# Dietary inclusion of hay of greater digestibility and water-soluble carbohydrate content increases performance of dairy cows, irrespective of concentrate type and breed

M. Coppa, Dominique Pomiès, Bruno Martin, Matthieu Bouchon, J.P. Renaud, Massoud Aoun, Bertrand Deroche, René Baumont

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M. Coppa, Dominique Pomiès, Bruno Martin, Matthieu Bouchon, J.P. Renaud, et al.. Dietary inclusion of hay of greater digestibility and water-soluble carbohydrate content increases performance of dairy cows, irrespective of concentrate type and breed. Animal, 2025, 19 (4), pp.101464. 10.1016/j.animal.2025.101464. hal-05043992

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# Dietary inclusion of hay of greater digestibility and water-soluble carbohydrate content increases performance of dairy cows, irrespective of concentrate type and breed

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M. Coppa<sup>a</sup>, D. Pomiès<sup>b</sup>, B. Martin<sup>b</sup>, M. Bouchon<sup>c</sup>, J.P. Renaud<sup>d</sup>, M. Aoun<sup>e</sup>, B. Deroche<sup>b,e</sup>, R. Baumont<sup>b,*</sup>

## ARTICLE INFO

**Article history:**
Received 14 June 2024
Revised 8 February 2025
Accepted 10 February 2025
Available online 17 February 2025

**Keywords:**
Dietary nitrogen efficiency
Holstein
Milk composition
Milk yield
Montbéliarde

## ABSTRACT

Water-soluble carbohydrate (WSC)-rich pasture or alfalfa hay, with high organic matter digestibility (OMd), are known to increase the voluntary dry matter intake (DMI) of dairy cows and diet digestibility, often resulting in higher dairy performance. However, little is known about the effect of highly digestible hays from botanically diversified natural grasslands, rich in WSC and their interactions with concentrate type on dairy performance. The present trial tested the effect on dairy cow performance of dietary inclusion of hay of greater digestibility and WSC content (high OMd, WSC-rich or low OMd, WSC-poor) from botanically diversified natural grasslands and its possible interactions with concentrate type with low or high starch degradation rate in cow breeds with different production potential. Twenty-eight Holstein and 28 Montbéliarde cows after the lactation peak were randomly allocated to four equivalent groups of 14 cows each. Cows were fed for 6 weeks with 5 kg DM/day of concentrate, 3 kg DM/day of the same 2nd cut hay and two different types of 1st cut hay (ad libitum). Two groups received the 1st cut hay characterised by a high (A) WSC content and high OMd and the two other groups received the 1st cut hay (B) with low WSC content and low OMd. One group per hay type received a wheat-based concentrate, with a high starch degradation rate and the other group received a maize-based concentrate, with a low starch degradation rate. Cows fed A hay diet ingested more WSC (+551 g/day) and had a higher WSC/CP ratio (+0.24) than B hay diet–fed cows. The resulting higher OM total tract apparent digestibility (+2.1%) of ingested diet improved milk yield (+1.4 kg/day), milk fat yield (+58 g/day) and feed conversion efficiency (+0.05 g milk /kg DMI) of cows fed A hay compared to B hay diets. The A hay diet also induced a lower milk urea (–91 mg/kg) and higher milk protein content (+1.1 g/kg) and yield (+59 g/day). The A hay diet milk was richer in de-novo synthesised fatty acids (FA) and poorer in FA derived from ruminal biohydrogenation of dietary polyunsaturated FA. The effect of concentrate type on dairy performance was not significant for either the A hay or B hay diets. Regardless of cow breed and concentrate type, including botanically diversified natural grasslands highly digestible and WSC-rich hay in cow diet improved milk yield, milk fat, and protein synthesis, and feed conversion efficiency.

© 2025 The Author(s). Published by Elsevier B.V. on behalf of The animal Consortium. This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/)).

## Implications

Several quality labelled dairy products led to hay-based diets during winter. Little is known about the effect on dairy cow performance of diet including highly digestible hay rich in water-soluble carbohydrates and its interactions with concentrate starch type, in cow breeds differing in milk production potential. Irrespective of the cow breed, such diets improved digestible dry matter intake,

dairy performance and dietary nitrogen efficiency, reducing milk urea and increasing milk protein. In mid-lactating cows, concentrate starch type had a negligible effect in improving dairy performance. Our findings could be useful in improving the nutritional efficiency of hay-based feeding systems for dairy cows.

## Introduction

Carbohydrate fractions contribute approximately 70% of DM in diets for lactating dairy cows (Oba, 2011). Among carbohydrates, fibre can limit voluntary dry matter intake (DMI) by physical fill

<footnote><sup>a</sup>Department of Agricultural, Forest and Food Sciences, University of Turin, Largo P. Braccini 2, 10095 Grugliasco, TO, Italy</footnote>

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

<footnote><sup>c</sup>INRAE, Herbipôle, 63122 Saint-Genès-Champanelle, France</footnote>

<footnote><sup>d</sup>PHILICOT, 1 Chemin du Moulin de la Ville, 71150 Chagny, France</footnote>

<footnote><sup>e</sup>IDENA, 21 rue du Moulin, 44880 Sautron, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: rene.baumont@inrae.fr (R. Baumont).</footnote>
and ferments slowly in the rumen (Allen, 1996). High water-soluble carbohydrate (WSC) forages have a higher organic matter digestibility (OMd) and energy density compared to low WSC forages that favour the voluntary DMI, increasing digestibility (Miller et al., 2001; Moorby et al., 2006) and often resulting in higher milk yield and fat and protein contents (Miller et al., 2001; Brito et al., 2008). Most of the WSCs can ferment very quickly in the rumen (Demarquilly et al., 1995; Boudon et al., 2001; Oba, 2011), thus supplying adequate energy for microbial growth favouring the incorporation of dietary N into microbial protein, and then reducing N excretion in urine and faeces and improving dietary N utilisation (Ellis et al., 2011). The effect of feeding highly digestible forages rich in WSC on dairy performance has mainly been studied with fresh herbage in grazing systems (reviewed by Edwards et al., 2007) or with alfalfa or ryegrass hay (reviewed by Klevenhusen and Zebeli, 2021). Several European Protected Designation of Origin cheese specifications include restrictions in cow feeding, indicating hay from permanent grassland as the main or the solely authorised forage base for winter feeding (Coppa et al., 2019). Thus, the improvement of dairy performance with highly digestible WSC-rich hay from botanically diversified natural grasslands is a key issue for quality-labelled dairy production systems, but little is known on this topic.

Furthermore, Klevenhusen and Zebeli (2021) highlighted that a favourable effect of highly digestible WSC-rich forages on milk yield and milk fat content is frequent, in particular with starch-rich diets, suggesting an interaction between the rapidity of degradation of forage WSC and that of starch supplemented with concentrates. The effect of feeding rapidly (i.e. from barley or wheat) or slowly (i.e. from maize) degradable starch on dairy performance is known (i.e. Moate et al., 2017; Ferland et al., 2018; Moate et al., 2019), but, to the best of our knowledge, no research has yet studied the interaction between the amount of WSC in forages and the starch degradation rate in the rumen, especially with hay-based diets. We hypothesise that cows fed low-degradation-rate maize starch, compared to high-degradation-rate wheat starch (Offner et al., 2003), showed improved dairy performance, thanks to synchronisation of the energy supply from WSC intake from hay and the rate of degradation of starch from concentrates with nitrogen supply in the rumen.

Finally, the effect on dairy performance of feeding highly digestible WSC-rich forages can change depending on the productivity level of the cows, according to the capacity of the diet to cover their energy requirements (Klevenhusen and Zebeli, 2021). However, there is a lack of knowledge on breed-dependent responses to the amount of WSC fed with forages and on its interaction with the rate of degradation of starch from concentrates. We hypothesise that breeds with high yield and energy requirements (like the Holstein breed) would benefit more from a highly digestible WSC-rich forage diet than less productive breeds (like the Montbéliarde breed).

Thus, the aim of this research was to test the effect on dairy cow performance of botanically diversified natural grasslands hay (high digestibility-WSC rich or low digestibility-WSC poor) and its possible interactions with concentrate starch degradation rate (low or high), in cow breeds differing in their milk production potential (Holstein vs Montbéliarde breeds).

Preliminary results were presented in abstract form at the 3rd EAAP Mountain Livestock Farming Systems Meeting, 5-7 June 2024, Clermont Ferrand, France.

## Material and methods

### Animals, diets and experimental design

The present study was carried out at the experimental farm of Herbipôle (INRAE, Marcenat, France, https://doi.org/10.15454/1.

5572318050509348E12) from December 2018 to March 2019. Twenty-eight Holstein (Ho) and 28 Montbéliarde (Mo) cows just after the lactation peak (28% primiparous cows per breed) were fed a common diet composed of 1st cut hay ad libitum, 3.0 kg of DM of 2nd cut hay and 5 kg DM/day of concentrates for 3 weeks (pre-experimental period). They were then randomly allocated to four groups of 14 cows each, balanced by breed, parity, and milk yield on days 4-6 after calving (20.2 ± 5.55 kg/day for Ho; 19.5 ± 4.67 kg/day for Mo), as a proxy of the milk yield at lactation pick (INRA 2018), BW at calving (696 ± 58.8 kg for Ho and 676 ± 67.5 kg for Mo), lactation stage (70 ± 23.6 DIM for Ho and 70 ± 22.3 DIM for Mo), and DM intake of a 1st cut hay during the pre-experimental period (12.9 ± 1.76 kg/day DM for Ho and 11.8 ± 1.61 kg/day DM for Mo). The four groups were individually fed with 5 kg DM/cow × day of concentrates, 3 kg DM/cow × day of the same 2nd cut hay fed as long stem during the pre-experimental period, but differed for two crossed factors: the type of 1st cut hay and concentrate composition. Two of the four cow groups were fed with 1st cut hay (ad libitum) fed as long stem characterised in particular by a high digestibility and WSC content (A), whereas the two other groups were fed 1st cut hay (ad libitum) with a low digestibility and WSC content (B). The A hay was harvested in the evening, whereas the B hay was harvested in the morning. Both 1st cut hays derived from two permanent mountain grasslands (Hay A: 80 ± 11% grasses, 10 ± 6% legumes and 10 ± 6% forbs; Hay B: 82 ± 11% grasses, 11 ± 5% legumes, 15 ± 6% forbs) harvested at a heading phenological stage and dried on field. One group per hay type received a concentrate based on wheat meal (CW), characterised by a high starch degradation rate (Offner et al., 2003) and the other received a concentrate based on maize meal (CM), characterised by a low starch degradation rate (Offner et al., 2003). The four groups were thus the hay A with concentrate CW, hay A with concentrate CM, hay B with concentrate CW, and hay B with concentrate CM diets. During the pre-experimental period, the concentrate fed to cows was composed of half CW and half CM concentrates. The detailed composition of concentrates is given in Table 1. The four groups were kept indoors for the entire experiment duration in a free-stall barn and had free access to water. Cows were milked in a herringbone milking parlour at 0700 and 1600 h. The experiment started after 3 weeks of adaptation to the experimental diets and then lasted 9 weeks.

### Sampling, measurement and analysis

#### Intake and feedstuffs

Cows were allocated in two separate pens according to the hay type fed (28 cows/pen). Pens were identical in size (244 m²), spatial organisation, physical characteristics, temperature (they are adjacent in the same barn), and management. The map of the

<table><caption>**Table 1**
Detailed composition of concentrates fed to dairy cows.</caption><thead><tr><th>Ingredient (% on concentrate DM)</th><th>CM</th><th>CW</th></tr></thead><tbody><tr><td>Barley meal</td><td>10.0</td><td>12.3</td></tr><tr><td>Maize meal</td><td>40.0</td><td></td></tr><tr><td>Wheat meal</td><td></td><td>40.0</td></tr><tr><td>Extruded soybean meal</td><td>16.0</td><td>12.0</td></tr><tr><td>Extruded soybean expeller</td><td>10.5</td><td>10.5</td></tr><tr><td>Extruded rapeseed expeller</td><td>8.2</td><td>20.0</td></tr><tr><td>Wheat bran</td><td>10.0</td><td></td></tr><tr><td>Cane molasses</td><td>3.0</td><td>3.0</td></tr><tr><td>Calcium carbonate</td><td>1.5</td><td>1.4</td></tr><tr><td>Rapeseed oil</td><td>0.5</td><td>0.5</td></tr><tr><td>Minerals and vitamins</td><td>0.3</td><td>0.3</td></tr></tbody></table>

Abbreviations: CM = concentrate based on maize meal, characterised by a low starch degradation rate; CW = concentrate based on wheat meal, characterised by a high starch degradation rate.
pens is given in Supplementary Material S1. The same researcher and technician staff were in charge of both pens. Cows were equipped with collars that allowed their automatic individual identification. Concentrates were individually distributed through an automatic dispenser (Delaval FSC400) recognising the collars and giving the concentrate type corresponding to the dietary treatment. Concentrate dispenser settings were identical in both pens. Individual concentrate intake was recorded. Intake of 1st cut and 2nd cut hay was individually recorded daily with automatic weighing troughs (BioControl AS, Rakkestad, Norway), recognising the collar when cows accessed to a trough. Troughs setting was performed before each distribution following emptying of the bins and was identical in the two pens: after cleaning the scales and emptying the bins, empty bins are put on the scales and the devices are set individually by putting a 25 kg weight on the top of each bin to control scale load. If needed, calibration to reach the exact corresponding weight is performed. Automatic weighting trough systems were chosen to monitor intake because they offered a barrier that provides some physical separation between adjacent cows (Supplementary Material S1), useful to reduce competition at the feed bunk (Huzzey et al., 2006). To avoid feed competition, all the cows within a pen have access to all troughs indifferently and the ratio between the number of cows and the number of troughs was set at 2:1. This ratio was set assuring that the sum of individual hay ingestion time stays lower than the available access time. Thus, 14 troughs per pen were available: 10 for the 1st cut hay and 4 for the 2nd cut hay. To limit the effect of hay sorting, troughs were filled up several times per day, trying to keep them almost full all day long. With the same aim, troughs were set to avoid cows ingesting more than 25% of their expected individual daily DMI in a 6 h period; when they exceeded the 25% of DMI per interval, their access to troughs was blocked for the rest of the interval. The DMI was calculated using the DM of the offered feedstuffs and of the refusals, analysed five times (Monday to Friday) per week, by oven-drying samples at 60 °C for 48 h. The dried samples were pooled by week and ground (1-mm screen, ZM 200 Retsch Mill) for the analysis of NDF, ADF (Van Soest et al., 1991; ISO 16472:2006), CP (N value × 6.25; Dumas methods: ISO 16634-1:2008), ash (by combustion at 550 °C for 6 h: ISO 5984:2014), gross energy (under oxygen combustion: ISO 9831:2004), starch (by hydrolysis by amyloglucosidase: ISO 15904), ether extract (petroleum ether extraction: ISO 6492:1999), and WSC (water extraction; Ould-Ahmed et al., 2015) contents. The DM enzymatic digestibility was determined by pepsin-cellulase hydrolysis (Aufrère et al., 2007). Nitrogen enzymatic degradability for concentrates was estimated by hydrolysis in protease for 1 h (Aufrère et al., 1989). Organic matter digestibility was calculated from the enzymatic digestibility of feedstuffs, according to Baumont et al. (2018a). The DM and theoretical starch degradability were estimated from INRA 2018 tables (Baumont et al., 2018b). The intake of chemical constituents was calculated by multiplying the intake of each feedstuff by its content of the constituent. The net energy balance for lactation was calculated by differences between the energy intake and requirements according to Sauvant et al. (2018a and 2018b).

## Total tract apparent digestibility and blood

Individual faecal samples were collected on 2 consecutive days per week, dried (60 °C for 72 h), ground (1-mm screen, ZM 200 Retsch Mill) and then pooled weekly for CP analysis, following the procedure described for forages. The OMd was estimated from faecal nitrogen content according to Peyraud (1998) as:

$$OMd (\%) = 0.908 - 3.088/(CP(g/kg)/10).$$

Blood samples were taken weekly from the tail vein after the morning milking with ethylenediaminetetraacetic acid-containing tubes

(Terumo). Samples were centrifuged at 1 200 × g for 20 min at 4 °C, and the plasma obtained was immediately stored at -20 °C, as described by Cremilleux et al. (2022). After thawing, the plasma concentrations of non-esterified fatty acids (FA) (Kit NEFA-HR2, Fujifilm WAKO), glucose, urea, and β-hydroxybutyrate (Kit 981379, 981818, 984325, respectively, all Thermo Scientific) were analysed on a chemistry analyzer (Arena 20XT Chemistry System, Thermo Scientific).

## Milk and performances

Daily, the milking order of the two groups of cows corresponding to the two pens was established maintaining constant the milking interval per group. The milk yield was recorded automatically at each milking. Milk samples were collected at four consecutive milkings per week. A sub-sample was preserved with bronopol at 4 °C and analysed for concentrations of fat, protein, casein, lactose, lipolysis, urea and SCC with a spectrometric method (MilkoScan FT6000, Foss). The fat and protein-corrected milk was calculated according to CVB (2008) as:

Fat and protein corrected milk (kg/day)

$$ = \text{milk yield (kg/day)} \times [0.337 \\ + 0.0116 \times \text{milk fat (g/kg milk)} \\ + 0.06 \times \text{milk protein (g/kg milk)}]. $$

During the last week of the experiment, another sub-sample was stored at -20 °C, lyophilised and then pooled by day according to milk yields obtained during morning and evening milking for fatty acid analysis. Milk lipids were methylated by adding 2 mL of 0.5 M sodium methoxide and 1 mL of methanolic HCl (5% HCl v/v in methanol) at 50 °C for 5 min. Milk FA were analysed by gas chromatography as described by Ferlay et al. (2010).

The BW and body condition score were measured once during the pre-experimental period and then again at the end of the experiment. The body condition score was graded on a scale from 0 to 5 (Bazin et al., 1984) by two independent trained assessors, and cows were concomitantly weighed on a scale (DeLaval, France).

Feed conversion efficiency was calculated by the ratio of daily Fat and protein-corrected milk to DMI.

## Statistical analysis

A GLM of ANOVA was performed on feedstuff chemical composition and characteristics data using SAS (Version 5.1; SAS Institute, 2009). A paired samples Student's T test was performed using SAS (Version 5.1; SAS Institute, 2009), to compare the chemical composition between offered hays and their refusals, aiming to exclude sorting effect on intake nutritive value.

The milk yield data were calculated on a daily basis, and then, all the animal-related data (intake, faeces, blood, and performance) were averaged within the experimental and within the pre-experimental periods. The animal was set as the experimental unit. Data were tested for normality (Shapiro-Wilk test - SAS 5.1) and log-transformed if necessary to achieve normality (blood non-esterified fatty acids and β-hydroxybutyrate, and milk lipolysis and SCC). Outliers were detected using the Dixon r22 ratio (Mini-tab, Version 18, State College, PA). All the animal-related data were processed using a mixed linear model in SAS (Version 5.1; SAS Institute, 2009) including hay type (A or B), concentrate type (CW or CM), breed (Ho or Mo) and the interactions hay type × concentrate type, hay type × breed, concentrate type × breed, hay type × concentrate type × breed. The triple interaction was never significant; thus, it was removed from the model. Lactation stage and the values of each variable measured during the pre-experimental periods, centred on the breed, were used as
covariates. The animal was a random factor. All data were presented in the tables as least square means. To compare means between groups for significant interactions, Tukey's posthoc test was applied. Effects were considered significant at a probability of $P < 0.05$, and as a tendency at $0.05 \le P < 0.10$. The code of the statistical procedure used is given in Supplementary Material S2.

## Results

### Feedstuff and cow diet compositions

The chemical composition of the different feedstuffs used in the experiment is given in Table 2. No differences in chemical composition between the offered hays and their refusals was observed (Supplementary Table S1). The A hay was richer in WSC (+54 g/kg DM) and numerically poorer in NDF and poorer in ADF (–40 and –43 g/kg DM, respectively), but the contents of all the other constituents were equivalent between the two 1st cut hays. As a consequence, A hay had a higher OMd (+4.5%) compared to B

hay. The CM concentrate had a lower CP content than the CW concentrate (–15 g/kg DM), but they did not differ in the content of any of the other constituents.

### Intake and total tract apparent digestibility

The DM and nutrient intake and total tract apparent digestibility according to the hay and concentrate types and to the breed are given in Table 3 and Supplementary Table S2. The cows fed A hay diet ingested more 1st cut hay (+2.1 kg DM/day), but less 2nd cut hay (–1.4 kg DM/day), resulting in total forages and total DM intakes that tended to be higher in cows fed A hay diet. Consequently, A hay diet–fed cows ingested more WSC (+0.55 kg/day) and more EE (+0.021 kg/day) than B hay diet–fed cows. They also had a higher WSC/CP and WSC/NDF ratios of the ingested diet (+0.24, and + 0.08, respectively). The estimated OMd total tract apparent digestibility and the digestible OM intake were higher for A hay diet (+2.1% and + 0.71 kg/day) than for B hay diet–fed cows.

<table><caption>**Table 2**

Chemical composition of the different feedstuffs fed to dairy cows during the experiment.</caption><thead><tr><th rowspan="2">Item</th><th colspan="2">1st cut hay type<fnref n="1" /></th><th rowspan="2">SEM</th><th rowspan="2">H<br>P-value</th><th rowspan="2">2nd cut hay</th><th colspan="2">Concentrate type<fnref n="2" /></th><th rowspan="2">SEM</th><th rowspan="2">C<br>P-value</th></tr><tr><th>A</th><th>B</th><th>CM</th><th>CW</th></tr></thead><tbody><tr><td>NDF (g/kg DM)</td><td>588</td><td>628</td><td>21.2</td><td>0.080</td><td>545</td><td>143</td><td>150</td><td>13.5</td><td>0.548</td></tr><tr><td>ADF (g/kg DM)</td><td>303</td><td>346</td><td>13.0</td><td>0.016</td><td>342</td><td>68</td><td>69</td><td>5.3</td><td>0.807</td></tr><tr><td>CP (g/kg DM)</td><td>88</td><td>102</td><td>8.6</td><td>0.117</td><td>114</td><td>253</td><td>268</td><td>5.9</td><td>0.035</td></tr><tr><td>EE (g/kg DM)</td><td>13.3</td><td>13.3</td><td>1.49</td><td>1.000</td><td>13.6</td><td>35.0</td><td>32.3</td><td>4.20</td><td>0.482</td></tr><tr><td>Starch (g/kg DM)</td><td></td><td></td><td></td><td></td><td></td><td>317</td><td>296</td><td>12.9</td><td>0.118</td></tr><tr><td>WSC (g/kg DM)</td><td>167</td><td>113</td><td>9.6</td><td>0.002</td><td>155</td><td>114</td><td>116</td><td>2.0</td><td>0.204</td></tr><tr><td>Ash (g/kg DM)</td><td>73</td><td>84</td><td>11.1</td><td>0.298</td><td>69</td><td>65</td><td>67</td><td>3.8</td><td>0.552</td></tr><tr><td>OMd (%)</td><td>63.3</td><td>58.8</td><td>2.4</td><td>0.017</td><td>57.8</td><td>91.2</td><td>91.3</td><td>0.45</td><td>0.597</td></tr><tr><td>GE (cal/g OM)</td><td>4 602</td><td>4 629</td><td>31.1</td><td>0.424</td><td>4 614</td><td>4 600</td><td>4 653</td><td>107.7</td><td>0.623</td></tr><tr><td>ED<sub>6</sub> N (%)</td><td></td><td></td><td></td><td></td><td></td><td>62.7</td><td>60.6</td><td>3.40</td><td>0.483</td></tr><tr><td>ED<sub>6</sub> DM (%)</td><td></td><td></td><td></td><td></td><td></td><td>68.9</td><td>83.7</td><td></td><td></td></tr><tr><td>ED<sub>6</sub> starch (%)</td><td></td><td></td><td></td><td></td><td></td><td>61.8</td><td>72.1</td><td></td><td></td></tr></tbody></table>

Abbreviations: H = hay type; C = Concentrate type; ED<sub>6</sub> = effective degradability; EE = Ether extract; GE = gross energy; OMd = Organic matter digestibility; WSC = water-soluble carbohydrates;

<table><caption>**Table 3**

DM and nutrient intakes and total tract apparent digestibility of dairy cows according to the hay and concentrate types and to the breed.</caption><thead><tr><th rowspan="2">Item</th><th colspan="2">Hay type<fnref n="1" /></th><th colspan="2">Concentrate type<fnref n="2" /></th><th colspan="2">Breed<fnref n="3" /></th><th rowspan="2">SEM</th><th colspan="5">P-value</th></tr><tr><th>A</th><th>B</th><th>CM</th><th>CW</th><th>Mo</th><th>Ho</th><th>H</th><th>C</th><th>Br</th><th>H × C</th><th>H × Br</th><th>C × Br</th></tr></thead><tbody><tr><td>Intake</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DM 1st cut hay (kg/day)</td><td>15.0</td><td>12.9</td><td>13.8</td><td>14.1</td><td>13.3</td><td>14.6</td><td>0.29</td><td>&lt;0.001</td><td>0.379</td><td>&lt;0.001</td><td>0.104</td><td>0.969</td><td>0.377</td></tr><tr><td>DM 2nd cut hay (kg/day)</td><td>0.8</td><td>2.2</td><td>1.5</td><td>1.5</td><td>1.5</td><td>1.5</td><td>0.01</td><td>&lt;0.001</td><td>0.557</td><td>0.531</td><td>0.332</td><td>0.649</td><td>0.311</td></tr><tr><td>DM forages (kg/day)</td><td>15.6</td><td>15.1</td><td>15.2</td><td>15.5</td><td>14.7</td><td>16.0</td><td>0.25</td><td>0.050</td><td>0.184</td><td>&lt;0.001</td><td>0.169</td><td>0.666</td><td>0.976</td></tr><tr><td>DM concentrate (kg/day)</td><td>4.8</td><td>4.8</td><td>4.8</td><td>4.8</td><td>4.8</td><td>4.8</td><td>0.19</td><td>0.911</td><td>0.944</td><td>0.837</td><td>0.899</td><td>0.887</td><td>0.794</td></tr><tr><td>DM total (kg/day)</td><td>20.4</td><td>19.9</td><td>20.0</td><td>20.3</td><td>19.5</td><td>20.8</td><td>0.31</td><td>0.079</td><td>0.241</td><td>&lt;0.001</td><td>0.156</td><td>0.695</td><td>0.899</td></tr><tr><td>OM (kg/day)</td><td>18.8</td><td>18.3</td><td>18.5</td><td>18.7</td><td>18.0</td><td>19.2</td><td>0.30</td><td>0.087</td><td>0.474</td><td>&lt;0.001</td><td>0.170</td><td>0.563</td><td>0.559</td></tr><tr><td>CP (kg/day)</td><td>2.8</td><td>2.8</td><td>2.7</td><td>2.8</td><td>2.7</td><td>2.8</td><td>0.55</td><td>0.991</td><td>0.007</td><td>0.043</td><td>0.646</td><td>0.864</td><td>0.715</td></tr><tr><td>NDF (kg/day)</td><td>9.8</td><td>9.9</td><td>9.7</td><td>9.9</td><td>9.4</td><td>10.2</td><td>0.13</td><td>0.603</td><td>0.198</td><td>&lt;0.001</td><td>0.064</td><td>0.426</td><td>0.979</td></tr><tr><td>WSC (kg/day)</td><td>3.05</td><td>2.50</td><td>2.75</td><td>2.79</td><td>2.68</td><td>2.86</td><td>0.048</td><td>&lt;0.001</td><td>0.311</td><td>&lt;0.001</td><td>0.150</td><td>0.996</td><td>0.536</td></tr><tr><td>EE (kg/day)</td><td>0.378</td><td>0.357</td><td>0.367</td><td>0.369</td><td>0.364</td><td>0.372</td><td>0.0076</td><td>0.016</td><td>0.833</td><td>0.241</td><td>0.704</td><td>0.679</td><td>0.672</td></tr><tr><td>Starch (g/day)</td><td>1 481</td><td>1 475</td><td>1 504</td><td>1 451</td><td>1 483</td><td>1 472</td><td>57.6</td><td>0.923</td><td>0.361</td><td>0.855</td><td>0.895</td><td>0.913</td><td>0.771</td></tr><tr><td>WSC/CP</td><td>1.08</td><td>0.84</td><td>0.98</td><td>0.94</td><td>0.95</td><td>0.97</td><td>0.026</td><td>&lt;0.001</td><td>0.009</td><td>0.043</td><td>0.961</td><td>0.757</td><td>0.652</td></tr><tr><td>WSC/NDF</td><td>0.31</td><td>0.23</td><td>0.27</td><td>0.27</td><td>0.27</td><td>0.27</td><td>0.002</td><td>&lt;0.0001</td><td>0.568</td><td>0.810</td><td>0.589</td><td>0.598</td><td>0.215</td></tr><tr><td>GE (kcal/day)</td><td>22 450</td><td>22 003</td><td>22 056</td><td>22 397</td><td>21 519</td><td>22 935</td><td>358.3</td><td>0.216</td><td>0.344</td><td>0.000</td><td>0.177</td><td>0.553</td><td>0.565</td></tr><tr><td>DOM (kg/day)</td><td>14.1</td><td>13.4</td><td>13.5</td><td>14.0</td><td>13.3</td><td>14.2</td><td>0.28</td><td>0.013</td><td>0.878</td><td>0.005</td><td>0.319</td><td>0.565</td><td>0.293</td></tr><tr><td>OMd (%)</td><td>75.0</td><td>72.9</td><td>74.3</td><td>73.6</td><td>74.1</td><td>73.8</td><td>0.33</td><td>&lt;0.001</td><td>0.053</td><td>0.265</td><td>0.159</td><td>0.200</td><td>0.458</td></tr></tbody></table>

Abbreviations: Br = Breed; C = Concentrate type; DOM = digestible organic matter; GE = gross energy; EE = ether extract; H = hay type; OM = organic matter; OMd = organic matter digestibility; WSC = water-soluble carbohydrate.

<footnote n="1">¹ A = 1st cut hay characterised by a high water-soluble carbohydrate content; B = 1st cut hay with a low content of water-soluble carbohydrates.</footnote>

<footnote n="2">² CM = concentrate based on maize meal, characterised by a slow starch degradability; CW = concentrate based on wheat meal, characterised by a fast starch degradability.</footnote>

<footnote n="1">¹ A = 1st cut hay characterised by a high water-soluble carbohydrate content; B = 1st cut hay with a low water-soluble carbohydrate content.</footnote>

<footnote n="2">² CM = concentrate based on maize meal, characterised by slow starch degradability; CW = concentrate based on wheat meal, characterised by fast starch degradability.</footnote>

<footnote n="3">³ Ho = Holstein cow breed; Mo = Montbéliarde cow breed.</footnote>
Concentrate type only affected CP intake, with CM-supplemented cows ingesting less CP than CW-supplemented cows (–0.15 g/day), but with a higher WSC/CP ratio of the ingested diet (+0.04).

The Mo cows ingested less 1st cut hay (–1.3 kg DM/day) than Ho cows, but the intake of 2nd cut hay and of concentrate did not differ between them. Consequently, Mo cows also had a lower total forage and total DM intake compared to Ho cows. The OM, CP, NDF, ADF and gross energy intakes were lower for Mo than Ho cows (–1.17, –0.11, –0.78 and –0.19 kg/day, and –1 416 kcal/day, respectively), as well as the WSC/CP ratio of the ingested diet (–0.02).

No significant interaction was observed between hay and concentrate types or breed.

### Blood metabolites, BW and body condition scores

The blood metabolite contents, BW and body condition score of dairy cows according to hay and concentrate types and breed are given in Table 4 and Supplementary Table S3. Blood metabolites were affected by diets according both to hay and concentrate types they included. The CM-supplemented cows had a higher non-esterified fatty acid content than CW-supplemented cows when fed A hay diet (0.075 vs 0.059 mmol/L: *P* = 0.025), but this difference was not significant when they were fed B hay diet (0.068 vs 0.080 mmol/L, respectively). The opposite trend was observed for β-hydroxybutyrate content, which was lower for CM than CW-supplemented cows when fed B hay diet (0.50 vs 0.60 mmol/L; *P* = 0.001), but the difference between the concentrate types was not significant when cows were fed B hay diet (0.56 vs 0.50 mmol/L for CM and CW, respectively). Blood glucose was higher in CM than in CW-supplemented cows fed B hay diet (0.60 vs 0.54 g/L; *P* < 0.001), but the opposite result was found when cows were fed A hay diet (0.53 vs 0.59 g/L for CM and CW, respectively). Blood urea was lower for CM than CW-supplemented cows when fed B hay diet (0.22 vs 0.28 g/L; *P* < 0.001), but this difference was not significant when cows were fed B hay diet (0.16 vs 0.14 g/L, respectively). Interactions between breed and hay or concentrate type were not significant.

Cows fed A hay diet had a higher body condition score (+0.15) than those fed B hay diet, but they had a similar BW. Breed had a significant effect on BW and body condition score, with Mo cows being lighter (–23 kg), but having a higher body condition score (+0.36).

Concentrate type did not affect BW or body condition score, and interactions between hay or concentrate type and breed were not significant.

### Milk yield, gross composition and feed conversion efficiency

The milk yield and gross composition and feed conversion efficiency of dairy cows according to the hay and concentrate types and to the breed are given in Table 5 and Supplementary Table S4. Cows fed A hay diet had a higher milk yield (+1.4 kg/day), milk protein content (+1.1 g/kg), fat and protein-corrected milk yield (+0.36).

<table><caption>**Table 4**
Blood metabolite contents, BW and body condition score of dairy cows according to the hay and concentrate types and to the breed.</caption><thead><tr><th rowspan="2">Item</th><th colspan="2">Hay type (H)<sup>1</sup></th><th colspan="2">Concentrate type (C)<sup>2</sup></th><th colspan="2">Breed (Br)<sup>3</sup></th><th rowspan="2">SEM</th><th colspan="6">P-value</th></tr><tr><th>A</th><th>B</th><th>CM</th><th>CW</th><th>Mo</th><th>Ho</th><th>H</th><th>C</th><th>B</th><th>H × C</th><th>H × B</th><th>C × B</th></tr></thead><tbody><tr><td>BW (kg)</td><td>678</td><td>678</td><td>677</td><td>678</td><td>666</td><td>689</td><td>3.5</td><td>0.859</td><td>0.759</td><td>&lt;0.001</td><td>0.389</td><td>0.930</td><td>0.358</td></tr><tr><td>BCS</td><td>1.71</td><td>1.58</td><td>1.64</td><td>1.65</td><td>1.82</td><td>1.46</td><td>0.050</td><td>0.011</td><td>0.933</td><td>&lt;0.001</td><td>0.811</td><td>0.213</td><td>0.884</td></tr><tr><td>Blood metabolites</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NEFA (mmol/L)</td><td>0.057</td><td>0.060</td><td>0.057</td><td>0.060</td><td>0.074</td><td>0.043</td><td>0.0076</td><td>0.354</td><td>0.574</td><td>0.588</td><td>0.025</td><td>0.576</td><td>0.187</td></tr><tr><td>BHB (mmol/L)</td><td>0.53</td><td>0.55</td><td>0.53</td><td>0.55</td><td>0.53</td><td>0.56</td><td>0.016</td><td>0.410</td><td>0.273</td><td>0.162</td><td>0.001</td><td>0.113</td><td>0.709</td></tr><tr><td>Glucose (g/L)</td><td>0.57</td><td>0.56</td><td>0.57</td><td>0.57</td><td>0.57</td><td>0.57</td><td>0.0129</td><td>0.252</td><td>0.971</td><td>0.804</td><td>&lt;0.001</td><td>0.137</td><td>0.180</td></tr><tr><td>Urea (g/L)</td><td>0.15</td><td>0.25</td><td>0.19</td><td>0.21</td><td>0.21</td><td>0.19</td><td>0.009</td><td>&lt;0.001</td><td>0.019</td><td>0.114</td><td>0.000</td><td>0.737</td><td>0.064</td></tr></tbody></table>

Abbreviations: BCS = body condition score; Br = Breed; BHB = β-hydroxybutyrate; C = Concentrate type; H = hay type; NEFA = non-esterified fatty acids.

¹ A = 1st cut hay characterised by a high water-soluble carbohydrate content; B = 1st cut hay with a low water-soluble carbohydrate content.

² CM = concentrate based on maize meal, characterised by slow starch degradability; CW = concentrate based on wheat meal, characterised by fast starch degradability.

³ Ho = Holstein cow breed; Mo = Montbéliarde cow breed.

<table><caption>**Table 5**
Milk yield and gross composition and feed conversion efficiency of dairy cows according to the hay and concentrate types and to the breed.</caption><thead><tr><th rowspan="2">Item</th><th colspan="2">Hay type (H)<sup>1</sup></th><th colspan="2">Concentrate type (C)<sup>2</sup></th><th colspan="2">Breed (Br)<sup>3</sup></th><th rowspan="2">SEM</th><th colspan="6">P value</th></tr><tr><th>A</th><th>B</th><th>CM</th><th>CW</th><th>Mo</th><th>Ho</th><th>H</th><th>C</th><th>B</th><th>H × C</th><th>H × B</th><th>C × B</th></tr></thead><tbody><tr><td>Milk yield (kg/day)</td><td>21.2</td><td>19.9</td><td>20.5</td><td>20.6</td><td>19.4</td><td>21.7</td><td>0.40</td><td>0.001</td><td>0.778</td><td>&lt;0.001</td><td>0.401</td><td>0.522</td><td>0.928</td></tr><tr><td>Milk fat (g/kg)</td><td>36.6</td><td>35.9</td><td>36.7</td><td>35.9</td><td>37.0</td><td>35.6</td><td>0.53</td><td>0.198</td><td>0.171</td><td>0.012</td><td>0.917</td><td>0.002</td><td>0.072</td></tr><tr><td>Milk protein (g/kg)</td><td>32.5</td><td>31.5</td><td>32.0</td><td>32.0</td><td>33.0</td><td>30.9</td><td>0.26</td><td>0.000</td><td>0.815</td><td>&lt;0.001</td><td>0.871</td><td>0.728</td><td>0.409</td></tr><tr><td>FPR</td><td>1.13</td><td>1.15</td><td>1.15</td><td>1.13</td><td>1.12</td><td>1.15</td><td>0.019</td><td>0.348</td><td>0.311</td><td>0.119</td><td>0.993</td><td>0.007</td><td>0.125</td></tr><tr><td>Lactose (g/kg)</td><td>49.6</td><td>49.6</td><td>49.6</td><td>49.6</td><td>49.4</td><td>49.9</td><td>0.23</td><td>0.968</td><td>0.788</td><td>0.034</td><td>0.310</td><td>0.772</td><td>0.620</td></tr><tr><td>Urea (mg/kg)</td><td>178</td><td>268</td><td>214</td><td>232</td><td>229</td><td>217</td><td>7.5</td><td>&lt;0.001</td><td>0.015</td><td>0.128</td><td>0.133</td><td>0.325</td><td>0.092</td></tr><tr><td>SCC (log 1 000 × mL)</td><td>154</td><td>82</td><td>92</td><td>144</td><td>115</td><td>121</td><td>41.8</td><td>0.093</td><td>0.607</td><td>0.224</td><td>0.998</td><td>0.710</td><td>0.838</td></tr><tr><td>FPCM (kg/day)</td><td>20.2</td><td>18.7</td><td>19.3</td><td>19.6</td><td>18.7</td><td>20.2</td><td>0.44</td><td>0.002</td><td>0.558</td><td>0.001</td><td>0.429</td><td>0.255</td><td>0.504</td></tr><tr><td>Fat yield (g/day)</td><td>771</td><td>713</td><td>742</td><td>742</td><td>716</td><td>768</td><td>19.1</td><td>0.004</td><td>0.979</td><td>0.009</td><td>0.583</td><td>0.074</td><td>0.347</td></tr><tr><td>Protein yield (g/day)</td><td>683</td><td>624</td><td>645</td><td>661</td><td>638</td><td>668</td><td>15.0</td><td>&lt;0.001</td><td>0.286</td><td>0.053</td><td>0.457</td><td>0.867</td><td>0.728</td></tr><tr><td>FCE on MY (kg milk /kg DMI)</td><td>1.05</td><td>1.00</td><td>1.03</td><td>1.03</td><td>1.00</td><td>1.05</td><td>0.019</td><td>0.011</td><td>0.981</td><td>0.008</td><td>0.051</td><td>0.429</td><td>0.408</td></tr><tr><td>FCE on FPCM (kg milk /kg DMI)</td><td>1.01</td><td>0.96</td><td>0.98</td><td>0.98</td><td>0.97</td><td>0.99</td><td>0.018</td><td>0.017</td><td>0.955</td><td>0.276</td><td>0.279</td><td>0.349</td><td>0.632</td></tr><tr><td>Net energy balance for lactation (kcal/day)</td><td>3 968</td><td>1 652</td><td>2 695</td><td>2 925</td><td>2 114</td><td>3 506</td><td>631.6</td><td>0.001</td><td>0.718</td><td>0.033</td><td>0.956</td><td>0.818</td><td>0.657</td></tr></tbody></table>

Abbreviations: Br = Breed; C = Concentrate type; DMI = DM intake; FCE = feed conversion efficiency; FPCM = fat and protein-corrected milk; FPR = fat-to-protein ratio; H = hay type; MY = milk yield; SCC = somatic cell count.

¹ A = 1st cut hay characterised by a high water-soluble carbohydrate content; B = 1st cut hay with a low water-soluble carbohydrate content.

² CM = concentrate based on maize meal, characterised by slow starch degradability; CW = concentrate based on wheat meal, characterised by fast starch degradability.

³ Ho = Holstein cow breed; Mo = Montbéliarde cow breed.
(+1.4 kg/day), fat yield (+58 g/day) and protein yield (+59 g/day), but they had a lower milk urea content (-91 mg/kg) than cows fed B hay diet. The A hay diet-fed cows also had a higher feed conversion efficiency on milk yield (+0.05 kg milk /kg DMI) and feed conversion efficiency on fat and protein-corrected milk (+0.04 kg milk /kg DMI) and a higher net energy balance for lactation (+2 317 kcal/day) than B hay diet-fed cows.

The Mo cows produced less milk (-2.3 kg/day), but richer in protein (+2.1 kg/g), with consequently a lower fat and protein-corrected milk (-1.6 kg/day) and fat yield (-52 g/day) than Ho cows. They also had a lower milk lactose content (-0.5 g/kg) and a lower feed conversion efficiency on milk yield (-0.05 kg milk/kg DMI), but similar feed conversion efficiency on fat and protein-corrected milk. The Mo cows had a lower net energy balance for lactation (-1 392 kcal/day) than Ho cows. The Mo cows had a higher milk fat content than Ho cows when fed A hay diet (38.2 vs 35.1 g/kg; P = 0.002), but the difference between breeds was not significant when cows were fed B hay diet (35.8 vs 36.1 g/kg for Mo and Ho, respectively). The milk fat to protein ratio was similar between Mo and Ho cows when fed B hay diet (1.14 vs 1.12), but was lower for Mo cows when fed B hay diet (1.10 vs 1.19, respectively: P = 0.007). No significant interactions between concentrate type and breed were observed.

## Milk fatty acid composition

The fatty acid composition of milk from dairy cows according to the hay and concentrate types fed and to their breed is given in Table 6 and Supplementary Table S5. The cows fed A hay diet

produced milk richer in SFA (+1.44 g/100 g FA), and in particular in saturated FA between C8:0 and C10:0 (+2.31 g/100 g FA) and between C10:0 and C15:0 (+0.5 g/100 g FA), and in n-6 FA (+0.12 g/100 g FA). It was also poorer in C18:0 (-1.4 g/100 g FA), MUFA (-1.3 g/100 g FA), in particular in total C18:1t isomers (-0.68 g/100 g FA), and in CLAc9t11 (-0.21 g/100 g FA). The A hay diet milk also had a higher C18:1t10/C18:1t11 ratio (+0.05).

The Mo cows produced milk richer in C14:0 (+0.68 g/100 g FA) and in CLAc9t11 (+0.13 g/100 g FA), but poorer in C17:0 (-0.03 g/100 g FA) than Ho cows. They also had a lower C14:1c9/C14:0 ratio (-0.023).

No significant effects of concentrate type or interactions between hay or concentrate types or breed were observed.

## Discussion

The aim of our research was to test the effect on dairy cow performance of hay intake from botanically diverse natural grasslands and its possible interactions with the rate of starch degradation in the concentrate. When studying feeding with cows that are not housed in individual pens, competition between cows can affect feeding behaviour, exposing dominated cows to the risk of reduced access to feed (Huzzey et al., 2006; Crossley et al., 2017). For this reason, we decided to feed hay A and B in separate pens, as including two hays of different quality in each pen could have reinforced the importance of hierarchy and social competition in access to mangers. However, it must be recognised that separating hay by pen confuses the effect of the hay with a potential effect of the pen. For this reason, it is essential to ensure identical physical

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

Fatty acid composition of milk from dairy cows according to the hay and concentrate types fed and to their breed.

<table><thead><tr><th rowspan="2">Fatty acids (g/100 g FA)</th><th colspan="2">Hay type (H)<sup>1</sup></th><th colspan="2">Concentrate type (C)<sup>2</sup></th><th colspan="2">Breed (Br)<sup>3</sup></th><th colspan="2">SEM</th><th colspan="6">P-value</th></tr><tr><th>A</th><th>B</th><th>CM</th><th>CW</th><th>Mo</th><th>Ho</th><th>H</th><th>C</th><th>B</th><th>H x C</th><th>H x B</th><th>C x B</th></tr></thead><tbody><tr><td>C4:0</td><td>3.01</td><td>3.09</td><td>3.09</td><td>3.01</td><td>3.04</td><td>3.06</td><td>0.109</td><td>0.452</td><td>0.478</td><td>0.828</td><td>0.701</td><td>0.382</td><td>0.638</td></tr><tr><td>C6:0</td><td>1.97</td><td>1.90</td><td>1.95</td><td>1.92</td><td>1.92</td><td>1.95</td><td>0.049</td><td>0.185</td><td>0.569</td><td>0.512</td><td>0.992</td><td>0.746</td><td>0.750</td></tr><tr><td>C8:0</td><td>1.11</td><td>1.01</td><td>1.07</td><td>1.05</td><td>1.06</td><td>1.06</td><td>0.026</td><td>&lt;0.001</td><td>0.652</td><td>0.820</td><td>0.679</td><td>0.820</td><td>0.274</td></tr><tr><td>C10:0</td><td>2.83</td><td>2.43</td><td>2.63</td><td>2.64</td><td>2.68</td><td>2.59</td><td>0.080</td><td>&lt;0.001</td><td>0.960</td><td>0.295</td><td>0.887</td><td>0.396</td><td>0.130</td></tr><tr><td>C12:0</td><td>3.60</td><td>2.99</td><td>3.29</td><td>3.30</td><td>3.36</td><td>3.23</td><td>0.106</td><td>&lt;0.001</td><td>0.862</td><td>0.245</td><td>0.740</td><td>0.380</td><td>0.116</td></tr><tr><td>C14:0</td><td>12.5</td><td>11.4</td><td>11.9</td><td>12.0</td><td>12.3</td><td>11.6</td><td>0.19</td><td>&lt;0.001</td><td>0.966</td><td>0.001</td><td>0.987</td><td>0.460</td><td>0.328</td></tr><tr><td>C15:0</td><td>1.24</td><td>1.28</td><td>1.24</td><td>1.27</td><td>1.23</td><td>1.28</td><td>0.034</td><td>0.269</td><td>0.393</td><td>0.186</td><td>0.975</td><td>0.724</td><td>0.168</td></tr><tr><td>C16:0</td><td>30.3</td><td>29.5</td><td>29.5</td><td>30.4</td><td>29.6</td><td>30.3</td><td>0.67</td><td>0.274</td><td>0.191</td><td>0.292</td><td>0.460</td><td>0.483</td><td>0.386</td></tr><tr><td>C16:1c9</td><td>1.52</td><td>1.39</td><td>1.44</td><td>1.46</td><td>1.42</td><td>1.48</td><td>0.090</td><td>0.149</td><td>0.848</td><td>0.504</td><td>0.141</td><td>0.661</td><td>0.565</td></tr><tr><td>C17:0</td><td>0.75</td><td>0.74</td><td>0.74</td><td>0.75</td><td>0.73</td><td>0.76</td><td>0.014</td><td>0.558</td><td>0.291</td><td>0.028</td><td>0.809</td><td>0.479</td><td>0.744</td></tr><tr><td>C18:0</td><td>8.2</td><td>9.6</td><td>9.0</td><td>8.8</td><td>8.6</td><td>9.2</td><td>0.04</td><td>&lt;0.001</td><td>0.564</td><td>0.097</td><td>0.309</td><td>0.339</td><td>0.922</td></tr><tr><td>C18:1t10</td><td>0.26</td><td>0.30</td><td>0.28</td><td>0.28</td><td>0.29</td><td>0.27</td><td>0.017</td><td>0.012</td><td>0.809</td><td>0.237</td><td>0.788</td><td>0.216</td><td>0.246</td></tr><tr><td>C18:1t11</td><td>1.09</td><td>1.61</td><td>1.40</td><td>1.30</td><td>1.40</td><td>1.31</td><td>0.062</td><td>&lt;0.001</td><td>0.115</td><td>0.167</td><td>0.810</td><td>0.579</td><td>0.674</td></tr><tr><td>C18:1c9 (+C18:1c10+C18:1t15)</td><td>19.9</td><td>20.8</td><td>20.6</td><td>20.2</td><td>20.8</td><td>20.0</td><td>0.449</td><td>0.058</td><td>0.349</td><td>0.082</td><td>0.732</td><td>0.277</td><td>0.149</td></tr><tr><td>C18:2n-6</td><td>1.41</td><td>1.27</td><td>1.35</td><td>1.34</td><td>1.32</td><td>1.36</td><td>0.047</td><td>0.005</td><td>0.836</td><td>0.381</td><td>0.240</td><td>0.171</td><td>0.716</td></tr><tr><td>C18:3n-3</td><td>0.61</td><td>0.58</td><td>0.60</td><td>0.59</td><td>0.58</td><td>0.61</td><td>0.027</td><td>0.333</td><td>0.702</td><td>0.279</td><td>0.829</td><td>0.138</td><td>0.570</td></tr><tr><td>CLAc9t11(+CLAt7c9-CLAt8c10)</td><td>0.62</td><td>0.83</td><td>0.76</td><td>0.69</td><td>0.79</td><td>0.66</td><td>0.040</td><td>&lt;0.001</td><td>0.084</td><td>0.002</td><td>0.516</td><td>0.121</td><td>0.332</td></tr><tr><td>Σ C10:0 to C15:0</td><td>23.4</td><td>21.1</td><td>22.3</td><td>22.3</td><td>22.6</td><td>22.0</td><td>0.38</td><td>&lt;0.001</td><td>0.903</td><td>0.144</td><td>0.896</td><td>0.633</td><td>0.426</td></tr><tr><td>Σ C8:0 to C10:0</td><td>3.94</td><td>3.44</td><td>3.70</td><td>3.69</td><td>3.74</td><td>3.65</td><td>0.102</td><td>&lt;0.001</td><td>0.940</td><td>0.379</td><td>0.829</td><td>0.469</td><td>0.143</td></tr><tr><td>SFA</td><td>68.8</td><td>67.4</td><td>67.8</td><td>68.4</td><td>67.8</td><td>68.4</td><td>0.60</td><td>0.021</td><td>0.309</td><td>0.355</td><td>0.799</td><td>0.137</td><td>0.113</td></tr><tr><td>MUFA</td><td>26.8</td><td>28.1</td><td>27.8</td><td>27.2</td><td>27.8</td><td>27.2</td><td>0.53</td><td>0.019</td><td>0.263</td><td>0.246</td><td>0.927</td><td>0.155</td><td>0.138</td></tr><tr><td>PUFA</td><td>3.66</td><td>3.77</td><td>3.77</td><td>3.65</td><td>3.74</td><td>3.68</td><td>0.105</td><td>0.312</td><td>0.271</td><td>0.548</td><td>0.805</td><td>0.069</td><td>0.458</td></tr><tr><td>BCFA</td><td>2.75</td><td>2.83</td><td>2.80</td><td>2.79</td><td>2.80</td><td>2.78</td><td>0.080</td><td>0.289</td><td>0.908</td><td>0.819</td><td>0.550</td><td>0.226</td><td>0.461</td></tr><tr><td>OCFA</td><td>1.44</td><td>1.46</td><td>1.43</td><td>1.46</td><td>1.42</td><td>1.48</td><td>0.042</td><td>0.673</td><td>0.467</td><td>0.150</td><td>0.894</td><td>0.825</td><td>0.200</td></tr><tr><td>Σ C18:1c</td><td>20.9</td><td>21.9</td><td>21.6</td><td>21.2</td><td>21.8</td><td>21.0</td><td>0.47</td><td>0.053</td><td>0.345</td><td>0.099</td><td>0.744</td><td>0.261</td><td>0.144</td></tr><tr><td>Σ C18:1t</td><td>2.07</td><td>2.75</td><td>2.49</td><td>2.33</td><td>2.49</td><td>2.33</td><td>0.097</td><td>&lt;0.001</td><td>0.114</td><td>0.113</td><td>0.691</td><td>0.468</td><td>0.476</td></tr><tr><td>Σ n-3 FA</td><td>0.88</td><td>0.87</td><td>0.88</td><td>0.87</td><td>0.86</td><td>0.90</td><td>0.033</td><td>0.668</td><td>0.659</td><td>0.268</td><td>0.764</td><td>0.170</td><td>0.545</td></tr><tr><td>Σ n-6 FA</td><td>1.90</td><td>1.77</td><td>1.85</td><td>1.82</td><td>1.80</td><td>1.87</td><td>0.055</td><td>0.029</td><td>0.673</td><td>0.197</td><td>0.217</td><td>0.190</td><td>0.693</td></tr><tr><td>n-3/n-6</td><td>2.16</td><td>2.05</td><td>2.10</td><td>2.12</td><td>2.11</td><td>2.11</td><td>0.055</td><td>0.054</td><td>0.708</td><td>0.901</td><td>0.123</td><td>0.610</td><td>0.684</td></tr><tr><td>C18:1t10/C18:1t11</td><td>0.236</td><td>0.186</td><td>0.204</td><td>0.218</td><td>0.211</td><td>0.211</td><td>0.0094</td><td>&lt;0.001</td><td>0.167</td><td>0.982</td><td>0.795</td><td>0.234</td><td>0.211</td></tr><tr><td>C14:1c9/C14:0</td><td>0.097</td><td>0.093</td><td>0.095</td><td>0.096</td><td>0.084</td><td>0.107</td><td>0.0052</td><td>0.430</td><td>0.793</td><td>&lt;0.001</td><td>0.924</td><td>0.335</td><td>0.278</td></tr></tbody></table>

Abbreviations: BCFA = branched-chain fatty acids; Br = Breed: C = Concentrate type; CLA = conjugated linoleic acid; FA = fatty acid; H = hay type; MUFA = monounsaturated fatty acids; OCFA = odd-chain fatty acids; PUFA = polyunsaturated fatty acids; SFA = saturated fatty acids.

¹ A = 1st cut hay characterised by a high water-soluble carbohydrate content; B = 1st cut hay with a low water-soluble carbohydrate content.

² CM = concentrate based on maize meal, characterised by slow starch degradability; CW = concentrate based on wheat meal, characterised by fast starch degradability.

³ Ho = Holstein cow breed; Mo = Montbéliarde cow breed.
characteristics, size, shape, climatic conditions and management for each pen, as was the case in our trial.

Individual variability in DMI and performance meant that a large number of individuals were needed to carry out the experiment, making it difficult to achieve the ideal density of one trough per cow. Nevertheless, to avoid feed competition limiting feed access time, the ratio of cows to troughs should be set so that the sum of individual hay ingestion times remains less than the available access time, as we did. In addition, the design of feeding barriers that provide some physical separation between adjacent cows is also a key strategy for reducing competition at the feed trough (Huzzey et al., 2006), as is the case with Biocontrol® self-weighing troughs. Furthermore, although the feeding behaviour of dairy cows can be affected by competition between individuals, dominated cows are able to compensate for lower feed access time by increasing feed intake rate, keeping DMI and production performance unchanged (Crossley et al., 2017). This confirms the potential to study the effect on individual DMI and performance in nutritional trials when cows are housed in groups.

### *Diets including highly digestible hay rich in water-soluble carbohydrates increase dairy performance of dairy cows*

Feeding dairy cows with botanically diversified natural grasslands A hay, highly digestible and rich in WSC, increased their dairy performance (milk yield, milk protein content, fat and protein yield and fat and protein-corrected milk). Similar results have been reported in the literature for WSC-rich alfalfa hay (Brito et al., 2008; Klevenhusen and Zebeli, 2021). A high content of WSC in hay resulted in a dilution of forage fibre (lower NDF and ADF; higher WSC/NDF intake, as in our trial), with a consequent increase of OMd (Fisher et al., 1999), which can partially explain the higher milk yield of A hay diet compared to B hay diet-fed cows. Brito et al. (2008) and Klevenhusen and Zebeli (2021) suggested that the higher performances of cows fed WSC-rich hay could also be related to a higher DMI, as function of a high OMd and probably a consequent fast transit rate. The lower physical fill of digestible WSC-rich forages does indeed favour voluntary DMI (Miller et al., 2001; Moorby et al., 2006; Kleefisch et al., 2017). In our experiment, A hay diet-fed cows ingested more 1st cut hay in a comparable intake time, resulting in a higher intake rate and in fewer intake events, compared to B hay diet-fed cows. Such an intake pattern expresses a preference for digestible WSC-rich hay, in agreement with the literature (Fisher et al., 1999; Horadagoda et al., 2009). However, this preference resulted in a reduction of 2nd cut hay intake by A hay diet compared to B hay diet-fed cows. Therefore, total DM, OM, CP and NDF intakes did not differ between A hay diet and B hay diet-fed cows, but DOM intake was higher for A hay diet than for B hay diet-fed cows, suggesting that DOM intake probably contributed to improve dairy performance in A hay diet-fed cows.

The higher intake of 1st cut hay by A hay diet-fed cows resulted in a higher EE intake, compared to B hay diet-fed cows. Lipid supplementation of dairy cow diets is known to potentially increase milk yield and milk fat content, but its effectiveness is related to the proportion of lipids in DMI (Dewanckele et al., 2020). The proportion of lipids in DMI was equivalent in our trial for the two diets (2.4% of DMI for both A hay diet and B hay diet-fed cows) and was low compared to the proportion able to affect milk yield or fat content (Glasser et al., 2008; Kliem and Shingfield, 2016; Martin et al., 2023).

Another factor responsible for the higher dairy performances of A hay diet-fed dairy cows might be their higher WSC intake and particularly their WSC/CP ratio of the ingested diet, compared to B hay diet-fed cows. Edwards et al. (2007) highlighted that a higher WSC/CP ratio in the diet supplied adequate energy for

microbial growth, allowing the incorporation of dietary N into microbial protein, thereby improving dietary N utilisation. This results in a reduction in urea N losses in blood, milk, urine and faeces (Ellis et al., 2011; Kokko et al., 2013), which is in agreement with the lower urea concentration in blood and milk for A hay diet compared to B hay diet-fed cows in our trial. A higher amount of bacterial protein leaving the rumen when cows are fed WSC-rich forages was shown in the literature to increase milk protein content and yield (Edwards et al., 2007; Ellis et al., 2011), as was seen with A hay diet-fed cows in our trial.

Diets rich in WSC are often associated with greater butyrate and propionate concentrations in the rumen (Sutoh et al., 1996; Oba, 2011). Butyrate is extensively metabolised by ruminal epithelial cells (Kristensen, 2005), and Sakata and Tamate (1978) have demonstrated that butyrate increases cell proliferation, resulting in greater feed efficiency in the case of greater butyrate concentration in rumen fluid (Guan et al., 2008). This hypothesis seems consistent with the higher feed conversion efficiency we observed for A hay diet than for B hay diet and underlies a more positive energy balance we observed for A hay diet-fed cows. Furthermore, we also observed a higher concentration of *de-novo* synthesised FA in the milk of A hay diet-fed cows, the synthesis of which in the mammary gland derives mainly from butyrate absorbed from the rumen (Chilliard et al., 2000). A higher *de-novo* synthesis in the mammary gland also results in a greater fat yield (Oba, 2011), as was the case for A hay diet compared to B hay diet-fed cows. Klevenhusen and Zebeli (2021) also suggested that WSC-rich diets can result in a higher body condition score for cows with milk yields lower than 20 kg/day, especially in mid-lactation, as was the case in our trial, because of a positive energy balance.

Another hypothesis explaining the higher *de-novo* synthesis of FA and fat yield of A hay diet compared to B hay diet-fed cows can be related to reduced trans-fatty acid production in the rumen because of incomplete biohydrogenation of unsaturated fatty acids (Oba, 2011). Indeed, we observed a lower concentration of C18:1 trans isomers in the milk produced by A hay diet than B hay diet-fed cows. Penner and Oba (2009) suggested that WSC-utilising microbes may perform biohydrogenation in the rumen and so reduce the concentration of C18:1 isomers. High amounts of C18:1t10 in the milk from B hay diet-fed cows may suggest an inhibitory effect of milk fat synthesis resulting in milk fat depression (Bauman and Griinari, 2003; Colman et al., 2013; Martin et al., 2021). However, milk fat content was equivalent for A hay and B hay diets. Similarly, also fat to protein ratio, which is known to be an indicator of milk fat depression related to C18:1t10 milk content Comino et al., 2015; Villot et al., 2020; Coppa et al., 2023), was unaffected by the type of hay included in the diets. The C18:1t10/C18:1t11 ratio was higher for A hay diet than for B hay diet-fed cows, and its values are lower than those considered able to induce milk fat depression (Villot et al., 2020). This suggests that the higher fat yield of A hay diet-fed cows is likely due to the higher milk yield for A hay diet-fed cows.

The lower concentration of C18:1 trans isomers, of CLA and, in general, of FA derived from intermediate steps of FA ruminal biohydrogenation in the milk produced by A hay diet than B hay diet-fed cows, might suggest a putative inhibitory effect of plant secondary metabolites on rumen microbial population (Buccioni et al., 2012; Renna et al., 2020; Cabiddu et al., 2022). Such metabolites are abundant in the dicotyledonous species of natural diversified grasslands (Buccioni et al., 2012; Renna et al., 2020; Cabiddu et al., 2022). Even if we did not measure the content of plant secondary metabolites in the hays used in our experiment, their proportion of forbs was low and similar between the hay types. Furthermore, the inhibitory effect of plant secondary metabolites on FA ruminal biohydrogenation is also known to be less pronounced with conserved than with fresh herbage (Coppa et al.,
2019), leading to consider such effect as marginal in our experi-
mental conditions.

Maize vs wheat-based concentrate inclusion in the diet had almost no effect on dairy performance and did not interact with hay type

The lack of effect of the concentrate type on dairy performance was partially unexpected, but can be explained in our conditions. The response of cows to starch degradation rate was shown to be greater when dietary NDF was rather low (de Ondarza et al., 2017). Indeed, Moate et al. (2017) and Nasrollahi et al. (2019) showed an increase in milk yield with starch fermentability in cows fed maize silage, though Miller et al. (2001) and Moorby et al. (2006) did not find any effect at pasture. Klevenhusen and Zebeli (2021) also suggest that the effect of carbohydrate degradability on dairy performance appears evident when milk yield is elevated enough not to allow poor WSC alone to completely cover the energy balance of cows. Thus, the low productivity level of dairy cows in our experiment (in line with those of Miller et al., 2001; Moorby et al., 2006, and consistent with the moderate nutritional quality, typical of natural grassland hays; INRA 2018) could also partially explain why we did not observe any effect of the type of concentrate, whereas Moate et al. (2017) and Nasrollahi et al. (2019) found significant effects with high-yielding cows. Concentrate type affected milk urea (and blood) content, being higher in CW diets. This could be due to the higher CP content of CW than CM concentrate, resulting in a higher CP intake of cows fed CW diets. Despite the higher CP intake of cows fed CW diets, the milk protein content was not affected.

The hypothesis that the WSC intake from A-hay diets and a low rate of starch degradation from CM concentrates synchronises the energy supply with the nitrogen supply in the rumen was not confirmed in our trial, probably because the advantages of such synchronisation are particularly evident for high-yielding cows (Klevenhusen and Zebeli, 2021), but this was not the case in our trial. The only exception was for blood glucose, which was higher for CM than for CW-supplemented cows when fed A hay diet. Most of the differences in blood metabolites between CM and CW-supplemented cows were observed for B hay diet only, CW-supplemented cows having higher concentrations of non-esterified fatty acids and β-hydroxybutyrate than CM-supplemented cows. This suggests that when hay is slowly digestible and poor in WSC, the starch degradation rate induces a higher production of volatile FA in the rumen, leading to an increase in blood β-hydroxybutyrate and non-esterified fatty acids (Oba, 2011). When hay is lowly digestible and poor in WSC, the role of synchronisation of the energy supply with the nitrogen supply in the rumen depends on starch: slow degradation of maize starch decreases N urea content in blood and milk, suggesting improved nitrogen utilisation efficiency (Ellis et al., 2011; Kokko et al., 2013). This is particularly interesting especially considering that CM concentrate had a lower CP content than CW, resulting in a higher CP intake for cow fed CW diets. However, the difference was probably too small to induce an effect on milk yield, protein content or protein yield. On the contrary, when diets are rich in WSC from highly digestible forages, the WSC concentration dilutes starch in the rumen, resulting in a lack of effect on dairy performance (Edwards et al., 2007).

The effects of inclusion of hay and concentrate types in cow's diet did
not change between breeds

The differences we observed between the Ho and Mo breeds in
terms of intake, feeding behaviour, BW, body condition score, dairy
performance and milk composition are well known and in agree-
ment with the literature (Martin et al., 2009; Ferlay et al., 2010;

Balandraud et al., 2019). However, hay and concentrate types seem to act similarly in Ho and Mo breeds, as their interactions with breed were not significant for almost all the studied variables. Consequently, the hypothesis that breeds with high yield and energy requirements (like the Holstein breed) would benefit more from a WSC-rich forage diet than less productive breeds (like the Montbéliarde breed) was not confirmed, but the extent of differences in milk yield between Ho and Mo in our trial was not huge.

Conclusion

In conclusion, our study shows that a diet including highly digestible and WSC-rich hay from botanically diversified permanent grasslands improves dairy performance. The resulting higher OMD of ingested diet and DOM intake improved milk yield, milk fat synthesis and feed conversion efficiency. Furthermore, a high WSC/CP ratio of the ingested diet resulted in low blood and milk urea, and in improved milk protein content and yield. In mid-lactation cows with moderate milk yield, the degradation rate of starch of the concentrate included in the diet seems to play a negligible role in improving dairy performance, regardless of the breed's production potential. The effects of the hay and concentrate types included in the diet did not differ between breeds.

Supplementary material

Supplementary Material for this article (https://doi.org/10.1016/j.animal.2025.101464) can be found at the foot of the online page, in the Appendix section.

Ethics approval

The protocol of this study was approved by the Ethics Commit-
tee of the Auvergne-Rhône-Alpes region and the French Ministry of
Higher Education, Research and Innovation (C2EA-02; Authorisa-
tion number: APAFIS APAFIS#17250-2015043014541577-v3). This
experiment was conducted at INRAE, Centre Auvergne-Rhône-
Alpes, France by the INRAE staff (grant agreement C1511401).

Data and model availability statement

None of the data were deposited in an official repository. Access
right to the data is confidential.

Declaration of Generative AI and AI-assisted technologies in the
writing process

During the preparation of this work the author(s) did not use
any AI and AI-assisted technologies.

Author ORCIDs

Mauro Coppa: https://orcid.org/0000-0003-2073-0599.

Dominique Pomiès: https://orcid.org/0000-0002-4043-4423.

Bruno Martin: https://orcid.org/0000-0003-2501-8306.

Matthieu Bouchon: https://orcid.org/0000-0002-7175-7233.

Jean-Pierre Renaud: none.

Massoud Aoun: none.

Bertrand Deroche: https://orcid.org/0000-0003-0424-0776.

René Baumont: https://orcid.org/0000-0002-3802-0651.

CRediT authorship contribution statement

M. Coppa: Writing – original draft, Methodology, Formal analy-
sis, Conceptualisation. D. Pomiès: Writing – review & editing,
**Methodology, Conceptualisation.** **B. Martin:** Writing – review & editing, Methodology, Conceptualisation. **M. Bouchon:** Writing – review & editing, Resources, Data curation. **J.P. Renaud:** Writing – review & editing, Resources. **M. Aoun:** Writing – review & editing, Resources. **B. Deroche:** Writing – review & editing, Validation, Investigation, Formal analysis, Data curation, Conceptualisation. **R. Baumont:** Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Data curation, Conceptualisation.

## Declaration of interest

None.

## Acknowledgments

The authors would like to thank PHILICOT for supplying hay, the University of Caen, and particularly Annette Morvan Bertrand for her help with WSC analysis. The authors especially thank the staff of the experimental Unit Herbipôle (INRAE, 15190 Marcenat, France; https://doi.org/10.15454/1.5572318050509348E12) and Lorène Salis, Aline Le Morvan, Frédéric Anglard and Lucy Garraud of UMR 1213 Herbivores (F-63122 Saint-Genès-Champanelle, France) for their help with data collection for animal measurements and analysis, as well as Emilie Tixier and Anne Ferlay (UMR 1213 Herbivores) for the fatty acid analysis and calculations. Linguistic revision was edited by David Marsh, a professional translator.

## Financial support statement

This research was funded by the French Association for Research and Technology (grant agreement no. 2016/0889) and IDENA.

## References

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