To improve sustainability, dairy farms can reduce protein-rich concentrate in the cows' diet providing fresh herbage produced on-farm. This study aimed to quantify effects of increasing the percentage of fresh herbage (0%, 25%, 50%, and 75%, on a dry matter [DM] basis) in a partial mixed ration-based diet on cow N use efficiency and excretion. The study was performed with five lactating cows, in a 4 × 4 Latin square design for four 3 week periods. Individual DM intake, milk yield, feces and urine excretions, and their N concentrations were measured daily. Dietary crude protein concentrations varied little among treatments (127 to 134 g/kg DM). DM intake and milk yield decreased linearly by 5.2 and 3.7 kg/day, respectively, while N use efficiency increased by 4.1 percentage points from 0% to 75% DM of fresh herbage in the diet. Urinary N was not influenced by the treatments, while fecal N decreased as the percentage of fresh herbage increased. This study highlights that replacing partial mixed ration with an increasing percentage of fresh herbage with slight changes in dietary N concentration increases N use efficiency and the percentage of urinary N in excreted N.

1 INTRODUCTION

To ensure sustainable production, dairy cattle farms must reduce their environmental impacts while increasing their feed self-sufficiency, especially in protein sources for feed. Ruminant production is known for its substantial contribution to greenhouse gas emissions and nitrogen (N) losses, such as nitrate and ammonia (NH₃) (European Environment Agency, 2019; Lesschen et al., 2011), as well as its consumption of resources that compete with human food (Mottet et al., 2017). Dairy cows are often fed conserved forage supplemented with protein-rich concentrates, including soybean meal, to balance energy and protein in the diet. However, soybean meal is an expensive feed that is imported to Europe mainly from South America and whose production is associated with negative environmental impacts (Prudêncio da Silva et al., 2010). One way to reduce consumption of soybean meal is to increase the supply of N from forage, especially fresh herbage, in the dairy cow diet. Fresh herbage is a feed produced on-farm that usually has higher N and protein concentrations than conserved forage (French National Institute for Agricultural Research [INRA], 2018; Ferreira et al., 2021), such as hay, which is generally more mature when harvested, or herbage silage for which proteins are partly degraded by fermentation during the storage process. Grazing enables low-cost N and protein supplies in the cow diets and the direct recycling of N excreted by cows for grassland fertilization. Furthermore, grasslands provide multiple ecosystem services, such as carbon sequestration, and help conserve biodiversity (Dumont et al., 2019, 2020).

When herbage is scarce and during dietary transitions (between grazing and winter period in barn), fresh herbage can be combined with conserved forage alone or mixed with concentrates as a partial mixed ration (PMR) to avoid risking a protein deficit when forage contains too little N. This can occur when the diet contains mainly corn silage and no protein supplementation, which drastically decreases dairy cow intake and can negatively influence cow production (Bryant & Donnelly, 1974; Ferreira et al., 2023). For this reason, most studies tested a combination of fresh herbage and corn silage in a PMR (Bargo et al., 2002; Miguel et al., 2014; Pastorini et al., 2019). Consequently, dry matter (DM) intake (DMI) and milk yield responses of dairy cows are well documented: Both frequently decrease as the percentage of fresh herbage in the diet increases. However, effects of combinations of fresh herbage and PMR on cow N use and excretion were poorly studied compared with those of herbage alone or a total mixed ration with only conserved forage and concentrates (Ferreira et al., 2021). We hypothesized that fresh herbage and PMR combinations may affect cow N use by varying the dietary N concentration and DMI (Castillo et al., 2000; Huhtanen et al., 2008; Huhtanen & Hristov, 2009), increasing the dietary degradable N concentration, and/or changing the protein supply in relation to the energy supply (Cantalapiedra-Hijar et al., 2014; Edouard et al., 2016, 2019; Souza & White, 2021).

Thus, the present study aimed to quantify changes in N use efficiency and N excretion when soybean meal and corn silage were gradually replaced with fresh herbage in the dairy cow diet. Fresh herbage was cut daily and offered indoors to study the effect of these feeding strategies on cow voluntary intake, nutrients digestion, and N excretion, as these variables are difficult to study at pasture.

2 MATERIALS AND METHODS

2.1 Treatments, experimental design, and cows

The experiment was conducted from April 2 to June 24, 2022, at IE PL, INRAE, Dairy nutrition and physiology (IE PL, 35650 Le Rheu, France; 10.15454/yk9q-pf68) with the agreement for animal housing number C-35–275-23. The experiment was performed following French and European Union legislation on animal experimentation and animal welfare. All procedures related to the care and management of animals were approved by an ethics committee of the French Ministry of Agriculture (Comité Rennais d'Ethique en matière d'Expérimentation Animale, No. 07), in agreement with French regulations (decree 2001-464, May 29, 2001) (approval number: APAFIS #35128-2022020318257178 v3).

Four treatments were compared—H0, H25, H50, and H75—which corresponded to an objective of 0%, 25%, 50%, or 75% fresh herbage on a DM basis in the diet, respectively. Treatments were compared using a 4 × 4 Latin square design balanced for potential carryover effects for four 3 week experimental periods. Each period included 15 days for adaptation to the treatment and 6 days for measurements.

The study was performed with five multiparous and ruminally cannulated Holstein Friesian cows. Cows were in midlactation at the beginning of the experiment (88 ± 34 days in milk) and had a mean body weight of 724 ± 57.8 kg. During the preexperimental period, from March 21 to April 1, cows were individually fed ad libitum, and their DMI and milk yield were 21.7 ± 1.96 and 39.2 ± 5.72 kg/day, respectively (mean calculated from five individual averages throughout 11 days, ±between-cow standard deviation). The preexperimental diet contained 38% fresh herbage on a DM basis, which was the mean percentage of fresh herbage in the four treatments, and 62% PMR. The PMR was composed of 85% corn silage and 15% soybean meal on a DM basis in all treatments.

2.2 Housing and feeding management

Cows were housed in tie stalls in a temperature-controlled and mechanically ventilated room, where they could see, smell, and hear each other throughout the entire experiment. They were milked twice a day in the room at 06:45 and 17:00 h. Cows were fed in individual troughs and had unlimited access to water and a salt lick. Two feeding management rules were observed throughout the experiment. First, total refusals (herbage and PMR) for the entire day had to be close to but higher than 10% of the offered diet. Thus, either fresh herbage or PMR was offered ad libitum. The second rule was to ensure that the diets ingested included the required percentages of fresh herbage. To this end, these percentages were checked daily, and the amounts of feed offered were adjusted depending on the daily DM concentration and individual intake of each feed. In general, for H0 and H25, PMR was offered ad libitum, while fresh herbage was restricted. Conversely, for H50 and H75, fresh herbage was fed ad libitum, while PMR was restricted.

Fresh herbage was cut once a day at 08:00 h using a mechanical mower with a cutter bar (Haldrup GmbH). The herbage was immediately offered at the trough or conserved in a cool room at 4°C to ensure that fresh herbage was offered throughout the day. For H0, PMR was offered twice a day, at 08:00 and 18:00 h. For H25, H50, and H75, fresh herbage was offered at 08:00, 09:30, 11:30, and 16:00 h (too large amounts to be offered all at once), and herbage refusals were removed at 17:45 h. The first two offerings of herbage were the most important, because they followed cows' natural feeding behavior. In the three treatments with fresh herbage, PMR was offered once a day at 18:00 h, and PMR refusals were removed and weighed at 07:45 h for all treatments. Consequently, the size of meals differed among treatments during the day. For example, cows fed the H25 diet received little feed in the morning (25%) and a large amount of feed in the evening (75%) and vice versa for cows fed the H75 diet. For the H0, cows had access to PMR all day long. For H25, H50, and H75, cows had 10 h per day to eat fresh grass and 14 h per day to eat PMR. Regardless of the treatment, the time the cows had to eat was therefore not a limiting factor for voluntary intake.

The fresh herbage came from a 2 ha grassland. The sward was managed to provide herbage with 30 to 35 days of regrowth (vegetative stage) to control as much as possible sward height and chemical composition throughout the experiment (Table 1). The precut sward height was measured on Days 14 and 18 of each period within the area to be mown with a rising platemeter (30 × 30 cm, 4.5 kg/m², Aurea Agrosciences, Blanquefort, France) and was between 8.7 and 21.2 cm. The sward was sown with a mixture of grasses (16 kg/ha of Lolium perenne L., Trybal cultivar; 8 kg/ha of L. perenne L., Ibisal cultivar; and 8 kg/ha of Festuca arundinacea Schreb., Philona cultivar) in September 2018. The botanical composition of fresh herbage was determined on one day per period from a representative 1 kg sample of freshly cut herbage. Herbage was composed mainly of grasses (98.6 ± 1.2% on a DM basis), with a predominance of L. perenne and no legume species. The grassland received 40 kg N/ha as ammonium nitrate at the end of February 2022 and then every 30–40 days.

TABLE 1. Chemical composition and nutritional value of feed offered to dairy cows.

Component	Fresh herbage				Corn silage	Soybean meal
Component	Period 1	Period 2	Period 3	Period 4	Corn silage	Soybean meal
DM, g/kg fresh weight	18.9	22.8	23.6	25.0	387 ± 5.7	895
OM, g/kg DM	916	928	931	913	960 ± 0.8	930
Ash, g/kg DM	84	72	69	87	40 ± 0.8	70
CP, g/kg DM	131	116	136	116	64 ± 2.9	511
NDF, g/kg DM	469	521	492	540	427 ± 20.2	115
ADF, g/kg DM	240	263	253	277	232 ± 12.7	66
UFL/kg DM	0.99	0.97	0.93	0.89	0.90 ± 0.015	1.26
PDI, g/kg DM	90	79	85	83	61 ± 0.4	220
RPB, g/kg DM	−11	−15	0	−17	−47 ± 2.4	235

Note: Means ± standard deviation for corn silage (n = 4).
Abbreviations: ADF, acid detergent fiber; CP, crude protein; DM, dry matter; NDF, neutral detergent fiber; OM, organic matter; PDI, truly digestible protein (INRA, 2018); RPB, rumen protein balance, CP intake minus nonammonia CP flowing from the duodenum (INRA, 2018); UFL, “unité fourragère lait” (1 UFL = 7.37 MJ of net energy for lactation; INRA, 2018).

2.3 Feed characteristics and intake calculations

Amounts of each feed offered and refused were weighed daily and dried in a ventilated oven for 48 h at 60°C to measure their DM concentration and calculate the daily DMI for each cow. To this end, a 1 kg sample of the fresh herbage and corn silage offered was dried daily, and three samples of 0.3 kg of soybean meal were dried each week. Similarly, a 1 kg sample of the herbage and PMR refused per cow was dried daily. The DM concentration of corn silage was corrected by considering the volatilization of fermentation products in the oven during drying (Dulphy et al., 1975). To this end, NH₃, volatile fatty acids, alcohols, and lactic acid in a frozen sample (−20°C) of corn silage were analyzed, and volatilization was calculated using the multiple regression of Dulphy et al. (1975). The volatilization correction factor was 18 g/kg DM.

Chemical analyses (organic matter [OM], crude protein [CP], neutral detergent fiber [NDF], and acid detergent fiber [ADF]) were performed per period on pooled lyophilized daily samples of the fresh herbage and corn silage offered on the six measurement days. The soybean meal offered was analyzed per period on pooled fresh daily samples. Chemical analyses of the herbage and PMR refused were performed per cow and period on pooled lyophilized daily samples on the same days. Nutrient intake (g/day) was then calculated per cow and period from amounts of nutrients offered and refused, thus considering potential feed preferences of each cow:

\mathrm{Nutrient}\ \mathrm{intake}=\sum \limits_{\mathrm{k}=1}^{\mathrm{n}}{\left[\left(\mathrm{OF}\times {\mathrm{NUT}}_{\mathrm{OF}}\right)-\left(\mathrm{RF}\times {\mathrm{NUT}}_{\mathrm{RF}}\right)\right]}_{\mathrm{k}},

with n the number of feeds in the diet (n = 2 for H0 and 3 for the other treatments); OF and RF the amount of feed offered and refused (kg DM/day), respectively; and NUT_OF and NUT_RF the nutrient concentration of feed offered and refused (g/kg DM), respectively.

Then, the concentration of truly digestible protein (PDI, in g/kg DM), the concentration of net energy for lactation (“unité fourragère lait” [UFL]; 1 UFL = 7.37 MJ/kg DM of net energy for lactation), and the rumen protein balance (RPB, in g/kg DM, as CP intake minus the nonammonia CP flowing from the duodenum) of each feed were calculated from their chemical analyses according to the INRA (2018) feeding system and using PrevAlim® software (https://www.inration-ruminal.fr/en/) (INRA, 2018). The PDI concentration was calculated from the feeds' CP and fermentable OM concentrations, the N degradability in the rumen, and the N digestibility; the UFL concentration was calculated mainly from the feeds' gross energy concentration and digestibility; and the RPB was calculated from the feeds' CP and fermentable OM concentrations and their N degradability in the rumen. The PDI and UFL concentrations and the RPB of the entire diets were then calculated from the nutritional values and DMI of each feed, considering the main sources of ruminal digestive interactions (Sauvant & Nozière, 2016), according to the INRA (2018) feeding system and using INRAtion® V5 software (INRA, 2018).

2.4 Milk yield and composition

Milk yield was recorded daily. Fat and protein concentrations were measured in fresh milk at each milking from Days 17 to 21. For each cow, milk N concentration was analyzed in 50 mL of fresh milk sampled on Day 19 from pooled morning and afternoon milk. A subsample of this pool was deproteinized by membrane ultrafiltration and frozen (−20°C) until analysis of urea concentration.

2.5 Feces and urine excretions, sampling, and digestibility calculations

Feces and urine excretions were determined individually by collecting all feces and urine for five consecutive days (Days 17–21). Feces were collected in a gutter behind the cow and regularly transferred to a closed bucket. Total amounts were weighed and sampled daily (2% of the feces excreted). Half of the sample was dried in a ventilated oven for 72 h at 60°C to determine the fecal DM concentration. The second half was frozen and lyophilized for chemical analyses (OM, N, NDF, and ADF) of pooled samples per cow and period. Daily samples were stored at 4°C in the same airtight container and frozen at −20°C on Day 21. Whole-tract digestibility of nutrients was calculated from the amount of each component ingested (intake, kg/day) and excreted in feces (fecal excretion, kg/day):

\mathrm{Digestibility}=\left(\mathrm{intake}-\mathrm{fecal}\ \mathrm{excretion}\right)/\mathrm{intake}.

To collect urine, each cow was equipped with a harness that held a tube around the vulva to drain urine into a plastic container. Urine was immediately acidified in the container with 500 mL of 20% H₂SO₄ to prevent ammonia volatilization. Urine was weighed and sampled daily (1% of the urine excreted). Daily samples were pooled per cow and period and stored at −20°C in the same container before analyzing N and urea.

2.6 Ruminal fermentation and plasma metabolites

Ruminal fermentation was assessed via ruminal pH and NH₃-N concentration kinetics based on 10 sampling times during the day (Day 20). Basal concentrations were determined at 07:45 and 17:45 h (before the first morning feeding and the evening feeding, respectively). The other samples were taken 1, 2, 3, and 5 h after these feedings (09:00, 10:00, 11:00, and 13:00 h, respectively, for morning herbage or PMR offering and 19:00, 20:00, 21:00, and 23:00 h, respectively, for evening PMR offering). At each time, 150 mL of ruminal fluid was sampled in the ventral sac via the cannula. The pH was measured immediately with a pH meter. Ruminal fluid was then filtered through six layers of muslin and frozen at −20°C (4 mL of ruminal fluid in 4 mL of 20% NaCl preservative). The weighted means of ruminal pH and NH₃-N concentration for the entire day were calculated based on the sampling times and intervals.

Blood metabolites were determined from blood sampled on Day 19 before the first morning feeding and the evening feeding (07:45 and 17:45 h, respectively) and then 3 h later (11:00 and 21:00 h, respectively). Blood was sampled in the caudal vein and centrifuged (8 mL, 2000×g at 4°C for 15 min). The plasma was then frozen at −20°C. The mean of the four plasma sampling times each day was then calculated for subsequent statistical analysis.

2.7 Chemical analyses

The lyophilized offered and refused feeds and feces were ground to 0.8 mm to analyze OM, NDF, ADF, and N. The OM concentration was measured by ashing in a muffle furnace at 550°C for 8 h (AOAC, 2019). Fiber concentrations (NDF and ADF) were analyzed sequentially with a Fibersac extraction unit (Ankom Technology) (AOAC, 2019; Van Soest et al., 1991). NDF concentration was assayed with a heat-stable amylase, and NDF and ADF concentrations were expressed exclusive of residual ash. Pepsin-cellulase digestibility of dried feeds was determined according to Aufrère and Michalet-Doreau (1988). The N concentration of the offered and refused feed, feces, urine, and milk was analyzed using the Dumas method (Leco Corporation analyzer) (AOAC, 2019). Urine, milk, and plasma urea concentrations were determined based on an enzymatic and colorimetric reaction assessed using a multiparameter analyzer (KONE Instruments 200 Corporation) (Talke & Schubert, 1965). The clearance rate of urea (volume of blood cleared per unit time, in L/h) was calculated as urinary urea excretion (g/day) divided by the plasma urea concentration (g/L). Milk fat and protein concentrations were measured using midinfrared spectrophotometry (Milkoscan, Foss Electric). Ruminal NH₃-N concentration was analyzed using the Berthelot colorimetric reaction method (KONE Instruments 200 Corporation) (Gordon et al., 1978).

2.8 Nitrogen use efficiency and balance calculations

Nitrogen use efficiency (%) was calculated by dividing the N in milk (g N/day) by N intake (g N/day). Unaccounted-for N was calculated as N intake minus the N in milk, excreted in feces and urine (g N/day), and retained (g N/day). The N retained (g N/day) was estimated from the UFL balance (UFL/day), assuming that 6 g N/UFL was retained by protein accretion or mobilization when the UFL balance was positive (weight gain) or negative (weight loss), respectively (INRA, 2018).

2.9 Statistical analysis

Data were averaged per cow and per period (n = 5 per treatment) and then analyzed using the following mixed model (SAS Institute, 2020; PROC MIXED):

{\mathrm{Y}}_{\mathrm{i}\mathrm{jk}}=\upmu +{\mathrm{Treatment}}_{\mathrm{i}}+{\mathrm{Period}}_{\mathrm{j}}+{\mathrm{Cow}}_{\mathrm{k}}+{\mathrm{e}}_{\mathrm{i}\mathrm{jk}},

with Y_ijk the analyzed variable, μ the overall mean, Treatment_i the fixed effect of the percentage of fresh herbage in the diet (3 df), Period_j the fixed effect of the experimental period (3 df), Cow_k the random effect of the cow, and e_ijk the residual error. Linear and quadratic responses to the percentage of fresh herbage in the diet were determined using orthogonal contrasts. The significant differences between treatments (p < 0.05) were determined with the Student test and the least significant difference procedure.

3 RESULTS

3.1 Intake, feed composition, diet composition, and digestibility

The CP concentration of fresh herbage and PMR averaged 125 (from 116 to 136 depending on the period) and 134 g/kg DM, respectively (Tables 1 and 2). As expected, the percentage of fresh herbage in the diet followed a regular interval among treatments. The DM refused averaged 13% of the DM offered and was not influenced by treatment (p > 0.05). Cows refused more fresh herbage than PMR, especially when the percentage of herbage was high. Obtaining the expected percentage of fresh herbage required restricting the amount of PMR offered, while fresh herbage was offered ad libitum, especially for H50 and H75. Consequently, almost no PMR was refused with the H75 diet. DMI was 22.6 kg/day for H0 and decreased linearly by 0.7 kg/day for every 10 percentage-point increase in fresh herbage in the diet (p < 0.01). From H0 to H75, the linear decrease in PMR intake resulted in a linear decrease in soybean meal intake, from 3.4 to 0.7 kg DM/day (p < 0.01).

TABLE 2. Effects of increasing the percentage of fresh herbage in the partial mixed ration (PMR) on dairy cow intake and diet composition and digestibility.

Variable	H0	H25	H50	H75	SEM	LIN	QUA
Variable	Treatment^†					Probability^‡
DM intake, kg/day	22.6^a	20.9^b	19.8^b	17.4^c	0.48	<0.001	0.479
Fresh herbage intake, kg DM/day	0.0^d	5.3^c	9.9^b	12.8^a	0.22	<0.001	<0.001
PMR intake, kg DM/day	22.6^a	15.6^b	9.9^c	4.6^d	0.50	<0.001	0.115
Soybean meal, kg DM/day	3.4^a	2.3^b	1.5^c	0.7^d	0.08	<0.001	0.117
Total DM refused, % DM offered	14.2	12.0	12.9	13.5	1.25	0.864	0.311
Fresh herbage refused, % DM herbage offered	0^c	9.0^b	20.3^a	17.4^a	1.45	<0.001	0.003
PMR refused, % DM mixed ration offered	14.5^a	12.5^a	3.6^b	0.4^b	2.10	<0.001	0.790
Diet DM, g/kg fresh weight	432^a	347^b	296^c	254^d	4.5	<0.001	0.002
Diet OM, g/kg DM	956^a	947^b	939^c	931^d	0.7	<0.001	0.940
Diet CP, g/kg DM	134^a	132^ab	128^bc	127^c	1.2	0.002	0.909
Diet NDF, g/kg DM	367^d	407^c	440^b	467^a	4.6	<0.001	0.202
Diet ADF, g/kg DM	200^d	217^c	231^b	242^a	2.4	<0.001	0.216
UFL/kg DM	0.89^c	0.91^bc	0.92^b	0.94^a	0.006	<0.001	0.595
PDI, g/kg DM	87^a	85^b	84^c	82^d	0.5	<0.001	0.930
PDI, g/UFL	98^a	94^b	91^c	88^d	0.4	<0.001	0.637
RPB, g/kg DM	−7.0	−6.4	−7.0	−6.6	1.11	0.888	0.953
DM digestibility, g/g	0.710^b	0.707^b	0.720^b	0.747^a	0.0061	0.002	0.038
OM digestibility, g/g	0.719^b	0.716^b	0.730^b	0.758^a	0.0062	0.002	0.036
NDF digestibility, g/g	0.514^d	0.581^c	0.651^b	0.714^a	0.0063	<0.001	0.726
ADF digestibility, g/g	0.539^d	0.594^c	0.662^b	0.721^a	0.0069	<0.001	0.769

Note: n = 5 data per treatment.
Abbreviations: ADF, acid detergent fiber; CP, crude protein; DM, dry matter; NDF, neutral detergent fiber; OM, organic matter; PDI, truly digestible protein (INRA, 2018); RPB, rumen protein balance, CP intake minus nonammonia CP flowing from the duodenum (INRA, 2018); UFL, “unité fourragère lait” (1 UFL = 7.37 MJ of net energy for lactation; INRA, 2018).
^† Treatments H0, H25, H50, and H75 correspond to objectives of 0%, 25%, 50%, and 75% of fresh herbage on a DM basis in the PMR diet, respectively. The PMR was composed of 85% corn silage and 15% soybean meal. In a given row, adjusted means with different superscript letters differ significantly between treatments (p < 0.05).
^‡ Probability of linear (LIN) and quadratic (QUA) effects of the treatment.

From H0 to H75, the dietary DM concentration decreased quadratically from 432 to 254 g/kg fresh weight (p < 0.01, Table 2). Dietary OM and CP concentrations decreased linearly by 3.4 and 1.1 g/kg DM, respectively, for every 10 percentage-point increase in fresh herbage in the diet (p < 0.01), while NDF and ADF concentrations increased linearly by 13.7 and 5.8 g/kg DM (p < 0.01). Dietary UFL concentration increased linearly as the percentage of fresh herbage increased (p < 0.01), while the PDI concentration and PDI:UFL ratio decreased linearly (p < 0.01). Conversely, the treatment had no influence on RBP (−7 ± 1.1 g/kg DM; p > 0.05). Whole-tract dietary DM and OM digestibilities were 0.035 and 0.036 higher, respectively, for H75 than for the other three diets (quadratic effect: p < 0.05). From H0 to H75, dietary NDF and ADF digestibilities increased linearly from 0.51 to 0.71 and from 0.54 to 0.72 g/g, respectively (p < 0.01).

3.2 Milk yield and composition

Milk yield was 30.3 kg/day for H0 and decreased linearly by 0.5 kg/day for every 10 percentage-point increase in fresh herbage in the diet (p < 0.01; Table 3). Milk fat concentration tended to be lower for H25 and H50 than for the other two treatments (quadratic effect: 0.05 < p < 0.1). From H0 to H75, milk protein concentration decreased linearly from 30.1 to 28.1 g/kg (p < 0.01).

TABLE 3. Effects of increasing the percentage of fresh herbage in the partial mixed ration (PMR) on dairy cow milk yield and composition, nitrogen use efficiency, and nitrogen partitioning.

Variable	H0	H25	H50	H75	SEM	LIN	QUA
Variable	Treatment^†					Probability^‡
Milk yield, kg/day	30.3^a	30.2^a	28.9^ab	26.6^b	0.74	0.006	0.163
Corrected milk yield^§, kg/day	29.1^a	27.9^a	26.9^ab	25.2^b	0.74	0.005	0.706
Milk fat concentration, g/kg	38.2	36.2	36.7	38.3	0.84	0.836	0.069
Milk protein concentration, g/kg	30.1^a	29.5^b	29.3^b	28.1^c	0.16	<0.001	0.130
N intake, g/day	484^a	441^b	408^b	353^c	11.5	<0.001	0.609
N in milk, g/day	143^a	138^ab	129^b	118^c	3.2	<0.001	0.345
N use efficiency, %	29.7^c	31.2^bc	31.8^b	33.8^a	0.57	<0.001	0.629
Fecal N, g/day	144^a	142^a	137^a	115^b	2.7	<0.001	0.005
Urinary N, g/day	108	107	116	122	7.2	0.146	0.668
Urinary N, % of N intake	22.8^b	24.7^b	28.9^b	35.2^a	1.64	<0.001	0.218
Urinary N, % of N excreted	43.0^b	43.3^b	45.9^b	51.5^a	1.40	0.002	0.098
Unaccounted-for N^¶, g/day	84^a	53^b	26^c	5^c	7.9	<0.001	0.554

Note: n = 5 data per treatment.
^† Treatments H0, H25, H50, and H75 correspond to objectives of 0%, 25%, 50%, and 75% of fresh herbage on a dry matter basis in the PMR diet, respectively. The PMR was composed of 85% corn silage and 15% soybean meal. In a given row, adjusted means with different superscript letters differ significantly between treatments (p < 0.05).
^‡ Probability of linear (LIN) and quadratic (QUA) effects of the treatment.
^§ Milk yield corrected for standard milk with concentrations of 40 g/kg fat and 31 g/kg protein (INRA, 2018).
^¶ Calculated from N intake, in milk, in feces, and in urine and corrected from an estimate of retained N. Retained N was estimated from the balance of net energy for lactation, assuming 6 g N retained per “unité fourragère lait” (Faverdin & Vérité, 1998). One “unité fourragère lait” equals to 7.37 MJ of net energy for lactation (INRA, 2018).

3.3 Nitrogen use efficiency and N partitioning

Nitrogen intake and N in milk were 484 and 143 g N/day for H0, respectively, and decreased linearly by 17.4 and 3.5 g N/day for every 10 percentage-point increase in fresh herbage in the diet (p < 0.01; Table 3). Consequently, N use efficiency increased linearly by 0.5 percentage points for every 10 percentage-point increase in fresh herbage (p < 0.01). Fecal N was 26 g N/day lower for H75 than for the other three diets (quadratic effect: p < 0.01), while urinary N was not influenced by the treatment (114 ± 7.2 g N/day; p > 0.05). The percentage of urinary N in N excreted in feces and urine was higher for H75 than for the other three diets (quadratic trend: 0.05 < p < 0.1). Unaccounted-for N decreased linearly by 11 g N/day for every 10 percentage-point increase in fresh herbage (p < 0.01).

3.4 Nitrogen metabolism components

From H0 to H75, mean ruminal pH decreased linearly from 6.46 to 6.31 (p < 0.05; Table 4), while mean ruminal NH₃-N concentration tended to increase from 63 to 89 mg N/L (0.05 < p < 0.1). Plasma urea concentration averaged 157 mg/L and was not influenced by treatment (p > 0.05). Milk urea concentration averaged 165 mg/L and tended to be lower for H25 and H50 than for the other two treatments (quadratic effect: 0.05 < p < 0.1). Urinary urea concentration was 7.4 g/L for H0 and decreased linearly by 0.26 g/L for every 10 percentage-point increase in fresh herbage in the diet (p < 0.05). Urinary urea N excretion (51 ± 6.0 g N/day) and the percentage of urea N in urinary N (44 ± 2.6%) were not influenced by treatment (p > 0.05). Clearance rate of urea and urine excretion increased linearly by 1.0 L/h and 1.2 kg/day, respectively, for every 10 percentage-point increase in fresh herbage (p < 0.05 and p < 0.01).

TABLE 4. Effects of increasing the percentage of fresh herbage in the partial mixed ration (PMR) on dairy cow ruminal pH and NH₃-N and on urea in plasma, milk, and urine.

Variable	H0	H25	H50	H75	SEM	LIN	QUA
Variable	Treatment^†					Probability^‡
Ruminal pH^§	6.46^a	6.46^a	6.37^ab	6.31^b	0.040	0.019	0.506
Ruminal NH₃-N concentration^§, mg/L	62.5	74.0	85.4	89.0	9.8	0.073	0.670
Plasma urea concentration^¶, mg/L	163	151	153	161	11.9	0.917	0.427
Milk urea concentration, mg/L	178	156	151	173	10.2	0.698	0.062
Urine urea concentration, g/L	7.38^a	6.58^ab	5.61^ab	5.57^b	0.548	0.032	0.510
Urinary urea N, g/day	46.6	46.7	47.9	61.4	6.01	0.131	0.306
Urinary urea N, % of urinary N	43.0^ab	43.5^ab	40.6^b	49.2^a	2.63	0.221	0.164
Clearance rate of urea, L/h	25.6^b	28.3^ab	27.7^ab	33.6^a	2.23	0.046	0.495
Urine excretion, kg/day	14.6^c	16.2^bc	19.3^b	23.8^a	1.2	<0.001	0.264

Note: n = 5 data per treatment.
^† Treatments H0, H25, H50, and H75 correspond to objectives of 0%, 25%, 50%, and 75% of fresh herbage on a dry matter basis in the PMR diet, respectively. The PMR was composed of 85% corn silage and 15% soybean meal. In a given row, adjusted means with different superscript letters differ significantly between treatments (p < 0.05).
^‡ Probability of linear (LIN) and quadratic (QUA) effects of the treatment.
^§ Weighted means of ruminal pH and NH₃-N concentration of the 10 sampling times during one day (07:45, 09:00, 10:00, 11:00, 13:00, 17:45, 19:00, 20:00, 21:00, and 23:00 h).
^¶ Mean of four plasma sampling times (07:45, 11:00, 17:45, and 21:00 h).

4 DISCUSSION

The objective of this study was to quantify the effects of increasing the percentage of fresh herbage in a corn silage-based diet on cow N use efficiency and N excretion. The close CP concentrations of fresh herbage and PMR enabled us to investigate the effects of partially replacing PMR with fresh herbage with no substantial change in the dietary CP concentration between treatments. The dietary CP concentration is known to have a strong effect on N use efficiency and urinary N (Castillo et al., 2000; Huhtanen & Hristov, 2009). Therefore, this study highlighted the effects of other factors than dietary CP concentration, such as DMI, on N flows when fresh herbage and PMR are combined in dairy cow diets. The strict observance of the percentages of feeds in the dairy cow diet enabled us to consider the effects of the combination of PMR and fresh herbage on voluntary intake and N flows.

4.1 Intake, diet digestibility, and consequences for milk yield

The linear decrease in DMI as the percentage of fresh herbage in the diet increased suggests no ingestive interactions between fresh herbage and PMR. This decrease in DMI is consistent with results of Bargo et al. (2002), Pérez-Ramírez et al. (2012), and Pastorini et al. (2019), who observed a decrease of 0.4–0.9 kg DM/day for every 10 percentage-point increase in fresh herbage for dairy cows fed PMR. The DMI may have decreased because fresh herbage has lower ingestibility than PMR, partly due to the low fill value of soybean meal, like for all concentrates when compared with forages (INRA, 2018), to cows' preference for PMR, or a combination of both. The fact that the amount of PMR offered had to be restricted to ensure that cows ate 50% or 75% fresh herbage suggests that they preferred PMR to fresh herbage. At the opposite, when no N-rich concentrate is provided, cows seem to prefer fresh herbage than corn silage (Bryant & Donnelly, 1974; Ferreira et al., 2023), probably to limit a deficit of dietary N.

The high and linear increase in dietary NDF and ADF digestibilities as the percentage of fresh herbage in the diet increased was due mainly to the digestibility of each feed, because fiber in fresh herbage is much more digestible than that in corn silage, and thus in PMR (Ferreira et al., 2021; INRA, 2018). Moreover, the diet digestibility may have increased thanks to the decrease in digestive interactions related to the decrease in DMI (INRA, 2018). The DM and OM digestibilities did not change from H0 to H50, despite the increase in dietary fiber digestibility. This was likely because the increase in dietary fiber concentration offsets the increase in fiber digestibility (INRA, 2018).

Despite the increase in diet digestibility and UFL concentration, the decrease in DMI led to a decrease in the net energy supply as the percentage of fresh herbage in the diet increased (20.1 to 16.4 UFL/day from H0 to H75). Consequently, milk yield and milk protein concentration decreased as the energy supply decreased (Coulon & Rémond, 1991). Bargo et al. (2002) and Pastorini et al. (2019) also observed a decrease in milk yield as the percentage of fresh herbage increased in the dairy cow diet, related to the decrease in DMI.

4.2 Nitrogen use efficiency

Dietary CP concentrations (127–134 g CP/kg DM) were lower than those usually recommended for lactating cows (140–160 g CP/kg DM; INRA, 2018) and very close among treatments. Under these conditions, the decrease in N intake as the percentage of fresh herbage in the diet increased was due mainly to the decrease in DMI. Consequently, the decrease in milk yield and milk protein concentration decreased the N in milk (143–118 g N/day from H0 to H75). The decrease in N intake as the percentage of fresh herbage increased exceeded the decrease in N in milk (by 17.4 and 3.5 g N/day for N intake and N in milk, respectively, for every 10 percentage-point increase in fresh herbage), which increased N use efficiency. The N use efficiency observed (~32%) agreed with that in studies that tested diets with a CP concentration similar to ours (Cantalapiedra-Hijar et al., 2014; Edouard et al., 2016; Yang et al., 2022). Logically, N use efficiency increased as N intake decreased (Edouard et al., 2016, 2019). Most previous studies indicated that dietary CP concentration was the main factor that influenced N use efficiency (Edouard et al., 2019; Huhtanen & Hristov, 2009). In the present study, increasing the percentage of fresh herbage in the diet influenced mainly N use efficiency through a decrease in DMI, because dietary CP concentration changed little among treatments. Linear responses of N intake, N in milk, and N use efficiency as the percentage of fresh herbage in the diet increased suggest no ingestive interactions between fresh herbage and PMR.

4.3 Nitrogen partitioning

In agreement with the literature, the decrease in DMI was the main cause of the decrease in fecal N, because these two variables are closely related (Huhtanen et al., 2008; INRA, 2018). The quadratic response of fecal N as the percentage of fresh herbage in the diet increased is consistent with the change in OM digestibility, which is strongly correlated with endogenous N excretion, the main component of fecal N (INRA, 2018).

In the literature, a decrease in N intake usually reduce urinary N (Huhtanen et al., 2008; Spanghero & Kowalski, 2021). In the present study, despite lower N intake caused by a decrease in DMI with increasing fresh herbage in the diet, urinary N did not change. This could be due to little variations in dietary CP concentration among treatments, known to be the main factor that influences urinary N (Huhtanen et al., 2008; Spek et al., 2013). Consequently, urinary N relative to N intake increased as the percentage of fresh herbage increased. One possible explanation is that, because fresh herbage contains more ash than PMR (De Boer et al., 2002; INRA, 2018; Table 1), cows fed more fresh herbage might have maintained their osmotic balance by increasing their clearance rate and urine excretion through renal regulation (Bannink et al., 1999; De Boer et al., 2002; Dijkstra et al., 2013; Faverdin & Vérité, 2003). Alternately, urinary N relative to N intake may have increased as the percentage of fresh herbage increased due to an imbalance between N degradation and use by ruminal microflora (Dijkstra et al., 2013). Partially replacing PMR with fresh herbage in the diet seemed indeed to influence ruminal fermentation in the present study (increasing trend in ruminal NH₃-N concentration and decreasing in ruminal pH as the percentage of fresh herbage increased).

Unaccounted-for N was high for H0 (84 g N/day), although it lays within the range given in the literature (from approximately −60 to 130 g N/day) and decreased as N intake decreased (Spanghero & Kowalski, 2021). At similar dietary CP concentrations, partially replacing PMR with fresh herbage changed the composition of cow manure, increasing urinary N and decreasing fecal N in excreted N. Urine has a high concentration of urea, which is a precursor of NH₃, with ~44% of urea N in urinary N observed in the present study (in agreement with Edouard et al., 2019, 2016, for similar dietary CP concentrations). Thus, feeding fresh herbage indoors could increase NH₃ emissions in the barn (Almeida et al., 2022). Conversely, when cows graze herbage during its growing periods, the urinary N is rapidly absorbed by grassland, which provides N fertilization and promotes direct N recycling with low NH₃ volatilization (<4% of the N) (Barré, 2001; Leterme et al., 2003).

This study therefore showed that the gradual replacement of PMR with fresh herbage decreased N losses and changed the composition of cow manure, increasing the percentage of urinary N in excreted N. This study highlights that partially replacing PMR with fresh herbage with slight changes in dietary N concentration increases N use efficiency but may shift N excretion toward urinary losses.

ACKNOWLEDGMENTS

This study was performed as part of a Ph.D. thesis (M. F.) supported by the French National Research Institute for Agriculture, Food and Environment (INRAE – PHASE division) and the Brittany Region. The experiment was conducted as a part of the EMIGRAZE project, supported by the French Environment and Energy Management Agency (ADEME). The authors are grateful to the staff of the INRAE PEGASE unit, at both the experimental dairy farm and the laboratory, who helped perform this study.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interests for this article.

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Volume95, Issue1

January/December 2024

e13965

This article also appears in:

Nutrition and feeding management of dairy cattle

Effects of replacing corn silage and soybean meal with an increasing percentage of fresh herbage on dairy cow nitrogen use efficiency and flows

Abstract

1 INTRODUCTION

2 MATERIALS AND METHODS

2.1 Treatments, experimental design, and cows

2.2 Housing and feeding management

2.3 Feed characteristics and intake calculations

2.4 Milk yield and composition

2.5 Feces and urine excretions, sampling, and digestibility calculations

2.6 Ruminal fermentation and plasma metabolites

2.7 Chemical analyses

2.8 Nitrogen use efficiency and balance calculations

2.9 Statistical analysis

3 RESULTS

3.1 Intake, feed composition, diet composition, and digestibility

3.2 Milk yield and composition

3.3 Nitrogen use efficiency and N partitioning

3.4 Nitrogen metabolism components

4 DISCUSSION

4.1 Intake, diet digestibility, and consequences for milk yield

4.2 Nitrogen use efficiency

4.3 Nitrogen partitioning

ACKNOWLEDGMENTS

CONFLICT OF INTEREST STATEMENT

REFERENCES

References

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Effects of replacing corn silage and soybean meal with an increasing percentage of fresh herbage on dairy cow nitrogen use efficiency and flows

Abstract

1 INTRODUCTION

2 MATERIALS AND METHODS

2.1 Treatments, experimental design, and cows

2.2 Housing and feeding management

2.3 Feed characteristics and intake calculations

2.4 Milk yield and composition

2.5 Feces and urine excretions, sampling, and digestibility calculations

2.6 Ruminal fermentation and plasma metabolites

2.7 Chemical analyses

2.8 Nitrogen use efficiency and balance calculations

2.9 Statistical analysis

3 RESULTS

3.1 Intake, feed composition, diet composition, and digestibility

3.2 Milk yield and composition

3.3 Nitrogen use efficiency and N partitioning

3.4 Nitrogen metabolism components

4 DISCUSSION

4.1 Intake, diet digestibility, and consequences for milk yield

4.2 Nitrogen use efficiency

4.3 Nitrogen partitioning

ACKNOWLEDGMENTS

CONFLICT OF INTEREST STATEMENT

REFERENCES

References

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