nmbu.no (K.S. Eikanger).</footnote>
dependency on imported products for protein ingredients.

Cereal grains are a favoured energy supplement for dairy cows. However, when fed in large quantities, their high starch content poses a challenge to the rumen environment by increasing short-chain fatty acids and lactate concentrations, decreasing rumen pH and thereby depressing fibre fermentation (Nikkhah, 2012). Grain processing is positively correlated with grain digestibility (Mathison, 1996), and modulating rumen pH through the degree of grain processing can therefore allow for greater inclusion of high-starch grains (Yang et al., 2001). Nonetheless, the lower protein content of cereal grains typically necessitates their combination with protein-rich ingredients to meet the nutritional requirements of high-yielding lactating dairy cows.

Supplying starch-rich diets with various alkalisating agents has proven beneficial by reducing the proportion of time with rumen pH below 5.8, aiming to reduce the risk of sub-acute rumen acidosis (SARA) (Bach et al., 2018; Razzaghi et al., 2021). Additionally, modulating the rumen microbiome's ability to synthesise microbial protein from non-protein nitrogen (NPN; i.e., urea) as an alternative nitrogen source in diets based on low-protein feed ingredients has proven to be a valuable strategy (Reid, 1953). While the use of feed-grade urea in dairy diets has been debated, and large variations in experiences tainted the motivation for its use (Kertz, 2010), Reid (1953) pointed out that greater bacterial protein synthesis can be achieved when feed-grade urea is fed together with starch as a microbial energy source in diets with low true protein content. Nonetheless, the rapid hydrolysis of urea to ammonia poses a risk of ammonia toxicity when the rate of hydrolysis exceeds the utilisation rate by microbes and the absorption over the rumen epithelium (Helmer and Bartley, 1971).

Significant advancements in our understanding of ruminant nitrogen metabolism over the past century (Schwab and Broderick, 2017) have led to the exploration of combining the potential of alkalisation technologies with NPN as a valuable nitrogen source for rumen microbes. Alkalisation in this case refers to an increase in pH and buffer capacity of the treated ingredients, reducing rumen acid loading and allowing for higher inclusion of acidogenic feed ingredients (i.e., high-starch ingredients such as barley (Rustomo et al., 2006)). This is achieved with ammoniation where a component mix of urea and selected enzymes (i.e., urease) are combined with high-starch ingredients. With moisture present, alkaline ammonium salts (i.e., ammonium bicarbonate) will form from urea. Depending on the degree of processing, ammonium salts are retained in the treated material, decreasing N solubility (Nikulina et al., 2018). This has resulted in emerging NPN alternatives to plant-based protein in ruminant diets.

The objective of this study was, thus, to assess the effect of replacing imported protein and carbohydrate ingredients by including alkalised barley as a NPN-source in pelleted concentrate diets with high inclusion of locally sourced ingredients, compared to: (1) a negative control containing untreated barley and added feed-grade urea instead of alkalised barley and (2) a positive control diet based on a typical commercial formulation including higher levels of imported ingredients with soya as the primary protein source. The study assessed impacts on feed intake, nutrient digestibility, and milk production in Norwegian Red (NRF) dairy cows. We hypothesised that delivering NPN through alkalised barley would improve dry matter intake (DMI) and fibre digestion, resulting in a higher milk production, and decreased nitrogen excretion in urine and faeces of dairy cows compared with added feed-grade urea. Secondly, it was hypothesised that a coarser pre-pelleting structure would improve energy and nitrogen utilisation of the alkalised barley compared with a finer pre-pelleting structure. Finally, we hypothesised that concentrate diets including locally sourced alkalised barley could replace a typical commercial soya-based concentrate without compromising feed intake and performance of NRF dairy cows.

## 2. Materials and methods

This experiment was performed at the Metabolism unit of the

Department of Animal and Aquacultural Sciences at the Norwegian University of Life Sciences during the spring of 2022. All animal procedures were performed following laws and regulations controlling the use of experimental animals and were approved by the national animal research authority under the Norwegian Food Safety Authority (Mattilsynet; FOTS ID: 28729).

### 2.1. Experimental design

Eight multiparous, rumen cannulated (Bar Diamond Inc., Parma, Idaho, USA; ø 10 cm), NRF cows with an average (± SD) milk yield (MY) of 30.5 ± 5.94 kg, at 67 ± 25 days in milk (DIM) and with a starting body weight of 629 ± 53 kg were used in the experiment. The experiment was conducted as a replicated 4 × 4 Latin square design, with four periods of 35 days, including 9 days of adaptation to experimental diets and a 26-day measurement period. Individuals were blocked in two squares based on MY, DIM, and parity, and then randomly allocated to a treatment sequence. The cows were housed in tie-stalls with rubber mats and sawdust bedding with access to individual feed and water troughs at all times.

### 2.2. Diets and feed management

Four pelleted concentrates were formulated with varying contents of locally produced ingredients (Table 1). Two concentrates with a high inclusion level of locally produced barley, containing alkalised barley (Alka grain 150 Barley, Strand Unikorn, Norway), with two different pre-pelleting particle sizes (i.e., Alka fine = Alka-F, and Alka coarse = Alka-C) were tested against a negative control with similar content of locally produced but untreated barley and feed-grade urea (Urea-F) replacing the portion of alkalised barley, and a positive control concentrate feed with a higher share of imported ingredients, including soya (Soya-F). All concentrates were planned to be roughly isonitrogenous and isoenergetic.

Alkalised barley was produced by mixing Home n' Dry pellets (Strand Unikorn, Norway/Dugdale Nutrition, UK), consisting of roughly equal parts of feed grade urea and whole fat soya, with whole barley grain in a 3:17 ratio and water to obtain ~ 18 % moisture. The mixed ingredients were thereafter covered by plastic for more than two weeks prior to being included in the pelleting process together with the remaining ingredients. The fine-structured diets were ground using a

<table><caption>**Table 1**

Ingredient composition of the concentrate feeds (g/kg).</caption><thead><tr><th></th><th>Treatment<sup>1</sup><br>Soya-F</th><th>Alka-C</th><th>Alka-F</th><th>Urea-F</th></tr></thead><tbody><tr><td>Rolled barley</td><td>-</td><td>427</td><td>427</td><td>717</td></tr><tr><td>Alka grain 150 Barley<sup>2</sup></td><td>-</td><td>200</td><td>200</td><td>-</td></tr><tr><td>Feed-grade urea</td><td>-</td><td>-</td><td>-</td><td>13</td></tr><tr><td>Ground barley</td><td>343</td><td>100</td><td>100</td><td>50</td></tr><tr><td>Ground oats</td><td>120</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Ground wheat</td><td>-</td><td>50</td><td>50</td><td>-</td></tr><tr><td>Wheat bran</td><td>133</td><td>70</td><td>70</td><td>63</td></tr><tr><td>Corn gluten meal</td><td>35</td><td>29</td><td>29</td><td>28</td></tr><tr><td>Ground corn</td><td>100</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Beet pulp</td><td>80</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Rapeseed expeller</td><td>-</td><td>30</td><td>30</td><td>33</td></tr><tr><td>Soya bean product</td><td>102</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Molasses</td><td>50</td><td>50</td><td>50</td><td>50</td></tr><tr><td>Other</td><td>38</td><td>44</td><td>44</td><td>47</td></tr></tbody></table>

<footnote n="1">¹ Concentrates: Soya-F = concentrate with imported soya and carbohydrate ingredients included as a positive control, Alka-C = concentrate with high levels of locally sourced ingredients including alkalised barley, with a coarse pre-pelleting structure, Alka-F = same as Alka-C with fine pre-pelleting structure equivalent to Soya-F, Urea-F = negative control based on same ingredients as Alka-C and Alka-F with untreated barley and feed-grade urea replacing the alkalised barley.</footnote>

<footnote n="2">² Alkalised barley.</footnote>
hammer mill equipped with a variable frequency drive set to a frequency of 36 Hz. For the coarse-structured diet, 42.7 % of the included barley was dry-rolled using roller gaps of 2.0, 1.5 and 1.2 mm to achieve a larger mean particle size. The remaining ingredients were processed as for the fine-structured diets. Particle size distribution was assessed by dry sieving of the pre-pelleting mash as described in Muqier et al. (2023). Briefly, for each feed, 100 g dry matter (DM) of material was sieved in triplicates through a series of 8 sieve sizes into 9 fractions (i.e., 3.16 mm, 2.50 mm, 2.00 mm, 1.40 mm, 1.00 mm, 0.80 mm, 0.50 mm, 0.25 mm and base-collector). Based on these fractions, the geometric mean particle size was calculated for each feed according to ASAE (1997). The geometric mean particle size was 1.14 mm for Alka-C and 0.49, 0.45, and 0.52 mm for Alka-F, Soya-F and Urea-F, respectively. Detailed particle size distributions for Alka-C and Alka-F are presented in supplementary materials (Figure S1. and Figure S2.).

Concentrate levels for the experiment were optimised before the first experimental period with Soya-F as the concentrate type, combined with a good quality grass silage available ad libitum using TINE OptiFør; the Nordic Feed Evaluation System (NorFor, 2011). Optimisations were based on individual requirements for maintenance and production. The chemical composition of the concentrate and grass silage is presented in Table 2. Each of the experimental concentrates replaced Soya-F on a weight-by-weight basis. For each period, the initially estimated experimental concentrate amount was reduced by 10 %, accounting for the advancing stage of lactation. The allocated concentrate and a common basal diet (i.e., grass silage) were fed to individual cows in three split portions daily, as 40 %, 30 % and 30 % of the total daily ration at 07:00, 14:00, and 19:00 h (local time), respectively, for the grass silage. The concentrate feeds were offered one hour later (i.e., 08:00, 15:00 and 20:00 h local time) in concentrate troughs adjacent to the individual main feed bunk. Grass silage was fed ad libitum, and the allowance was calculated based on the previous week's intake targeting a minimum 5 % refusal rate.

The conserved grass silage cut in 2021 was prepared twice weekly by

<caption>**Table 2**</caption>

Chemical composition and estimated energy and protein values for the common grass silage and individual concentrates.

<table><thead><tr><th></th><th>Grass silage</th><th colspan="4">Treatment</th></tr><tr><th></th><th></th><th>Soya-F</th><th>Alka-C</th><th>Alka-F</th><th>Urea-F</th></tr></thead><tbody><tr><td colspan="6"><strong>Chemical composition, g/kg DM unless otherwise given</strong></td></tr><tr><td>Dry matter,%</td><td>30.5</td><td>86.1</td><td>85.8</td><td>85.7</td><td>85.9</td></tr><tr><td>Ash</td><td>81.4</td><td>70.6</td><td>78.8</td><td>70.2</td><td>89.7</td></tr><tr><td>Crude protein</td><td>158</td><td>180</td><td>173</td><td>184</td><td>178</td></tr><tr><td>Fat</td><td>33.9</td><td>26.5</td><td>19.7</td><td>22.7</td><td>23.2</td></tr><tr><td>NDFom</td><td>535</td><td>194</td><td>167</td><td>178</td><td>178</td></tr><tr><td>ADFom</td><td>311</td><td>77.1</td><td>58.1</td><td>59.2</td><td>63.3</td></tr><tr><td>WSC</td><td>43.3</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Starch</td><td>-</td><td>358</td><td>373</td><td>394</td><td>424</td></tr><tr><td>restCHO</td><td>147</td><td>169</td><td>188</td><td>151</td><td>106</td></tr><tr><td colspan="6"><strong>Estimated energy and protein values</strong></td></tr><tr><td>OMD, g/kg OM</td><td>72.7</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>NEL<sub>20</sub>, MJ/kg DM</td><td>6.2</td><td>6.23</td><td>6.19</td><td>6.19</td><td>6.23</td></tr><tr><td>AAT<sub>20</sub>, g/kg DM</td><td>80.9</td><td>108</td><td>98</td><td>98</td><td>99</td></tr><tr><td>PBV<sub>20</sub>, g/kg DM</td><td>36.7</td><td>6</td><td>17</td><td>17</td><td>17</td></tr></tbody></table>

Feeds: Grass silage = common diet of conserved grass silage, Soya-F = concentrate with imported soya and carbohydrate ingredients included as a positive control, Alka-C = concentrate with high levels of locally sourced ingredients including alkalised barley, with a coarse pre-pelleting structure, Alka-F = same as Alka-C with fine pre-pelleting structure equivalent to Soya-F, Urea-F = negative control based on same ingredients as Alka-C and Alka-F with untreated barley and feed-grade urea replacing the alkalised barley; NDFom = ash-corrected neutral detergent fibre, ADFom = ash-corrected acid detergent fibre, WSC = water-soluble carbohydrates, restCHO = residual carbohydrates, OMD = organic matter digestibility estimated by near-infrared spectroscopy, AAT<sub>20</sub> = standard feed value for amino acids absorbed in the small intestine at 20 kg of DMI/d, NEL<sub>20</sub> = standard feed value for net energy lactation at 20 kg of DMI/d, PBV<sub>20</sub> = standard feed value for protein balance in the rumen at 20 kg of DMI/d.

chopping and mixing for 20 min in a Siloking Duo 1814 mixing wagon (Kverneland, Bryne, Norway). Each batch of mixing was preserved using 4 L per ton wet feed of a propionic acid-based preservative (GrasAAT FEED, Addcon Nordic AS, Porsgrunn, Norway). The silage was thereafter stored at 4°C until used up.

## 2.3. Sampling and recording

Daily feed intake was calculated after weighing daily orts before morning feeding. Daily individual drinking water intake was recorded automatically using water meters (Sensus residia, AxFlow AS, Oslo, Norway). Concentrate and grass silage samples were taken at the beginning of each period and the end of each week within a period, resulting in five samples for each treatment per period. Samples were stored at -20°C until prepared for analysis. Grass silage samples were pooled into one composite sample per treatment per period whereas concentrates were pooled into two samples per type representing early and late stages of the experiment.

Cows were milked twice daily (07:00 and 19:00 h local time), and individual milk yields were recorded for each milking using a DeLaval milk meter MM6 (DeLaval Inc., Tumba, Sweden). Milk samples were taken for both morning and evening milkings once a week during the measurement periods. Samples for analysis were preserved with Bro-nopol tablets (2-Bromo-2-nitropane-1,3 diol, Broad Spectrum Microtab II; Advanced Industries Inc, MA, USA) and stored at 4°C awaiting next-day transport to the TINE SA milk laboratory (Brumunddal, Norway).

Faeces and urine were collected for individual cows over 72 h at the end of each period (i.e., d 32-34) for estimation of digestibility and nitrogen balance. For the duration of the total collection, the cows were monitored at all times and faeces and urine were primarily collected directly in separate buckets to avoid cross-contamination. Faeces collected over 24 h were weighed and mixed before a 10 % subsample was taken and kept at -20°C until the end of each collection period. The composite sample was then thawed, and samples of 500 g were taken in duplicates both for DM determination at 103°C and freeze-drying for further analysis. Urine was preserved with 10 % H<sub>2</sub>SO<sub>4</sub> (ca 1 L per 20 L urine) aiming to keep the pH below 4. The pH was regularly controlled throughout the collection period. After each 24 h period, the total urine volume was weighed, and 10 % was taken out and pooled as a composite sample per cow and kept at -20°C. At the end of each collection period, the composite samples were thawed, and duplicates of 10 ml samples were taken for analysis.

Blood samples were taken from the jugular vein 90 min post concentrate feeding in the morning at day 35 in each period. Samples taken for serum biochemical analysis were allowed to clot for approximately one hour before centrifugation at 1000 g for 10 min. The serum was transferred to cryovials (2 ml; CryoELITE) and kept at -80°C until thawed for analysis.

The cows were weighed once while they were fed a common diet, i. e., before starting the first period, and thereafter on day 35 in each period. To minimize the effect of variation in rumen fill during a day, all cows were weighed at a similar time point each time (i.e., 11:00 h).

## 2.4. Chemical analyses

Concentrate samples were dried at 45°C whereas silage samples were dried at 60°C in preparation for milling. The samples were milled to pass a 1.00 mm screen, using a cutting mill (SM 200, Retsch GmbH, Haan, Germany). A smaller portion of the concentrate samples was also milled to pass a 0.5 mm screen for starch analysis using a Retsch ultra centrifugal mill (Retsch ZM100, Gemini BV, The Netherlands). All samples were dried at 103°C overnight to determine DM content (ISO, 1999), with ash being determined by incineration at 550°C (ISO, 2022). The nitrogen content was analysed using a Kjeltec 2400/2460 Auto Sampler System (Foss Analytica, Hilleroed, Denmark) performed according to AOAC method 2001.11 (Thiex et al., 2002), and crude protein (CP) was
calculated as 6.25 x Kjeldahl nitrogen. Crude fat content was analysed by accelerated solvent extraction using a Dionex™ ASE 200 Accelerated Solvent Extractor (Thermo Scientific, Waltham, USA). Neutral detergent fibre (NDF) content was determined according to (Mertens 2002), using sodium sulphite and alpha-amylase, with an Ankom²²⁰ fibre analyser (ANKOM Technology, Fairport, NY, USA). Acid detergent fibre (ADF) was determined using AOAC Method 973.18 (AOAC, 2000), without acetone washing of samples. Both NDF and ADF samples were corrected for residual ash content. Concentrate samples were prepared for total starch analysis using AACC method 76–13.01 (AACC, 2000), hydrolysing starch to glucose, before determining the concentration by colourimetric spectrometry using an RX DaytoNA⁺ spectrometer (Randox Laboratories Ltd, Crumlin, UK). Silage samples were analysed for water-soluble carbohydrates as described by Randby et al. (2010). Residual carbohydrates (g/kg DM) were calculated as 1000 minus the sum of ash, CP, NDF, starch/water-soluble carbohydrates and crude fat.

Milk samples were analysed for protein, fat, lactose, free fatty acids (FFA), urea and somatic cell count (SCC) with a Bentley FTS/FCM Combisystem mid-infrared milk analyser (Bentley Instruments Inc., MN, USA). Energy corrected MY (ECM, kg) was calculated individually based on MY (kg) and milk chemical composition (g/kg) according to Sjaunja et al. (1990) as MY × [(38.30 × fat content + 24.20 × protein content +16.54 × lactose content + 20.70)/3,140].

Freeze-dried faeces samples were analysed in the same manner as for concentrate feeds, and urine samples were analysed for Kjeldahl-N (Kjeltec 2400; Foss Tecator, Höanäs, Sweden) using AOAC Method 2001.11 (Thiex et al., 2002).

Blood serum samples were analysed for serum metabolites. Glucose, total protein, β-hydroxybutyric acid (BHB), serum urea nitrogen (SUN), creatinine and total bilirubin were analysed with a Siemens Atellica CH biochemistry analyser. In preparation, the RANBUT kit from Randox (Randox Laboratories Ltd, Crumlin, UK) was used for BHB. Atellica kits (Siemens Healthcare Diagnostics Inc., NY, USA) were used for glucose, total protein, SUN, creatinine and total bilirubin. Albumin and globulins were measured using serum electrophoresis (Sebia Capillarys 2, Sebia, Surrey, UK). The analysis was performed at the central laboratory at the Norwegian University of Life Sciences, Ås.

## 2.5. Statistical analysis

Feed intake, milk yield, ECM and milk composition parameters, including nutrient intake per kg metabolic BW (BW<sup>0.75</sup>); calculated as g/kg of the mean individual BW between weights measured at the end of a period and the corresponding previous recording), feed use efficiency (FUE; calculated as ECM per kg DMI), and nitrogen use efficiency (NUE; calculated as nitrogen excreted in milk as percentage of dietary nitrogen consumed) were averaged per cow per week within a period and subjected to statistical analysis using a mixed effects model in R (R Core Team 2023 edition 4.2.3). Individual cows were treated as a random effect, while treatment, period, and measurement week within a period (i.e., week of measurement period, excluding the 9-days of adaptation) were included as fixed effects. For the remaining parameters: total tract digestibility of nutrients, and serum metabolites, which had a limited number of measurements per cow, a restricted model excluding the effect of week within a period was used. An autoregressive (AR [1]) covariance structure was selected based on Bayesian information criterion (BIC) (Wolfinger, 1996).

The full model used was as follows:

$$Y_{ijkl} = \mu + \text{Treatment}_i + \text{Period}_j + \text{Week}_k(\text{Period}_j) + \text{Cow}_l + e_{ijkl}$$

where $Y_{ijkl}$ = response variable (e.g., DMI); $\mu$ = overall mean; $\text{Treatment}_i$ = fixed effect of concentrate type i (i.e., Soya-F, Alka-C, Alka-F, Urea-F); $\text{Period}_j$ = fixed effect of period j (j = 1 to 4); $\text{Week}_k(\text{Period}_j)$ = fixed effect of measurement week k (k = 1 to 4) within period j; $\text{Cow}_l$ = random effect of cow l (l = 1 to 8); and $e_{ijkl}$ = residual error. Data are presented as

estimated marginal means. A custom contrasts matrix was defined for comparing the mean of the two concentrates including alkalised barley (Alka-C and Alka-F) with Soya-F (i.e., Soya-F vs. Alka-diets) and Urea-F (i.e., Urea-F vs Alka-diets) and the effect of pre-particle size for Alka-diets (Alka-C vs. Alka-F) using the R 'emmeans' contrasts() function without adjustments. Differences were considered statistically significant when $P < 0.05$, and trends were apparent when $0.05 \le P < 0.10$.

# 3. Results

## 3.1. Nutrient intake and total tract digestibility

The average daily intake of feed and individual nutrients is presented in Table 3. The total DMI (i.e., silage plus concentrate) did not differ between cows fed Alka-diets and Soya-F but were significantly higher for cows fed Urea-F than for those fed Alka-diets ($P = 0.02$). Concentrate pre-pelleting particle size did not influence total DMI for Alka-diets. Intake of grass silage made up 61–62 % of the cows total feed intake and silage DMI or the proportion of silage DMI to total DMI were not statistically different for any of the contrasts. There was no statistical difference found in organic matter (OM) intake for the three contrasts. Further, the intake of NDF corrected for ash (NDFom) were significantly lower for Alka-diets compared to Soya-F ($P = 0.03$) or Urea-F ($P = 0.001$). Pre-pelleting structure did not affect NDFom intake. Crude protein intake did not differ between the Alka-diets and Soya-F. A significantly higher CP intake was, however, observed for Urea-F compared to the Alka-diets ($P = 0.02$), as well as for Alka-F compared with Alka-C ($P < 0.001$). The intake of starch was found significantly different for all contrasts ($P < 0.001$), with a higher intake for Alka-diets compared with Soya-F and a lower intake compared to Urea-F. Starch intake was lower for Alka-C compared to Alka-F.

Overall the calculated intake of DM, OM, NDFom, CP and starch per kg metabolic BW (g/kg BW<sup>0.75</sup>) followed a similar pattern as the gross daily intake of the respective nutrients. No difference was found between cows fed Alka-diets or Soya-F for intake of DM and OM per kg metabolic BW. Soya-F tended to have a higher intake of NDFom ($P = 0.07$) and CP ($P = 0.07$) than the Alka-diets, whilst the Alka-diets had a significantly higher starch intake ($P < 0.001$) than the Soya-F. Urea-F had a significantly higher intake of DM ($P = 0.02$), NDFom ($P = 0.003$), CP ($P = 0.02$) and starch ($P < 0.001$) per kg metabolic BW compared to the Alka-diets. However, OM intake per kg metabolic BW did not differ between the Alka-diets and Urea-F. Intake of DM and NDFom per kg metabolic BW were not affected by pre-pelleting particle size of the Alka-diets. A tendency ($P = 0.07$) towards a lower intake of OM, as well as a significantly lower intake of CP and starch ($P < 0.001$) were however observed with an Alka-C compared to Alka-F.

Daily intake of drinking water (L/day) and water consumed per unit DMI (L/kg DMI) did not differ between Alka-diets and Soya-F. However, drinking water intake was significantly lower for Alka-diets compared to Urea-F ($P < 0.001$). The intake of drinking water for cows fed Alka-C did not differ from cows fed Alka-F. A significantly higher ($P = 0.04$) water intake per kg DMI was found for Alka-C relative to Alka-F.

Total tract nutrient digestibility (%) for DM, OM, NDFom, and CP did not differ for the three contrasts (Table 4). Starch digestibility was however found to be significantly lower ($P < 0.001$) for Alka-diets in contrast to Soya-F or Urea-F, and for Alka-C compared to Alka-F ($P < 0.001$).

## 3.2. Milk production and composition

A summary of milk yield and milk composition is presented in Table 5. Results showed no difference in MY and ECM production between cows fed Alka-diets and Soya-F, whereas Alka-diets had a significantly higher MY ($P = 0.003$) and ECM ($P = 0.005$) production compared to Urea-F. Pre-pelleting particle size did not influence MY or ECM production between Alka-C and Alka-F. Alka-diets gave a
<caption>**Table 3**</caption>

Nutrient and water intake in early-lactation by NRF dairy cows given grass silage and concentrates with a higher share of imported ingredients, including soya (Soya-F), or concentrate with higher share of local ingredients, including alkalised barley with fine (Alka-F) or coarse (Alka-C) pre-pelleting structure, or with added feed-grade urea (Urea-F).

<table><thead><tr><th></th><th>Treatment</th><th></th><th></th><th></th><th></th><th>Contrasts (P-value)</th><th></th><th></th></tr><tr><th></th><th>Soya-F</th><th>Alka-C</th><th>Alka-F</th><th>Urea-F</th><th>SE</th><th>Soya-F vs. Alka-diets<fnref n="1" /></th><th>Urea-F vs. Alka-diets<fnref n="1" /></th><th>Alka-C vs. Alka-F</th></tr></thead><tbody><tr><td><strong>Intake, kg/d</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Total DM</td><td>23.6</td><td>23.5</td><td>23.6</td><td>23.9</td><td>0.73</td><td>0.86</td><td>0.02</td><td>0.68</td></tr><tr><td>Silage DM</td><td>14.4</td><td>14.4</td><td>14.5</td><td>14.7</td><td>0.59</td><td>0.81</td><td>0.10</td><td>0.63</td></tr><tr><td>Silage DM:Total DM, kg/kg</td><td>0.61</td><td>0.62</td><td>0.62</td><td>0.62</td><td>0.02</td><td>0.58</td><td>0.59</td><td>0.51</td></tr><tr><td>OM</td><td>21.8</td><td>21.6</td><td>21.8</td><td>21.8</td><td>0.68</td><td>0.79</td><td>0.50</td><td>0.14</td></tr><tr><td>NDFom</td><td>9.54</td><td>9.20</td><td>9.31</td><td>9.64</td><td>0.295</td><td>0.03</td><td>0.001</td><td>0.24</td></tr><tr><td>CP</td><td>3.94</td><td>3.86</td><td>3.94</td><td>3.95</td><td>0.129</td><td>0.11</td><td>0.02</td><td>&lt;0.001</td></tr><tr><td>Starch</td><td>3.86</td><td>3.99</td><td>4.25</td><td>4.45</td><td>0.26</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td><strong>Intake per unit BW<sup>0.75</sup>, g/kg</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DM</td><td>186</td><td>185</td><td>186</td><td>187</td><td>4.76</td><td>0.97</td><td>0.02</td><td>0.47</td></tr><tr><td>OM</td><td>171</td><td>170</td><td>172</td><td>172</td><td>4.41</td><td>0.61</td><td>0.49</td><td>0.07</td></tr><tr><td>NDFom</td><td>74.4</td><td>72.3</td><td>73.3</td><td>74.7</td><td>1.63</td><td>0.07</td><td>0.003</td><td>0.17</td></tr><tr><td>CP</td><td>31.0</td><td>30.3</td><td>31.0</td><td>31.0</td><td>0.85</td><td>0.07</td><td>0.02</td><td>&lt;0.001</td></tr><tr><td>Starch</td><td>30.4</td><td>31.3</td><td>33.6</td><td>35.2</td><td>2.03</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td><strong>Drinking water</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>L/day</td><td>90.1</td><td>91.3</td><td>89.7</td><td>95.8</td><td>3.62</td><td>0.70</td><td>&lt;0.001</td><td>0.10</td></tr><tr><td>L/kg DMI</td><td>3.83</td><td>3.88</td><td>3.80</td><td>4.01</td><td>0.11</td><td>0.67</td><td>&lt;0.001</td><td>0.04</td></tr></tbody></table>

<caption>**Table 4**</caption>

Nutrient digestibility by cows fed grass silage and concentrates with a higher share of imported ingredients, including soya (Soya-F), or concentrate with higher share of local ingredients, including alkalised barley with fine (Alka-F) or coarse (Alka-C) pre-pelleting structure, or with added feed-grade urea (Urea-F).

<table><thead><tr><th></th><th>Treatment</th><th></th><th></th><th></th><th></th><th>Contrasts (P-value)</th><th></th><th></th></tr><tr><th></th><th>Soya-F</th><th>Alka-C</th><th>Alka-F</th><th>Urea-F</th><th>SE</th><th>Soya-F vs. Alka-diets<fnref n="1" /></th><th>Urea-F vs. Alka-diets<fnref n="1" /></th><th>Alka-C vs. Alka-F</th></tr></thead><tbody><tr><td><strong>Digestibility, %</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DMD</td><td>72.5</td><td>72.3</td><td>72.5</td><td>72.6</td><td>0.39</td><td>0.92</td><td>0.68</td><td>0.65</td></tr><tr><td>OMD</td><td>73.1</td><td>73.0</td><td>73.1</td><td>73.1</td><td>0.40</td><td>0.99</td><td>0.94</td><td>0.71</td></tr><tr><td>NDFomD</td><td>62.8</td><td>62.1</td><td>61.3</td><td>62.8</td><td>1.25</td><td>0.26</td><td>0.24</td><td>0.46</td></tr><tr><td>CPD</td><td>71.0</td><td>71.1</td><td>71.2</td><td>71.8</td><td>0.69</td><td>0.77</td><td>0.15</td><td>0.71</td></tr><tr><td>StarchD</td><td>98.43</td><td>96.73</td><td>98.63</td><td>98.86</td><td>0.22</td><td>&lt;0.001</td><td>&lt;0.001</td><td>&lt;0.001</td></tr></tbody></table>

<caption>**Table 5**</caption>

Milk yield and milk composition from NRF dairy cows in early-lactation given grass silage and concentrates with a higher share of imported ingredients, including soya (Soya-F), or concentrate with higher share of local ingredients, including alkalised barley with fine (Alka-F) or coarse (Alka-C) pre-pelleting structure, or with added feed-grade urea (Urea-F).

<table><thead><tr><th></th><th>Treatment</th><th></th><th></th><th></th><th></th><th>Contrasts (P-value)</th><th></th><th></th></tr><tr><th></th><th>Soya-F</th><th>Alka-C</th><th>Alka-F</th><th>Urea-F</th><th>SE</th><th>Soya-F vs. Alka-diets<fnref n="1" /></th><th>Urea-F vs. Alka-diets<fnref n="1" /></th><th>Alka-C vs. Alka-F</th></tr></thead><tbody><tr><td><strong>Yield, kg/d</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MY</td><td>30.2</td><td>29.2</td><td>29.7</td><td>28.4</td><td>1.70</td><td>0.18</td><td>0.003</td><td>0.56</td></tr><tr><td>ECM<fnref n="2" /></td><td>32.2</td><td>31.3</td><td>32.3</td><td>30.5</td><td>1.77</td><td>0.68</td><td>0.005</td><td>0.23</td></tr><tr><td><strong>Milk composition %</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fat</td><td>4.43</td><td>4.58</td><td>4.65</td><td>4.49</td><td>0.099</td><td>0.02</td><td>0.09</td><td>0.45</td></tr><tr><td>Protein</td><td>3.67</td><td>3.62</td><td>3.69</td><td>3.69</td><td>0.060</td><td>0.32</td><td>0.007</td><td>&lt;0.001</td></tr><tr><td>Lactose</td><td>4.55</td><td>4.53</td><td>4.51</td><td>4.50</td><td>0.062</td><td>0.38</td><td>0.06</td><td>0.29</td></tr><tr><td><strong>Milk component yield, kg/d</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fat</td><td>1.33</td><td>1.33</td><td>1.36</td><td>1.27</td><td>0.075</td><td>0.55</td><td>0.03</td><td>0.49</td></tr><tr><td>Protein</td><td>1.11</td><td>1.06</td><td>1.09</td><td>1.05</td><td>0.063</td><td>0.03</td><td>0.16</td><td>0.05</td></tr><tr><td>Lactose</td><td>1.38</td><td>1.34</td><td>1.34</td><td>1.31</td><td>0.092</td><td>0.28</td><td>0.03</td><td>0.77</td></tr><tr><td><strong>Other variables</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>FFA<fnref n="3" />, mmol/L</td><td>0.42</td><td>0.44</td><td>0.43</td><td>0.42</td><td>0.035</td><td>0.37</td><td>0.43</td><td>0.45</td></tr><tr><td>MUN<fnref n="4" />, mg/dL</td><td>12.4</td><td>12.9</td><td>12.9</td><td>12.8</td><td>0.61</td><td>0.08</td><td>0.47</td><td>0.71</td></tr><tr><td>SCC<fnref n="5" /></td><td>265</td><td>150</td><td>263</td><td>203</td><td>105</td><td>0.78</td><td>0.30</td><td>0.30</td></tr><tr><td>FUE<fnref n="6" />, kg ECM/kg DMI</td><td>1.36</td><td>1.33</td><td>1.36</td><td>1.26</td><td>0.050</td><td>0.57</td><td>&lt;0.001</td><td>0.37</td></tr></tbody></table>

<footnote n="1">¹ Alka-diets = Alka-C+Alka-F. DM = dry matter, OM = organic matter, NDFom = ash-corrected neutral detergent fibre, CP = crude protein, DMI = dry matter intake.</footnote>

<footnote n="1">¹ Alka-diets = Alka-C+Alka-F-DMD = dry matter digestibility, OMD = organic matter digestibility, NDFomD = digestibility of ash-corrected neutral detergent fibre, CPD = crude protein digestibility, StarchD = starch digestibility.</footnote>

<footnote n="1">¹ Alka-diets = Alka-C+Alka-F.</footnote>

<footnote n="2">² ECM = energy-corrected milk yield as described by (Sjaunja et al., 1990).</footnote>

<footnote n="3">³ FFA = free fatty acids.</footnote>

<footnote n="4">⁴ MUN = milk urea nitrogen.</footnote>

<footnote n="5">⁵ SCC = somatic cell count (cells x 10³/ml), means are estimated with original data, whereas statistics were performed on log (log+1) transformed data.</footnote>

<footnote n="6">⁶ FUE = feed use efficiency.</footnote>
significantly higher milk fat content compared to Soya-F (P = 0.02), with a tendency towards a higher milk fat content compared to Urea-F (P = 0.09). Pre-pelleting particle size did not influence milk fat content between the two Alka-diets. No differences were found for milk protein content between Alka-diets and Soya-F. Milk from cows fed Alka-diets had a higher protein content than milk from Urea-F fed cows (P = 0.007). Furthermore, Alka-F produced a higher milk protein content than Alka-C (P < 0.001). For milk lactose, no significant differences were seen between Alka-diets and Soya-F, but Alka-diets tended to give a higher milk lactose content than Urea-F (P = 0.06). No difference was observed between the Alka-C and Alka-F for milk lactose content.

Milk fat yield did not differ between Alka-diets and Soya-F. Alka-diets gave a significantly higher fat yield compared to Urea-F (P = 0.03). No difference was observed for milk fat yield between the two Alka-diets. Milk protein yield was significantly lower for Alka-diets compared to Soya-F (P = 0.03). Alka-diets and Urea-F did not differ in milk protein yield whilst a strong tendency (P = 0.05) was observed between the two Alka-diets with a lower protein yield for Alka-C compared to Alka-F. Lactose yield did not differ between Soya-F and Alka-diets, whilst a significantly higher lactose yield was found for Alka-diets compared to Urea-F (P = 0.03). No difference was seen between the two Alka-diets for milk lactose yield. The concentrations of FFA and SCC did not differ for the contrast included. Milk urea nitrogen (MUN) tended (P = 0.08) to be greater for Alka-diets compared to Soya-F, with no differences observed between Alka-diets and Urea-F or between Alka-C and Alka-F.

Feed use efficiency, calculated as kg ECM per kg DMI was not different between the Alka-diets and Soya-F. The Alka-diets had higher FUE (P < 0.001) compared to Urea-F, and no significant difference was observed between the Alka-F and Alka-C.

### 3.3. Nitrogen utilisation efficiency and serum metabolites

Data on nitrogen partitioning, as assessed during the total collection period, is presented in Table 6. The intake of dietary N did not differ statistically for the contrasts made. Alka-diets had similar urine output as Soya-F, whereas a significantly lower urine output was observed for the Alka-diets compared to Urea-F (P = 0.04). Alka-C had a significantly higher urine output compared to Alka-F (P < 0.001). Estimated urinary, faecal, and milk N output (g/d) and the total cumulative N excreted through these routes showed no significant differences for the contrasts made. Nitrogen not recovered in the above routes did not differ between the Alka-diets compared with Soya-F or Urea-F, but was significantly lower for Alka-C compared to Alka-F (P = 0.04). When looking at the

portion of N recovered in the different fractions as a percentage of the dietary N intake, no significant differences were observed for the contrasts between Alka-diets and Soya-F or Urea-F for urine N, faecal N and milk N, total N excreted or N not recovered. Alka-C tended to have a higher output of N in urine relative to N intake (P = 0.06) compared to Alka-F, whereas no differences were seen for faecal or milk N. The cumulative N excreted relative to N intake were significantly higher (P = 0.04) for Alka-C compared to Alka-F and N not recovered were consequently significantly lower (P = 0.04) for Alka-C compared to Alka-F.

### 3.4. Serum metabolites

Data on serum metabolites is presented in Table 7. Serum urea nitrogen was significantly higher for Alka-diets compared to Soya-F (P = 0.03) but tended to be lower than that of the Urea-F (P = 0.09). Pre-pelleting particle size did not influence SUN between Alka-diets. No difference were found for serum total protein between Alka-diets and Soya-F or Urea-F. Alka-C fed cows did however have a lower total protein serum concentration compared to cows fed Alka-F (P = 0.002). A higher mean albumin concentration for Alka-diets compared to Soya-F (P = 0.02) and a strong tendency towards a higher concentration compared to Urea-F (P = 0.05) were evident together with a significantly lower albumin concentration for Alka-C compared to Alka-F (P = 0.006). Creatinine were found to be significantly higher for Alka-diets in contrast to Soya-F (P = 0.02), but no differences were found for the contrast between Alka-diets and Urea-F or between Alka-C and Alka-F. Glucose and BHB serum concentrations were not different for Alka-diets compared to Soya-F or Urea-F. Alka-C and Alka-F had similar serum creatinine concentrations, but Alka-C had significantly lower (P = 0.002) serum glucose concentration compared to Alka-F. Total bilirubin concentrations were similar for Alka-diets and Soya-F, while tending to be lower for Alka-diets compared to Urea-F (P = 0.07). No difference in total bilirubin was found between Alka-C and Alka-F.

## 4. Discussion

### 4.1. Feed intake

Including feed grade urea in dairy cow diets have been reported to negatively affect DMI to various degrees, depending on inclusion levels, form of administration, and feeding frequency (Kertz, 2010). The recent network meta-analysis by Simoni et al. (2023) did however not reveal any negative effects of feeding isonitrogenous total mixed rations with feed grade urea or slow-release urea partially replacing plant protein.

<caption>**Table 6**</caption>

Nitrogen utilisation of NRF dairy cows in early-lactation given grass silage and concentrates with a higher share of imported ingredients, including soya (Soya-F), or concentrate with higher share of local ingredients, including alkalised barley with fine (Alka-F) or coarse (Alka-C) pre-pelleting structure, or with added feed-grade urea (Urea-F).

<table><thead><tr><th rowspan="2"></th><th colspan="4">Treatment</th><th colspan="3">Contrasts (P-value)</th></tr><tr><th>Soya-F</th><th>Alka-C</th><th>Alka-F</th><th>Urea-F</th><th>SE</th><th>Soya-F vs. Alka-diets<sup>1</sup></th><th>Urea-F vs. Alka-diets<sup>1</sup></th></tr></thead><tbody><tr><td>N intake, g/d</td><td>628</td><td>615</td><td>630</td><td>633</td><td>21.3</td><td>0.48</td><td>0.22</td></tr><tr><td>Urine output, kg/d</td><td>31.4</td><td>33.2</td><td>30.0</td><td>33.2</td><td>1.03</td><td>0.75</td><td>0.04</td></tr><tr><td><u>N excretion, g/d</u></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Urine N</td><td>189</td><td>196</td><td>186</td><td>196</td><td>10.2</td><td>0.69</td><td>0.33</td></tr><tr><td>Faecal N</td><td>182</td><td>177</td><td>181</td><td>180</td><td>8.35</td><td>0.32</td><td>0.79</td></tr><tr><td>Milk N</td><td>170</td><td>166</td><td>165</td><td>163</td><td>7.77</td><td>0.19</td><td>0.55</td></tr><tr><td>Total N excreted</td><td>539</td><td>540</td><td>532</td><td>540</td><td>17.5</td><td>0.72</td><td>0.66</td></tr><tr><td>N not recovered</td><td>88.7</td><td>73.9</td><td>93.7</td><td>92.4</td><td>10.9</td><td>0.42</td><td>0.22</td></tr><tr><td><u>N excretion as % of N intake</u></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Urine N</td><td>30.1</td><td>32.4</td><td>30.2</td><td>31.3</td><td>1.71</td><td>0.27</td><td>0.98</td></tr><tr><td>Faecal N</td><td>29.0</td><td>28.8</td><td>28.6</td><td>28.4</td><td>0.73</td><td>0.60</td><td>0.48</td></tr><tr><td>Milk N<sup>2</sup></td><td>27.0</td><td>26.8</td><td>26.1</td><td>25.7</td><td>0.85</td><td>0.24</td><td>0.24</td></tr><tr><td>Total N excreted</td><td>86.0</td><td>88.3</td><td>85.1</td><td>85.6</td><td>1.60</td><td>0.37</td><td>0.24</td></tr><tr><td>N not recovered</td><td>14.0</td><td>11.7</td><td>14.9</td><td>14.4</td><td>1.60</td><td>0.37</td><td>0.24</td></tr></tbody></table>

<footnote n="1">¹ Alka-diets = Alka-C+Alka-F.</footnote>

<footnote n="2">² Milk N = apparent nitrogen use efficiency (NUE).</footnote>
<caption>**Table 7**</caption>

Serum metabolites of NRF dairy cows in early-lactation given grass silage and concentrates with a higher share of imported ingredients, including soya (Soya-F), or concentrate with higher share of local ingredients, including alkalised barley with fine (Alka-F) or coarse (Alka-C) pre-pelleting structure, or with added feed-grade urea (Urea-F).

<table><thead><tr><th rowspan="2"></th><th colspan="5">Treatment</th><th colspan="3">Contrasts (P-value)</th></tr><tr><th>Soya-F</th><th>Alka-C</th><th>Alka-F</th><th>Urea-F</th><th>SE</th><th>Soya-F vs. Alka-diets<fnref n="1" /></th><th>Urea-F vs. Alka-diets<fnref n="1" /></th><th>Alka-C vs. Alka-F</th></tr></thead><tbody><tr><td><strong>Serum metabolites</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>SUN<fnref n="2" />, mg/dL</td><td>14.2</td><td>15.0</td><td>15.0</td><td>15.5</td><td>0.39</td><td>0.03</td><td>0.09</td><td>0.89</td></tr><tr><td>Total protein, g/L</td><td>76.8</td><td>74.7</td><td>78.1</td><td>74.7</td><td>1.83</td><td>0.91</td><td>0.18</td><td>0.002</td></tr><tr><td>Albumin, g/L</td><td>34.3</td><td>34.9</td><td>36.6</td><td>34.6</td><td>0.91</td><td>0.02</td><td>0.05</td><td>0.006</td></tr><tr><td>Creatinine, µmol/L</td><td>69.3</td><td>72.5</td><td>73.1</td><td>71.4</td><td>2.49</td><td>0.02</td><td>0.29</td><td>0.72</td></tr><tr><td>Glucose, mmol/L</td><td>3.30</td><td>3.03</td><td>3.35</td><td>3.16</td><td>0.12</td><td>0.21</td><td>0.59</td><td>0.002</td></tr><tr><td>BHB<fnref n="3" />, mmol/L</td><td>1.00</td><td>1.14</td><td>1.05</td><td>0.97</td><td>0.079</td><td>0.28</td><td>0.14</td><td>0.32</td></tr><tr><td>Total bilirubin, µmol/L</td><td>4.79</td><td>4.99</td><td>5.24</td><td>5.49</td><td>0.22</td><td>0.14</td><td>0.07</td><td>0.23</td></tr></tbody></table>

Similarly, dry matter intake in the present study was not found to differ between treatments including soya or alkalised barley as a protein source. Previous studies including diets with ammoniated barley reoprted no influence on DMI when feeding lactating Holstein cows high moisture barley with varying levels of ammoniation in a total mixed ration (Robinson and Kennelly, 1988). Belanche et al. (2021) also observed a similar DMI by sheep fed alkalised barley or barley with added urea. The lower DMI by Alka-diets relative to Urea-F observed here could be attributed to an additive effect of a numerically higher silage intake and a higher concentrate DMI due to slight variation in the moisture content (Table 2) of the concentrates fed with quantitative (w/w) replacement for Soya-F.

In Norway, dairy cattle are typically fed concentrates and roughage separately, unlike the norm in many parts of the world where total mixed ration is used. The quality of the silage and ingredients in the Soya-F concentrate used in this trial are representative of Norwegian diets for high-yielding cows, confirming the potential of the Alka-diets in practical feeding. There are limited studies that include alkalised barley in dairy cow diets and the available studies typically focus on the effect of non-treated versus urea-treated barley (Rode et al., 1986; Robinson and Kennelly, 1988). As our study aimed to evaluate the full replacement of soya protein with NPN from alkalised barley, it is challenging to make direct comparisons with previously reported studies.

The differences observed in intake of different nutrient fractions per day and per kg metabolic BW are justified by the slight variation in the chemical composition of concentrate feeds (Table 2).

## 4.2. Digestibility

A previous study assessing in vitro DM degradation, utilizable CP, and metabolizable energy of pelleted concentrates equivalent to Soya-F and Alka-F found no differences in in vitro DM degradation between these diets (Muqier et al., 2023). Based on the present results, this also holds up for in vivo DM digestibility. On the other hand, an increase in digestibility of CP and NDF was observed when partially replacing soya meal with slow-release urea in a study by Grossi et al. (2021). Various slow-release urea formulations such as lipid- or polymer-coated urea have been intensively investigated in recent years as alternatives to conventional feed-grade urea. These formulations aim to slow down the release of urea, thereby reducing the risk of ammonia toxicity caused by rapid urea hydrolysis and prolong post-intake N availability (Ma and Faciola, 2024). Similarly, a slower ammonia release has been theorized when N is captured in the cellular matrix of grains (Srivastava and Mowat, 1980). In addition, the alkalisation process is also anticipated to disrupt the lignin structure (Chen et al., 2013), induce partial delignification, and break glycosidic bonds of polysaccharides, thereby reducing polymerisation and causing fibre swelling. These structural changes can enhance the availability of lignocellulosic biomasses to

rumen microbes (Chesson, 1988; Kim et al., 2016), potentially improving fibre digestibility. Consequently, higher fibre digestibility was hypothesised for diets including alkalised barley in contrast to added feed grade urea. Yet, this was not found in the current study and may only be evident in the portion of Alka grain 150 alone (Table 1), rather than the whole diet. Similarly, Belanche et al. (2021) observed no differences in in sacco OM degradability or in vivo total tract digestibility with sheep when comparing ammoniated barley against barley with added feed-grade urea. While in another study, (Rode et al., 1986) urea-treated high-moisture barley was found to have a greater DM disappearance than ammoniated high-moisture barley in sacco up until 18 hours of incubation. By 24 hours, disappearance rates were similar, and by 36 h, ammoniated barley showed greater disappearance. The authors speculated a slower enzymatic degradation of starch with the ammonia treatment, as observed by Srivastava and Mowat (1980) with increasing ammoniation of high-moisture corn or with ammoniated high-moisture corn compared to feed-grade urea-treated corn (Mowat et al., 1981).

A previous study by Gallo et al. (2016) found in vitro starch degradation to linearly decrease with increasing particle size, corresponding with the present results of Alka-C having a lower starch digestibility compared to Alka-F with a finer pre-pelleting particle size. This is also in agreement with Yang et al. (2001) who observed no difference in fibre digestibility between fine and coarse-structured diets, but a lower total tract digestibility of starch with increasing particle size in vivo.

## 4.3. Milk production and composition

In the current experiment, the Alka-diets demonstrated production responses similar to that of Soya-F. In contrast, Urea-F produced less milk than Alka-diets, suggesting that the form of NPN delivered affected N and energy metabolism.

Partially replacing plant protein with slow-release urea has shown different results regarding MY and composition. A meta-analysis by Simoni et al. (2023) analysing the effect of feed-grade urea and slow-release urea inclusion in dairy cattle diets revealed no effect on MY and composition. However, adding alkalising agents in a high-starch low-NDF diet increased MY, ECM yield and fat composition in a study by Razzaghi et al. (2021).

The observed increase in MY and ECM production with alkalised barley compared to barley with added feed-grade urea cannot be directly attributed to differences in nutrient intake or overall diet digestibility. However, variations in blood serum metabolites measured 90 min post-concentrate feeding suggested differences in nutrient metabolism between the dietary treatments. Serum concentrations of albu- min and total protein were within the normal range of the reference intervals for healthy dairy cows (Alberghina et al., 2011) for all treatments and indicated that the cows were not deprived of protein.

<footnote n="1">¹ Alka-diets = Alka-C+Alka-F.</footnote>

<footnote n="2">² SUN = serum urea nitrogen.</footnote>

<footnote n="3">³ BHB = β-hydroxybutyric acid.</footnote>
However, blood total protein and albumin reflect protein absorption in the intestines and have been shown to increase linearly with CP intake (Law et al., 2009). Interestingly, the observed differences between Alka-diets and Urea-F appear to contradict this relationship with a higher CP intake for Urea-F without an increase in blood serum total protein and a strong tendency towards a lower blood serum albumin. Serum urea nitrogen concentrations reflect both the availability of N for ruminal recycling and the presence of excess rumen-available N not captured by rumen microbes (Law et al., 2009). The tendency toward higher SUN for Urea-F, combined with lower serum albumin, suggested that Alka-diets may support a more efficient microbial protein synthesis. Ammoniated barley has previously been found to increase urinary excretion of purine derivatives (i.e., proxies for rumen microbial crude protein synthesis), compared to barley with added urea (Belanche et al., 2021), further strengthening this theory.

A greater intestinal protein absorption is also indicated for Alka-F compared to Alka-C. The difference in serum total protein and albumin of 4.4 and 4.6 % respectively is however not fully explained by a 1.56 % lower CP intake by Alka-C. Nikulina et al. (2018) demonstrated increased N solubility with reduced particle size of ammoniated barley and corn, indicating a higher retention of N in larger particles.

Nitrogen capturing or rumen microbial protein synthesis is more effective when readily available energy is available (Calsamiglia et al., 2010). All treatments included here supported the cows' energy requirements for milk production, as indicated by serum glucose and BHB concentrations, which were within the range for healthy early lactation cows as reported by Hussein et al. (2020). Elevated total bilirubin levels in the blood are associated with poor energy balance and risk of metabolic disorders (Russell and Roussel, 2007). Although the contrast between Urea-F and Alka-diets showed a tendency towards a higher serum bilirubin levels for Urea-F, all groups had total bilirubin serum concentrations in the lower range of the 6.41±5.03 interval reported by Andjelić et al. (2022) in healthy Holstein dairy cows in similar lactation stages, as well as by Hussein et al. (2020).

## 4.4. Nitrogen balance

Libera et al. (2021) reported a higher MUN concentration when partially replacing soya with ammoniated triticale and oats. However, in the current experiment, we only observed numerical differences well within the normal range of expected MUN (Jonker and Kohn, 2001). Contrary to the findings by Haig et al. (2002), where positive correlation was observed between urinary N output and soluble intake protein, we did not observe differences in urinary N output between Soya-F and Alka-diets. Galo et al. (2003) also found a greater urinary N output for Holstein dairy cows fed a diet containing a controlled-release urea product compared to the isonitrogenous control diet. The absence of differences in N lost through excretion in urine and faeces or NUE between Urea-F and Alka-diets do not align with the previously discussed assumption of a lower N utilisation for microbial protein synthesis. However, the higher water intake as well as the greater urine output by Urea-F compared to the Alka-diets could be, as suggested by Galo et al. (2003), stimulated by a need to excrete more nitrogen due to an unsynchronised release of rumen urea and carbohydrate degradation. The assumed shorter but greater post-feeding increase in rumen NH₃-N after intake of Urea-F, could lead to a periodic increase and subsequent considerable decrease in glomerular filtration (Eriksson and Valtonen, 1982). This could stimulate a greater recycling of urea to the rumen explaining the observed similar urinary N excretion. The cost of rumen microbial maintenance might also be higher for diets with more rapid available N, resulting in less microbial protein per kg digested OM (Galo et al., 2003). Furthermore, hepatic urea synthesis represents a metabolic burden (Huntington and Archibeque, 1999; Salami et al., 2021), which can reduce available energy for milk synthesis and thereby lead to a reduced FUE.

## 5. Conclusion

In the current study, pelleted concentrate feeds including alkalised barley present as a viable alternative to concentrates including imported soya protein and carbohydrate ingredients for dairy cows fed grass silage as a basal diet, demonstrating similar utilisation and performance. High-starch concentrates including alkalised barley improved feed use efficiency compared to high-starch concentrates with added feed-grade urea, as evidenced by increased ECM production. This improvement occurred despite no differences in digestibility of the total diet or N utilisation. A coarser pre-pelleting structure of the alkalised diet did not display any specific advantage over the finer pre-pelleting structure, with performance being affected by poorer starch availability. Further investigation into digestibility kinetics and the effect of alkalised grains on rumen parameters are essential to understanding the mechanisms herein and for optimising its use and potential.

### Notes

Data is available upon request to the corresponding author.

### CRediT authorship contribution statement

K.S. Eikanger: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. M. Eknæs: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Methodology, Investigation, Conceptualization. J.K. Sommerseth: Writing – review & editing, Resources, Methodology, Conceptualization. I.J. Karlengen: Writing – review & editing, Resources, Methodology, Conceptualization. I. Schei: Writing – review & editing, Resources, Methodology, Conceptualization. M. Silberberg: Writing – review & editing, Resources, Methodology, Conceptualization. A. Kidane: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

I.J. Karlengen works for the company providing the concentrate feeds for the experiment reported.

The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

We thank the staff members of the metabolism unit at the Norwegian University of Life Sciences for their help and facilitation with the experiment, animal care and help during sampling. This study was funded by the Norwegian Research Council through the project "Alka-line Grain Technology: increased Norwegian Ingredients in cattle diets and Effects on milk production, Animal Health and GHG Emissions" (Project #302341 – AlkaNor), with support from industrial partners (Dugdale Nutrition, Norgesfør, Strand Unikorn and TINE). We would also like to thank Paul Sayle and Alan Sayle with Dugdale Nutrition/ Sayle-agri International for their input, and comments, and for sharing their knowledge about Home'n Dry.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.livsci.2025.105790.
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