Animal Feed Science and Technology 293 (2022) 115454

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Energy utilization and milk fat responses to rapeseed oil when fed
to lactating dairy cows receiving different dietary forage to
concentrate ratio
A. Razzaghi a, b, H. Leskinen a, S. Ahvenjärvi a, H. Aro c, A.R. Bayat a, *
a
Animal Nutrition, Production Systems, Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland
b
Innovation Center, Ferdowsi University of Mashhad, 9177948974 Mashhad, Iran
c
HKScan Corporation, PO Box 50, Lemminkäisenkatu 48, FI-20521 Turku, Finland

A R T I C L E I N F O A B S T R A C T

Keywords: We evaluated energy and N utilization, performance, and milk fatty acid (FA) profile using grass
Energy balance silage-based diets when rapeseed oil (RO) was included in high- or low-forage diets. Four
Forage to concentrate ratio multiparous Nordic Red cows averaging 101 ± 16 days in milk at the beginning of the study were
Milk fatty acid profile
randomly assigned to a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treat­
Rapeseed oil
ments. Each 21-d period consisted of a 14-d diet adaptation period and 7-d collection period.
Cows were fed the following diets comprised total mixed rations based on grass silage with forage
to concentrate (FC) ratio of 35:65 and 65:35 containing 0 or 50 g/kg of RO. Significant FC × RO
interactions were observed for milk yield, milk protein and lactose yields, milk fat concentration,
and milk proportions of trans-11 18:1, trans-10 18:1, trans-10, cis-12 18:2, and saturated FA.
Feeding low-forage diet was effective in increasing milk yield compared with the high-forage diet,
and the RO supplementation increased it further (P ≤ 0.01). A similar pattern was observed for
the yields of milk protein and lactose. Supplementing the low-forage diet with RO reduced milk
fat concentration by 19% relative to other diets without affecting milk fat yield. The proportion of
N intake lost as urine decreased (P ≤ 0.05) with the RO supplementation of low-forage diet
without affecting energy and N balances. Nutrient intakes were greater (P ≤ 0.01) in cows fed
low-forage diet, whereas RO decreased (P < 0.05) protein, starch, and fiber intakes. Methane
production, expressed as a proportion of energy intake, decreased with low-forage compared with
high-forage diets and this variable declined similarly by RO supplementation of both diets (P <
0.01). The milk proportions of trans-10 18:1 and trans-10, cis-12 CLA increased (P ≤ 0.01) by RO
supplementation of the low-forage but not high-forage diet. However, RO supplementation of
both high- and low-forage diets increased (P < 0.01) total trans FA and decreased saturated FA
proportions, even though the changes were more profound in low-forage diet (P ≤ 0.01). In
addition, RO increased (P < 0.01) cis monounsaturated FA in milk for both high- and low-forage
diets. Overall, the low-forage diets had lower methane emissions and RO increased partitioning of

Abbreviations: BH, biohydrogenation; BW, body weight; CLA, conjugated linoleic acids; CP, crude protein; DE, digestible energy; DM, dry matter;
DMI, DM intake; ECM, energy-corrected milk; FA, fatty acid; FAME, fatty acid methyl esters; FC ratio, forage-to-concentrate ratio; HF, high-forage
dietiNDF indigestible neutral detergent fiber; LF, low-forage diet; ME, metabolizable energy; MFD, milk fat depression; NDF, neutral detergent fiber
expressed exclusive of residual ash; NFC, non-fibre carbohydrates; NRC, National Research Council; SD, standard deviation; TMR, total mixed
ration; UFA, unsaturated fatty acid; VFA, volatile fatty acid.
* Corresponding author.
E-mail address: alireza.bayat@luke.fi (A.R. Bayat).

https://doi.org/10.1016/j.anifeedsci.2022.115454
Received 7 January 2022; Received in revised form 12 September 2022; Accepted 23 September 2022
Available online 27 September 2022
0377-8401/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
A. Razzaghi et al. Animal Feed Science and Technology 293 (2022) 115454

N towards milk secretion (P ≤ 0.01) without influencing energy or N balances. According to the
results, RO supplementation did not compromise intake of nutrients with low-forage diets con­
taining 150 g/kg starch, and oil could be preferentially used to improve milk production and milk
fat quality accompanied by a reduction in methane energy loss.

1. Introduction

In dairy cow rations, modifying forage to concentrate (FC) ratio or supplementation with unsaturated fat affect energy and nitrogen
(N) utilization and potentially mitigates enteric methane emissions from ruminants (Sterk et al., 2011; Nichols et al., 2019; Morris
et al., 2020). Diet digestibility and energy utilization are partly related to dietary NDF concentration and NDF digestibility from forage
sources (Huhtanen et al., 2009). Also, forage source, and dietary lipid and starch contents are known to affect energy and nitrogen (N)
utilization in lactating dairy cows (Sterk et al., 2011; Nichols et al., 2019; Morris et al., 2020). Increasing dietary starch content rises
energy supply in the rumen, which supports microbial growth and rumen outflow of microbial protein (Roman-Garcia et al., 2016;
White et al., 2016) whereas lipids are modified extensively in the rumen and utilized mainly as post-absorptive energy sources, which
may be directly transferred to milk fat (Harvatine et al., 2009; Boerman et al., 2015). Full-fat rapeseed and rapeseed oil, containing the
greatest content of unsaturated fatty acid (UFA) especially oleic acid (cis-9 18:1) and linoleic acid (cis-9, cis-12 18:2), have been
investigated for their effects on lactation performance (Kliem et al., 2019), fatty acid (FA) profile (Welter et al., 2016), and enteric
methane emissions (Beauchemin et al., 2009) in dairy cows.
A high proportion of concentrate (Piperova et al., 2002), or a combination of dietary high proportion of concentrate and UFA
(Griinari et al., 1998) have been outlined as dietary factors inducing milk fat depression (MFD; Bauman and Griinari, 2003). The lower
milk fat concentration and yield often reduce energy demand for milk production in cows resulting in enhanced adipose de novo FA
synthesis, which accounts for, at least in part, the increased tissue energy during MFD (Moore et al., 2004; Harvatine et al., 2009).
Dietary FC ratio and plant oil supplements affect rumen fermentation pathways, resulting in different volatile fatty acid profile and
thereby methane production (Beauchemin et al., 2020), which may alter energy or N partitioning (Aguerre et al., 2011; Bayat et al.,
2017). With respect to differential effects of energy sources on microbial protein synthesis, production of fermentation acids in the
rumen, and post-absorptive metabolism, they are expected to affect both milk production and components differently (Nichols et al.,
2019; Morris et al., 2020). Furthermore, increasing energy supply from lipids may increase the efficiency at which ME is converted into
milk energy through the direct transfer of dietary FA into milk (Hammon et al., 2008).
Energy from lipids and forage or starch may interact to affect N and energy partitioning and interactions likely exist between FC
ratio and lipid supplement, but these interactions are not entirely understood. The main objective of the present study was to quantify
the effects of high- and low-forage diets with or without rapeseed (Brassica napus) oil (RO) supplementation (i.e., providing simul­
taneous glucogenic and lipogenic nutrients) on milk production, energy and N metabolism, efficiency of energy utilization, and milk
fat biohydrogenation (BH) intermediates of Nordic Red cows in mid-lactation. We hypothesized that supplement of RO would increase
the energy utilization for milk production, especially when added to a low-forage diet, in addition to altering milk FA profile. Besides,
we hypothesized that lower dietary FC ratio would have positive effects on N metabolism by dietary N use efficiency.

2. Materials and methods

This experiment was conducted at Luke’s research barn, Jokioinen, Finland in 2019. Animal care and all experimental procedures
used in this study were approved by the National Ethics Committee (ESAVI/24435/2018, Hämeenlinna, Finland) in accordance with
the guidelines established by the European Community Council Directive 2010/63/EU (EU 2010) for animal experiments and com­
plied with the ARRIVE guidelines.

Table 1
Chemical composition of feed ingredients.
Item Grass silageA Concentrate (High forage diet) Concentrate (Low forage diet) Rapeseed oil

Dry matter, g/kg 266 877 875 1000

Organic matter, g/kg DM 942 914 931 1000
Crude protein, g/kg DM 132 191 195 0
Neutral detergent fiber, g/kg DM 549 271 276 0
Ether extract, g/kg DM 33.7 35.1 37.0 824.1
Starch, g/kg DM 13.8 219 232 0
Water soluble carbohydrates, g/kg DM 32.6 66.4 66.7 0
Gross energy, MJ/kg DM 17.9 18.2 18.7 39.0
A
Mean fermentation characteristics of experimental silage: pH, 4.09; in DM (g/kg) lactic acid, 65.4; acetic acid,18.0; propionic acid, 0.25; butyric
acid, 1.03; soluble N (g/kg of total N), 597; ammonium N (g/kg of total N), 71.7.

2
A. Razzaghi et al. Animal Feed Science and Technology 293 (2022) 115454

2.1. Cows, experimental design, and treatments

Four multiparous Nordic Red dairy cows were assigned randomly to treatment sequences in a 4 × 4 Latin square design with a 2 × 2
factorial arrangement of treatments. Cows had (mean ± SD) 632 ± 46 kg body weight, 3.5 ± 0.58 parity, and 101 ± 16 days in milk
with milk yield of 38.8 ± 1.9 kg/d at the beginning of the experiment. Each experimental period lasted for 21 d consisting of 14 d for
adaptation and 7 d for sample and data collection. Treatments comprised diets based on grass silage (FC ratio 65:35 or 35:65 on a DM
basis) containing either no added oil [high-forage diet (HF) and low-forage diet (LF)] or 50 g/kg diet DM of RO [high-forage diet plus
oil (HFO) and low-forage diet plus oil (LFO); Tables 1 and 2]. The semi-purified rapeseed oil (AvenaKantvik Ltd, Kirkkonummi,
Finland) was stored in 4◦C until incorporated into the low or high FC ratio diets and RO replaced concentrate ingredients. Treatments
were supplemented with 50 g/kg DM of a UFA source (rapeseed oil) based on our previous experiments (Bayat et al., 2017, 2018) to
induce maximum effects on milk FA and enteric methane production without compromising the DMI of the cows. The appropriate
amount of rapeseed oil for each cow was weighed daily and mixed thoroughly with the TMR. The forage was restrictively fermented
grass silage prepared from primary growth of mixed timothy (Phleumpratense) and meadow fescue (Festuca pratensis) swards, grown at
Jokioinen (60◦ 49’N, 23◦ 28’E) and treated at harvest with a formic acid-based ensiling additive (5 L/ton, AIV 2 Plus, Eastman/Taminco
Finland Oy, Finland). Before ensiling in the bunker silo, the grass was wilted on the field to reach about 30% DM. Experimental diets
were offered ad libitum to result in 10% refusals and formulated to meet requirements for ME and protein of lactating cows producing
40 kg milk/d (Finnish Feed Evaluation System; Luke, 2021). Diets contained grass silage (as the only forage source), rapeseed meal,
rolled barley, ground oats, molassed sugar beet pulp, and a premix of minerals and vitamins. Chemical composition of grass silage,
supplemental concentrates and rapeseed oil is presented in Table 1. The grass silage had a relatively high quality as indicated by its
fermentation characteristics and chemical composition. The HF and LF concentrate pellets used to make dietary treatments with 65:35
and 35:65 FC ratios (on DM basis), respectively had similar formulation except vitamin and mineral premix which for LF was half of
that in HF concentrate. This resulted in differences in diet energy and N contents due to differences in FC ratio between HF and LF
groups (Table 2). The diets were prepared as TMR to avoid selection of dietary components (i.e., silage and concentrate pellets) and
maintain the desired FC ratio, and offered daily at 0600, 0900, 1600, and 1900 h. Cows were housed in free stalls in the barn during
adaptation period and in the respiration chambers during the sampling period with free access to water and salt block and were milked
at 0700 and 1645 h in a 2 × 6 auto tandem parlor milking system.

2.2. Measurements and chemical analysis

Daily feed intake and milk yield were recorded throughout the experiment, but only measurements made between day 15 and 21 of
each experimental period were used for statistical analysis. During this period, representative samples of silage and concentrates were
collected daily, composited, and stored frozen (− 20 ◦ C) until submitted for chemical composition [dry matter (DM), ash, crude protein
(CP), neutral detergent fiber (using sodium-sulfite and heat-stable α-amylase and corrected for ash, NDF), ether extract (EE), starch,

Table 2
Formulation and chemical composition of experimental total mixed ration.
TreatmentsA

Item HF HFO LF LFO

Feed ingredients, g/kg DM
Grass silage 650 650 350 350
Rolled barley 78.8 67.5 150 138
Ground oats 78.8 67.5 150 138
Molassed sugar beet pulp 78.8 67.5 149 137
Rapeseed meal 101 86.4 190 176
Rapeseed oilB – 50 – 50
Vitamin and mineral premixC 13.0 11.1 11.7 10.8
Chemical composition, g/kg DM unless stated
Dry matter, g/kg as-fed 479 486 662 668
Organic matter 932 936 935 938
Crude protein 153 143 173 163
Neutral detergent fiber 452 438 372 358
Forage neutral detergent fiber 357 357 192 192
Potentially degradable NDFD 360 350 286 276
Ether extract 34.2 73.6 35.9 75.2
Starch 86.0 75.0 155 144
Water soluble carbohydrates 44.0 41.0 55.0 51.0
Gross energy, MJ/kg DM 18.0 19.1 18.4 19.4
A
Refers to diets with forage to concentrate (FC) ratio 65:35 with no additional lipid (HF); FC ratio 65:35 containing 50 g/kg diet DM of rapeseed oil
(HFO); FC ratio 35:65 with no additional lipid (LF); and FC ratio 35:65 containing 50 g/kg diet DM of rapeseed oil (LFO).
B
Rapeseed oil containing (g/100 g total fatty acids) 16:0 (4.06), 18:0 (1.85), cis-9–18:1 (58.4), cis-11–18:1 (3.38) and cis-9, cis-12–18:2 (19.5) as
major components and gross energy content 41.4 MJ/kg of DM.
C
Premix (Mahti-Mira, Vilomox Finland, Paimio) declared as containing (g/kg) Ca (220), Na (105), and Mg (64) and (mg/kg) vitamins A (156), D (52),
E (1144), CuSO4 (1180), MnSO4 (1025), ZnO (2127), Ca (IO3)2 (66), Na2SeO3 (33), Se (37), and CoCO3 (520).
D
Potentially degradable NDF.

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A. Razzaghi et al. Animal Feed Science and Technology 293 (2022) 115454

and water-soluble carbohydrates] determinations as described by Ahvenjärvi et al. (2018). Gross energy (GE) in samples of silage,
concentrates, oil supplement, and excreta was determined by bomb calorimetry (1108 Oxygen bomb, Parr Instrument, Illinois).
Indigestible NDF (iNDF) of silage, concentrate, and feces was determined by day 12 of ruminal incubation using nylon bags (60 × 120
mm, pore size 0.017 mm) followed by NDF analysis. Two fistulated dairy cows in mid-lactation fed grass silage-based diets (55:45 FC
ratio on DM basis) were used for the iNDF determination. Dry matter content of silage was corrected for the loss of volatiles according
to Huida et al. (1986).
Four open-circuit respiratory chambers (21.5 m3) were used to measure gas exchanges (oxygen, carbon dioxide, and methane) of
the cows individually. The measuring system is described in detail elsewhere (Bayat et al., 2022). Briefly, concentrations of the gases in
the inlet and exhaust airflow were measured by a computer-controlled system using dedicated analyzers (Oxymax, Columbus In­
struments, Columbus, OH). Air outflow for each chamber was measured by HFM-200 mass flow meter with a laminar flow element
capable of measuring up to 3000 L/min (Teledyne Hastings Instruments, Hampton, VA). Absolute gas exchanges were calculated by
multiplying air flow and gas concentration differences under standard temperature and pressure conditions. Gas recovery tests were
conducted before the experiments by releasing 3 levels of carbon dioxide, nitrogen (for diluting oxygen), and methane mimicking
minimum, average and maximum gas exchanges by the cows based on our previous experiments. The recovery rates were 103.1 ± 5.1,
100.1 ± 0.04, and 100.7 ± 1.95% for carbon dioxide, oxygen, and methane, respectively.
Total-tract apparent digestibility coefficients were determined by total fecal collection over a 72-h interval starting at 1000 h on
d 17 of each experimental period. Excreted feces were weighed, thoroughly mixed, subsampled (5%, wt/wt), and stored at − 20 ◦ C
before chemical analysis. Urine was separated from feces by means of a lightweight harness and flexible tubing attached to the vulva,
and collected in plastic canisters containing 500 mL of 5 M sulfuric acid. Collection vessels were changed at 12-h intervals and daily
samples (5%, wt/wt) were taken and stored at − 20 ◦ C. Milk samples were taken for 3 consecutive days from d 17 (evening) to 20
(morning) of each period, treated with a preservative (Bronopol, Valio Ltd., Helsinki, Finland) and stored at 4 ◦ C for analysis of milk
fat, crude protein, and lactose by infrared analysis (MilkoScan 133B, Foss Electric, Hillerød, Denmark). Daily milk composition was
calculated according to morning and evening milk yields. For analysis of milk FA composition using GC, milk was sampled during d 18
and 19 (evening and morning, respectively), pooled according to milk yield, and stored at − 20 ◦ C without preservative.

2.3. Lipid analysis

Fatty acid methyl esters of lipid in feed and milk samples were prepared as described by Shingfield et al. (2003). Briefly, fat in 1 mL
of milk was extracted twice with a mixture of ammonia, ethanol, diethyl ether, and hexane (0.2:1:2.5:2.5, vol/vol). Fatty acids were
then methylated with methanolic sodium methoxide in the presence of methyl acetate. Total FAME profile was determined using a GC
(6890 N, Agilent Technologies, Santa Clara, CA) equipped with a CP-Sil 88 column (100 m × 0.25 mm i.d., 0.2 µm film thickness,
Agilent Technologies) and flame ionization detector with a temperature gradient program (Shingfield et al., 2003). Identification was
based on retention time comparisons with authentic FAME standards (Larodan Fine Chemicals AB, Malmö, Sweden; Nu-Chek Prep Inc.,
Elysian, MN; Sigma-Aldrich) and previous milk samples verified by GC-MS (6890 and 5973, Agilent Technologies) and silver ion HPLC
(Shingfield et al., 2003). Hydrogen was used as a carrier gas starting from 206.8 kPa initial pressure and gradually raised after 50 min
to a final pressure of 310.3 kPa.

2.4. Calculations and statistical analysis

Potentially digestible NDF was calculated as NDF – iNDF. Energy intake and energy excretion in feces and urine were calculated by
multiplying DMI or feces and urine DM by their respective GE contents. Energy-corrected milk was calculated as ECM = milk (kg/d) ×
[38.3 × fat (g/kg) + 24.2 × crude protein (g/kg) + 16.54 × lactose (g/kg) + 20.7]/3140; Sjaunja et al. (1990)). Energy secretion in
milk was calculated by multiplying ECM by 3.14. Heat production of the cows was calculated based on the exchanged oxygen, carbon
dioxide, and methane and urinary N excretion measured in the respiratory chambers using the following equation: Heat Production
(MJ/d) = 16.18 × O2 + 5.16 × CO2 – 5.90 × urinary N – 2.42 × CH4 where the gases are expressed in liters per day and urinary N is
expressed in gram per day (Brouwer, 1965). Methane energy was calculated using the conversion factor of 55.24 kJ for every gram of
methane (Kriss et al., 1930). Energy balance was calculated as the difference between energy intake and energy excretion as feces,
urine, methane, milk, and heat. Daily ME intake was calculated as the difference between GE intake and energy excretions as feces,
urine, and methane. Nitrogen balance was calculated as the difference between N intake and excretion in feces, urine, and milk.
Before statistical analysis, all data were tested for normality of distribution using Proc Mixed (version 9.4, SAS Institute Inc., Cary,
NC). The DM intake and the yield of milk and milk components were averaged before statistical analysis. Measurements of intake,
energy and N utilization, milk production, and milk FA composition were analyzed by ANOVA for a 4 × 4 Latin square design with a 2
× 2 factorial arrangement of treatments, with a statistical model that included the fixed effects of period, FC ratio, RO supplementation
and their interaction, and the random effect of cow using the PROC MIXED procedure of SAS. Least-square means with their standard
errors are reported and the effects of FC ratio, RO, and their interaction were declared significant at P ≤ 0.05. Probabilities at 0.05 ≤ P
≤ 0.10 were considered as a trend. The means were compared together using Tukey test when the interaction was significant.

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A. Razzaghi et al. Animal Feed Science and Technology 293 (2022) 115454

3. Results

3.1. Nutrient composition of diets

The ingredient and chemical composition of the experimental diets are reported in Table 2. Experimental diets contained different
concentrations of CP (148 vs. 168 g/kg DM) and GE(18.5 vs. 18.9 MJ/kg DM) for HF than LF diets, respectively. As expected, the EE
concentration was greater (74.4 vs. 35.0 g/kg DM) for diets supplemented with RO than those without RO, and the starch concen­
tration was greater for LF than HF diets (149 vs. 81 g/kg DM basis); as a consequence, fiber fractions exhibited the opposite trend due
to changes in barley and oats proportions. The variation in dietary FA was mostly driven by the replacement of barley and oats with RO
and altering dietary FC ratio. Silage had high quality, in terms of nutritive value and fermentation characteristics.

3.2. Nutrient intakes and lactation performance

Dry matter intake increased by 8% (P < 0.05) for LF than HF diets (Table 3). Intakes of OM, CP, and starch were greater (P ≤ 0.01),
and that of NDF and pdNDF were lower (P < 0.01) for LF than HF diets. Supplementing RO tended to decrease (P = 0.09) DMI for HF
diet. In addition, RO led to lower (P < 0.05) intakes of CP, starch, and NDF. Further, EE intake increased (P < 0.01) by supplementing
RO whereas GE intake was not affected. Dietary FA intakes were significantly affected by the interaction between RO and FC ratio (P ≤
0.05); in particular, intake of cis-9 18:1, cis-9, cis-12 18:2, and cis-9, cis-12, cis-15 18:3 increased in LFO. Also, intake of total FA was
greater (P < 0.01) with RO-supplemented diets compared with the diets without the supplement, specifically, cows fed LFO diet had
greatest daily FA intake than the rest of the cows (P < 0.05 for interaction of FC and RO).
The LF diet increased yields of milk, milk protein, and lactose compared with HF, and RO increased them only when supplemented
to LF diet (P < 0.05 for FC × RO interaction). In addition, milk fat concentration decreased in LFO diet (P = 0.026 for FC × RO
interaction). The yields of ECM (P < 0.01) and milk fat (P < 0.05), and concentration of milk protein (P < 0.05) were greater for LF
than HF diets (Table 4). Feed efficiency, calculated as kg of milk per kg of DMI, improved (P < 0.01) by feeding LF diet to dairy cows
compared with HF and by supplementing RO, similar pattern was observed (P = 0.017). However, treatments had no effect (P ≥ 0.12)
on the feed efficiency calculated as ECM/DMI.

Table 3
Effect of dietary forage to concentrate ratio and rapeseed oil on nutrient and fatty acids intake of lactating cows fed grass silage-based diets.
TreatmentsA P-valueB

Item HF HFO LF LFO SEM FC RO FC × RO

Intake, kg/d
Dry matter 24.7 22.3 25.8 25.6 0.83 0.015 0.091 0.13
Organic matter 23.1 20.8 24.1 24.1 0.78 0.014 0.11 0.13
Crude protein 3.76 3.20 4.43 4.17 0.120 < 0.01 < 0.01 0.18
Neutral detergent fiberC 11.3 9.83 9.73 9.31 0.389 0.013 0.020 0.13
Forage NDF 9.01 8.05 5.22 5.17 0.310 < 0.01 0.084 0.11
pdNDFD 8.99 7.86 7.50 7.20 0.312 < 0.01 0.023 0.13
Ether extract 0.85c 1.56b 0.92c 1.87a 0.048 < 0.01 < 0.01 0.022
Starch 2.06 1.64 3.91 3.60 0.082 < 0.01 < 0.01 0.50
Gross energy, MJ/d 447 423 475 497 15.8 < 0.01 0.93 0.12
Fatty acid, g/d
14:0 2.59b 2.81ab 2.32c 2.90a 0.101 0.28 < 0.01 0.052
16:0 104c 127b 121b 160a 4.9 < 0.01 < 0.01 0.052
18:0 8.17c 24.8b 9.38c 30.2a 0.81 < 0.01 < 0.01 0.010
cis-9 18:1 87.8d 631b 155c 813a 20.6 < 0.01 < 0.01 < 0.01
cis-11 18:1 14.4d 43.6b 25.5c 61.5a 1.43 < 0.01 < 0.01 0.014
cis-9, cis-12 18:2 171d 321b 252c 448a 11.7 < 0.01 < 0.01 0.035
cis-9, cis-12, cis-15 18:3 164c 231b 109d 209a 7.9 < 0.01 < 0.01 0.024
20:0 5.27b 10.0a 3.87c 10.2a 0.32 0.055 < 0.01 0.031
cis-11 20:1 2.19d 11.9b 3.60c 15.5a 0.39 < 0.01 < 0.01 0.010
Σ SFA 151c 198b 159c 233a 7.2 0.010 < 0.01 0.036
Σ MUFA 123d 716b 203c 925a 23.3 < 0.01 < 0.01 0.010
Σ PUFA 338c 564b 365c 675a 19.7 < 0.01 < 0.01 0.023
Σ Fatty acids 615c 1484b 728.6c 1841a 49.7 < 0.01 < 0.01 0.013
A
Refers to diets with forage to concentrate (FC) ratio 65:35 with no additional oil (HF); FC ratio 65:35 containing 50 g/kg diet DM of rapeseed oil
(HFO); FC ratio 35:65 with no additional oil (LF); and FC ratio 35:65 containing 50 g/kg diet DM of rapeseed oil (LFO). Values are LS means and
pooled SEM for n = 4.
B
FC, effect of forage to concentrate ratio in the diet; RO, effect of rapeseed oil supplement; FC × RO, interaction of forage to concentrate ratio and
rapeseed oil supplement.
C
aNDFom
D
Potentially digestible NDF.

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A. Razzaghi et al. Animal Feed Science and Technology 293 (2022) 115454

Table 4
Effect of dietary forage to concentrate ratio and rapeseed oil on milk yield and milk composition of lactating cows fed grass silage-based diets.
TreatmentsA P-valueB

Item HF HFO LF LFO SEM FC RO FC × RO

Yield, kg/d
Milk 31.2c 30.5c 36.2b 40.6a 1.47 < 0.01 0.038 0.011
ECMC 33.3 32.9 39.4 39.4 1.95 < 0.01 0.82 0.82
Fat 1.43 1.40 1.64 1.49 0.095 0.028 0.24 0.22
Protein 1.12c 1.08c 1.37b 1.46a 0.070 < 0.01 0.26 0.020
Lactose 1.40c 1.37c 1.63b 1.85a 0.080 < 0.01 0.040 0.014
Concentration, g/kg
Fat 44.9a 45.8a 45.2a 36.6b 1.79 0.032 0.054 0.026
Protein 36.2 35.6 37.8 36.1 1.64 0.036 0.020 0.20
Lactose 45.0 45.0 45.1 45.5 4.3 0.13 0.090 0.16
Urea, mg/dL 23.9 19.2 30.3 22.7 1.97 < 0.01 < 0.01 0.22
Feed efficiency
Milk/DMI 1.26 1.38 1.41 1.58 0.045 < 0.01 0.017 0.56
ECM/DMI 1.35 1.48 1.53 1.53 0.067 0.12 0.33 0.35
A
Refers to diets with forage to concentrate (FC) ratio 65:35 with no additional lipid (HF); FC ratio 65:35 containing 50 g/kg diet DM of rapeseed oil
(HFO); FC ratio 35:65 with no additional lipid (LF); and FC ratio 35:65 containing 50 g/kg diet DM of rapeseed oil (LFO). Values are LS means and
pooled SEM for n = 4.
B
FC, effect of forage to concentrate ratio in the diet; RO, effect of rapeseed oil supplement; FC × RO, interaction of forage to concentrate ratio and
rapeseed oil supplement.
C
Energy-corrected milk calculated as described by Sjaunja et al. (1990).

3.3. Energy and nitrogen metabolism

The effects of diets on variables related to energy utilization and excretion are reported in Table 5. Dietary treatments had no effect
(P ≥ 0.22) on milk energy secretion as a proportion of either GE or ME intake, urine energy excretion as a proportion of GE intake, heat
production, and energy balance. Feeding LF diets increased (P < 0.01) GE, DE and ME intakes and energy metabolizability (ME to GE
intake) while it decreased feces (P < 0.05) and methane (P < 0.01) energy excretion as a proportion of GE intake compared with HF
diets. Further, supplementing diets with RO caused a decrease (P < 0.01) in methane energy excretion as a proportion of GE intake
compared with un-supplemented diets. Daily methane emission was lowered by RO supplementation and the response was stronger
with HF diet (P < 0.05 for the interaction of FC and RO). Methane yield was lowered (P < 0.01) by both greater concentrate ratio and
dietary RO supplement while methane intensity tended to be reduced by RO more profoundly when supplemented to HF than LF diet

Table 5
Effect of dietary forage to concentrate ratio and rapeseed oil on energy and N metabolism, methane and carbon dioxide emissions, and oxygen
consumption of lactating cows fed grass silage-based diets.
TreatmentsA P-valueB
C
Item HF HFO LF LFO SEM FC RO FC × RO
Energy
Gross energy intake, MJ/d 447 423 475 497 15.8 < 0.01 0.93 0.12
DE intake, MJ/d 288 265 316 326 11.3 0.003 0.49 0.13
ME intake, MJ/d 244 227 272 285 9.7 0.003 0.83 0.14
Proportion of energy intake, kJ/MJ
Feces 354 374 335 344 8.5 0.016 0.12 0.54
Urine 32.0 32.7 32.0 29.7 1.60 0.25 0.31 0.24
Methane 66.1 56.0 60.0 52.2 2.40 < 0.01 < 0.01 0.26
Milk 234 245 261 249 11.4 0.22 0.94 0.34
Heat 294 296 299 285 9.3 0.56 0.29 0.17
Milk energy/ME intake 429 456 457 433 22.5 0.92 0.93 0.29
ME intake/GE intake 547 537 573 574 7.8 < 0.01 0.61 0.47
Energy balance, MJ/d 8.24 -1.40 6.63 19.9 10.31 0.32 0.85 0.25
Methane
Emission, g/d 535a 428c 516a 470b 27.7 0.25 < 0.01 0.015
Yield, g/kg DMI 21.6 19.2 20.0 18.3 0.81 < 0.01 < 0.01 0.33
Intensity, g/kg ECM 16.0 13.0 13.2 12.0 0.64 < 0.01 < 0.01 0.087
O2 consumption, g/d 8612 8291 9258 9320 467 < 0.01 0.29 0.14
CO2 production, g/d 14,374 13,133 15,628 15,203 711 < 0.01 < 0.01 0.11
A
Refers to diets with forage to concentrate (FC) ratio 65:35 with no additional lipid (HF); FC ratio 65:35 containing 50 g/kg diet DM of rapeseed oil
(HFO); FC ratio 35:65 with no additional lipid (LF); and FC ratio 35:65 containing 50 g/kg diet DM of rapeseed oil (LFO). Values are LS means and
pooled SEM for n = 4.
B
FC, effect of forage to concentrate ratio in the diet; RO, effect of rapeseed oil supplement; FC × RO, interaction of forage to concentrate ratio and
rapeseed oil supplement.
C
DE, digestible energy; ME, metabolizable energy; GE, gross energy.

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A. Razzaghi et al. Animal Feed Science and Technology 293 (2022) 115454

(P = 0.09 for the interaction of FC and RO). Both oxygen consumption and carbon dioxide production we lower (P < 0.01) with LF
compared with HF diets and RO reduced (P < 0.01) carbon dioxide production but not oxygen consumption.
Supplement of RO resulted in decreased urine N as a proportion of N intake when included in LF, but not HF diet (P = 0.05 for FC ×
RO interaction; Table 6). Dietary treatments had no effect on N balance while feeding greater amount of concentrates increased (P <
0.01) N intake but decreased (P < 0.05) fecal N as a proportion of N intake compared with feeding lower amount of concentrates.
However, supplementing diets with RO decreased (P = 0.01) N intake and increased (P < 0.05) N secreted in milk as a proportion of N
intake.

3.4. Milk fatty acid composition

The effects of dietary treatments on milk FA profile are reported in Table 7. The RO supplement resulted in greater decreases in 8:0,
10:0, and 12:0 concentrations in milk fat when included in LF than HF diets (P < 0.01 for FC × RO interaction). A tendency was found
for the interaction (P < 0.10) between inclusion of RO and FC ratio on 4:0, 14:0 and trans-9 14:1 concentrations in milk fat; in
particular, 4:0 and trans-9 14:1 decreased in HFO diet. Rapeseed oil supplementation increased the milk proportion of 18:0 in both LF
and HF diets with more pronounced increase in HFO diet (P = 0.07 for FC × RO interaction). Dietary inclusion of RO increased the
concentration of trans-11 18:1 more in cows fed HF diets, than those fed LF diets (P < 0.05 for FC × RO interaction). Furthermore, RO
increased concentrations of trans-10 18:1 and trans-10, cis-12 18:2 in milk fat when included in LF diet but did not change the pro­
portion of these FA in milk fat when included in HF diet (P ≤ 0.01 for FC × RO interaction). Trans-10 18:1 was the major 18:1 in­
termediate for LFO treatment, whereas trans-11 18:1 was the most abundant 18:1 intermediate for HFO diet. Milk fat trans 18:1, trans
18:2, and total trans-FA concentrations were greater (P < 0.01 for FC × RO interaction) with LFO whereas SFA proportion was lower (P
< 0.05 for FC × RO interaction). In addition, decreasing FC ratio increased (P < 0.01) n-6/n-3 ratio in milk fat. Rapeseed oil decreased
(P < 0.01) the concentrations of 16:0, cis 16:1, total 16:1, cis-9, cis-12 18:2, cis 18:2, and cis PUFA and increased (P < 0.01) the
proportions of trans-16:1, cis-9 18:1, cis-9, trans-11 CLA, total CLA, cis MUFA, and cis UFA in milk fat.

4. Discussion

Our aim was to evaluate the effect of typical Nordic grass silage-based diets differing in lipogenic or glucogenic nutrients and UFA
source on energy and N partitioning. To achieve this, we did not simply replace one ingredient for another but the whole concentrate
ingredients replaced for the grass silage (except vitamin and mineral premix) which resulted in differences in diet energy and N
contents due to the differences in forage and concentrate intakes. Two FC ratios (anisonitrogenic and anisoenergetic) were obtained by
replacing grass silage with grains and sugar beet pulp. Based on how the diets were formulated, we expected to cause a greater increase
in milk production, without compromising feed intake and milk components, when RO was supplemented to low- (moderate starch
concentration) compared with high-forage (low starch concentration) diets.

4.1. Nutrient intakes

In our study, the HF and LF diets differed mainly in their starch, NDF, and CP contents. Consequently, the feed intake difference
originated primarily from different composition of the carbohydrate fractions i.e., NDF content. The dietary NDF content was 445 and
365 g/kg on DM basis for HF and LF groups, corresponding to NDF intakes of 1.7% and 1.5% of BW, respectively. Multiple mechanisms
regulate DMI of ruminants, but DMI generally increases with reducing diet NDF, especially dietary forage NDF content (Allen, 2000).
The higher DMI of cows fed LF diets is consistent with the previous studies that evaluated alfalfa silage-based diets with 35:65 and
60:40 FC ratios (Yang and Beauchemin, 2007) or grass silage-based diets with 30:70 and 70:30 FC ratios (Saliba et al., 2014). Similarly,
Sterk et al. (2011) reported that shifting from a high-forage to a low-forage (65:35 vs. 35:65 FC ratio) diet increased DMI and milk yield
in dairy cows. This result could be related to dietary NDF concentration and longer retention time of forage particles restricting the
flow of digesta through the gastrointestinal tract (Allen, 2000). Moreover, cows fed LF diets had a greater dietary protein intake

Table 6
Effect of dietary forage to concentrate ratio and rapeseed oil on nitrogen metabolism of lactating cows fed grass silage-based diets.
TreatmentsA P-valueB

Item HF HFO LF LFO SEM FC RO FC × RO

Nitrogen intake, g/d 601 511 709 667 19.2 < 0.01 < 0.01 0.18
Proportion of nitrogen intake, g/kg
Milk 293 334 304 343 15.3 0.41 0.012 0.98
Feces 365 358 345 342 6.7 0.029 0.51 0.92
Urine 334ab 341ab 347a 322b 16.3 0.67 0.22 0.052
Nitrogen balance, g/d 5.19 -15.0 3.61 -3.66 9.148 0.61 0.18 0.50
A
Refers to diets with forage to concentrate (FC) ratio 65:35 with no additional lipid (HF); FC ratio 65:35 containing 50 g/kg diet DM of rapeseed oil
(HFO); FC ratio 35:65 with no additional lipid (LF); and FC ratio 35:65 containing 50 g/kg diet DM of rapeseed oil (LFO). Values are LS means and
pooled SEM for n = 4.
B
FC, effect of forage to concentrate ratio in the diet; RO, effect of rapeseed oil supplement; FC × RO, interaction of forage to concentrate ratio and
rapeseed oil supplement.

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A. Razzaghi et al. Animal Feed Science and Technology 293 (2022) 115454

Table 7
Effect of dietary forage to concentrate ratio and rapeseed oil on fatty acid profile in milk of lactating cows fed grass silage-based diets.
TreatmentsA P-valueB

FAC, g/100 g of total FA HF HFO LF LFO SEM FC RO FC × RO

4:0 4.03 3.82 3.81 3.24 0.157 < 0.01 0.005 0.083
8:0 1.40b 0.99c 1.73a 0.93d 0.038 < 0.01 < 0.01 < 0.01
10:0 3.04b 1.92d 4.19a 1.94c 0.086 < 0.01 < 0.01 < 0.01
12:0 3.40b 2.08c 4.85a 2.29c 0.098 < 0.01 < 0.01 < 0.01
14:0 12.4 9.1 13.2 9.2 0.17 0.017 < 0.01 0.061
trans-9 14:1 0.016 0.011 0.016 0.015 0.0020 0.057 0.017 0.078
cis-9 14:1 1.00b 0.61c 1.02a 0.88ab 0.080 < 0.01 < 0.01 0.017
16:0 34.1 20.3 30.0 18.0 0.92 0.010 < 0.01 0.34
Σ cis 16:1 1.99 1.46 1.94 1.49 0.118 0.79 < 0.01 0.36
Σ trans 16:1 0.115 0.218 0.121 0.264 0.0113 0.047 < 0.01 0.105
Σ 16:1 2.11 1.68 2.06 1.75 0.117 0.69 < 0.01 0.17
18:0 9.4 18.0 9.2 15.4 1.06 0.045 < 0.01 0.069
cis-9 18:1D 16.5 25.3 15.1 26.5 0.92 0.53 < 0.01 0.24
Σ cis 18:1 16.4 26.7 16.2 28.2 0.94 0.34 < 0.01 0.20
trans-11 18:1 0.646b 1.778a 0.607b 0.976b 0.1200 < 0.01 < 0.01 0.014
trans-10 18:1 0.189b 0.513b 0.290b 3.249a 0.1594 < 0.01 < 0.01 < 0.01
Σ trans 18:1 1.83c 5.03b 2.03c 7.68a 0.232 < 0.01 < 0.01 < 0.01
Σ 18:1 18.3c 31.7b 18.2c 35.9a 0.88 0.016 < 0.01 0.015
cis-9, cis-12 18:2E 1.12 0.90 1.63 1.44 0.045 < 0.01 < 0.01 0.52
Σ cis 18:2 1.16 0.97 1.67 1.52 0.046 < 0.01 < 0.01 0.50
Σ trans 18:2 0.913c 1.648b 0.886c 2.015a 0.1076 0.063 < 0.01 0.038
Σ 18:2 2.07c 2.62b 2.56b 3.53a 0.145 < 0.01 < 0.01 0.045
cis-9, trans-11 18:2F 0.250 0.675 0.263 0.602 0.0623 0.59 < 0.01 0.45
trans-10, cis-12 18:2 0.001b 0.001b 0.002b 0.007a 0.0008 < 0.01 0.061 0.013
Σ CLA 0.338 0.748 0.352 0.707 0.0579 0.77 < 0.01 0.54
cis-9, cis-12, cis-15 18:3G 0.402a 0.295b 0.346ab 0.340ab 0.0255 0.80 0.031 0.049
20:0 0.147 0.286 0.125 0.227 0.0168 0.025 < 0.01 0.22
Σ cis 20:1 0.220 0.305 0.224 0.377 0.0239 0.13 < 0.01 0.16
Σ trans 20:1 0.021 0.068 0.027 0.083 0.0033 0.010 < 0.01 0.16
Σ 20:1 0.241 0.372 0.251 0.460 0.0259 0.073 < 0.01 0.13
Σ Unidentified 0.105 0.087 0.095 0.081 0.0119 0.41 0.14 0.83
Σ cis MUFA 20.6 29.7 20.4 31.6 1.05 0.24 < 0.01 0.15
Σ cis PUFA 1.97 1.56 2.51 2.16 0.060 < 0.01 < 0.01 0.42
Σ cis UFA 22.6 31.3 22.9 33.7 1.10 0.077 < 0.01 0.15
Σ SFA 74.2a 61.5b 73.6a 56.0c 1.12 < 0.01 < 0.01 0.013
Σ transFA 3.11c 7.13b 3.35c 10.2a 0.28 < 0.01 < 0.01 < 0.01
Σ trans FA + SFA 77.3 68.7 77.0 66.2 1.10 0.078 < 0.01 0.15
Σ OBCFA 4.14 3.18 4.16 3.10 0.131 0.76 < 0.01 0.65
Ratio
cis-9 14:1/(cis-9 14:1 +14:0) 0.075b 0.063c 0.072bc 0.086a 0.0068 0.019 0.78 < 0.01
cis-9 18:1/(cis-9 18:1 +18:0) 0.623a 0.584b 0.622a 0.635a 0.0238 0.004 0.050 < 0.01
n-6/n-3 FA 2.12 2.20 3.35 3.25 0.110 < 0.001 0.92 0.44
A
Refers to diets with forage to concentrate (FC) ratio 65:35 with no additional lipid (HF); FC ratio 65:35 containing 50 g/kg diet DM of rapeseed oil
(HFO); FC ratio 35:65 with no additional lipid (LF); and FC ratio 35:65 containing 50 g/kg diet DM of rapeseed oil (LFO). Values are LS means and
pooled SEM for n = 4.
B
FC, effect of forage to concentrate ratio in the diet; RO, effect of rapeseed oil supplement; FC × RO, interaction of forage to concentrate ratio and
rapeseed oil supplement.
C
FA, fatty acid; CLA, conjugated linoleic acid; OBCFA, odd- and branched-chain FA.
D
Co-elutes with trans-13 18:1 and trans-15 18:1.
E
Co-elutes with cis-9, cis-1518:2and cis-9 19:1
F
Co-elutes with an unidentified FA.
G
Co-elutes with trans-15 20:1 and trans-16 20:1.

(approximately 0.82 kg per day) than those fed HF diets and this difference can improve feed intake (M’Hamed et al., 2001).
We also observed a tendency for negative effect of RO supplementation on feed intake especially for cows fed HFO, even though
interaction effect failed to reach statistical significance. The effect of oils in various forms (oil, oilseed cake, or crushed oilseeds) on feed
intake of dairy cows has been generally negative (Glasser et al., 2008; Beauchemin et al., 2009). A concentration of > 500 g/d from
sunflower oil or linseed oil tended to reduce DMI (Shingfield et al., 2008; Saliba et al., 2014) when included in the high-, but not
low-concentrate diet. Despite lower feed intake in HFO relative to HF, they had similar GE intake, while no effect on nutrients intake
was observed by supplementing RO to LF diet except for EE intake. Moreover, results from Benchaar et al., (2012, 2015) indicate that
using vegetable oil at rates ranging from 3.5% to 5.5%, reduces intake and DM digestibility in cereal forage (e.g., corn silage) but not in
high fiber based-diets (e.g., legume or grass forages). Therefore, it appears that the effect of diet energy density and energy intake from
the oil supplement is different in diets containing various levels of starch, crude protein, and due to forage type. In addition, dietary
addition of RO increased the intake of UFA (i.e., cis-9 18:1, cis-9, cis-12 18:2, and cis-9, cis-12, cis-15 18:3), and this increase led to a

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greater magnitude with LF compared with HF.

4.2. Production responses

It should be noted that due to the small number of cows and the relatively short experimental periods, the present study was not
designed to measure production responses and results should be interpreted cautiously. Therefore, only production variables with
significant effects are discussed herein.
The observed increase in milk yield with LF compared with HF diets could be explained by a greater intake of DM having higher
nutrient density i.e., concentrates. The significant interaction between FC and RO on milk yield, reflected as the highest milk yield for
LFO diet compared with other diets, is likely a response to difference in GE supply with dietary starch and oil. In our study, energy
density and total GE intake were greater for LF diets; hence more energy was available in form of glucogenic or both glucogenic and
lipogenic for milk production especially when RO was included in LF diet, with no compromise in feed intake. In an experiment in
which 50 g/kg DM of sunflower oil was supplemented to a starch-rich diet (>300 g/kg of DM), feed intake tended to decrease (Ventto
et al., 2017) without influencing milk production.
Although milk fat concentration decreased with LFO compared with the other 3 diets, milk fat yield was not different. Thus,
decreased milk fat concentration could have originated from a dilution effect due to an increase in milk yield (~8 kg/d) with feeding
LFO versus other diets. However, ECM yield was higher with LF compared with HF diets, regardless of RO supplementation. We
observed that LFO diet decreased milk fat concentration by 19% whereas increased the concentration of trans-10 18:1, a good indicator
for BH-induced MFD, in milk fat by 89% relative to other diets. However, the change in milk fat yield of LFO cows in the current study
is not a typical MFD condition i.e., remarkable reduction in milk fat yield with no changes in milk yield and other milk components as
proposed by Bauman and Griinari (2003), while cows having a relatively high milk fat content (37–46 g/kg). Although the concen­
tration of trans-10 18:1 FA in milk fat from LFO was much greater than the mean suggested by Matamoros et al. (2020) to induce MFD
(3.25 vs. 1.39 as percentage of FA), the cows had similar milk fat yield compared with other diets. As trans intermediates (i.e., trans-10
and trans-10, cis-12 CLA) are not the only regulating factors for milk fat yield, it appears that the greater feed intake with LF diets
provides enough substrates for more milk fat synthesis. In contrast, it seems that the high dietary NDF concentration (on average 445
g/kg DM) in HF diets prevented decreases in milk fat concentration and yield when RO was included in the diet at 50 g/kg DM as
suggested by Kliem et al. (2019). However, Saliba et al. (2014) compared diets differing in FC ratios (30:70 and 70:30) plus linseed oil
supplement, which were different in dietary energy content, and observed that adding 30 g/kg linseed oil to both diets containing 120
and 366 g/kg starch decreased milk fat concentration. Further, Benchaar et al. (2015) indicated that diets containing mainly cereal
forages (e.g., corn silage) are more sensitive to the depressive effects of vegetable oil on ruminal degradation of NDF and milk fat
content than diets based on legume or grass silages. This result might be attributed to the forage source used and replacing strategy
compared with our study (corn silage vs. grass silage). In lactating cows, the efficacy of linseed oil for reducing methane emissions was
reported to be more profound when included in diets based on corn silage than red clover silage (Benchaar et al., 2015). In contrast,
decreases in methane emissions following feeding of extruded linseeds were found to be similar in cows fed diets based on grass hay or
corn silage (Martin et al., 2016).
Yield of milk protein increased in cows fed LF compared with HF while it increased further when RO was added to the LF diet. The
increased milk protein yield with LF in the current study may be multifaceted. First, this increase may be related to the greater CP
concentration in LF diets than the HF diets, which along with the 8% greater DM intake, both contribute to greater protein available for
milk protein synthesis. Martineau et al. (2013) reported that milk protein responded positively to canola meal when cows were fed
grass forages as in our study cows fed LF consumed a diet with greater rapeseed meal proportion than HF diets. Second, greater milk
protein yield with LF compared with HF diets arises mainly from greater milk yield in association with greater starch intake which are
expected to promote microbial protein synthesis (Sterk et al., 2011; Roman-Garcia et al., 2016). Specially, feeding diets with more
starch compared with NDF as an energy source result in increased net splanchnic release of amino acids, post-hepatic availability of
amino acids, and mammary utilization of essential amino acids for milk protein synthesis (Cantalapiedra-Hijar et al., 2014; White
et al., 2016). Additionally, the greater energy content may have caused the increased milk protein production (RO supplement in our
study) as described by Morris et al. (2020). Not only energy supply, but also the nature of energy supply (glucogenic vs. ketogenic
nutrients) can influence milk protein synthesis (Emery, 1978). Milk lactose yield increased for LF compared with HF, and RO sup­
plementation increased it further when included in the LF diet. As milk lactose is the major driver of milk yield due to osmotic pressure,
increased lactose synthesis typically increases milk yield (Cant et al., 1993). Furthermore, reduction in de novo synthesis of FA in the
mammary gland has the potential to reduce the glucose demand for fat synthesis. If glucose supply is adequate, the “spared” glucose
could then be used by other tissues or for lactose synthesis in the mammary gland resulting in increased milk yield (Voigt et al., 2005)
as observed in the current study. It seems that, in our study, increase in efficiency of feed utilization for milk production with RO
supplementation to the LF diet could be related to the increase in energy concentration of the diet and the concomitant decline in
methane energy losses, which apparently supported milk production.

4.3. Energy and nitrogen utilization

Although our objectives included measurements of energy utilization towards body reserves, BW changes are not reported in the
current experiment because changes in BW may not necessarily reflect changes in tissue energy (NRC, 2001) and due to the short
experimental periods, the measured BW changes are not reliable. Forage-to-concentrate ratio (65:35 vs. 35:65) and RO supplemen­
tation (0 vs. 50 g/kg, on DM basis) did not change the energy balance of the cows. However, we observed a reduced methane energy

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excretion as a proportion of energy intake when concentrate was substituted for forage. In agreement with our results, Bayat et al.
(2017) reported lower methane energy excretion as a proportion of energy intake from cows fed high-concentrate compared with
low-concentrate (304 vs. 126 g/kg starch on a DM basis, respectively). Similarly, Lovett et al. (2003) using heifers reported a sig­
nificant reduction in methane energy excretion as a proportion of energy intake when concentrate proportion increased in the diet.
Rapeseed oil reduced methane energy excretion as a proportion of energy intake (kJ/MJ) by 3.0% and 2.7% per additional 10 g/kg
supplemental oil with HF and LF diets, respectively. Beauchemin and McGinn (2006) found a significant reduction in methane loss as
proportion of GE when feeding a diet with 46 g/kg rapeseed oil added to heifers but observed a depression in feed intake. It appears
that both forage type and FC ratio are effective on methane energy excretion responses to oil addition to the diet. In lactating cows,
Benchaar et al. (2015) reported supplementation of the silage-based diets with 4% DM of linseed oil caused decrease in enteric
methane production expressed as a percentage of GE intake, with a more pronounced decrease for cows fed corn silage- than red clover
silage-based diets (23 vs. 11%). While increasing the FC ratio also reduced ruminal methane emissions in the reports from lactating
cows where forages were replaced by concentrates in the diet (Ferris et al., 1999; Aguerre et al., 2011). Bayat et al. (2017) showed that
sunflower oil was more effective in reducing methane emissions when added to low- than high-concentrate diets, whereas in the
current study this variable reduced similarly in cows fed HFO and LFO diets. In the current study, the main changing factor in
experimental diets is grass silage whereas changes in starch content are marginal for lactating dairy cows (8 vs. 14.5% diet DM).
Therefore, changes in grass silage ratios and fermentation quality can affect NDF digestion, rumen fermentation, methane production,
and finally energy utilization (Huhtanen et al., 2009). We highlight that the grass silage used in our experiment had relatively high
quality as indicated by its chemical composition. This has minimized the difference between silage and supplemental concentrate
quality that consequently might have caused minimum differences in energy metabolism when the silage was replaced by concen­
trates. We hypothesized that dietary oil supplementation would increase the efficiency of ME utilization for milk production because
incorporation of dietary fat into milk is more energetically efficient than de novo lipogenesis (Baldwin et al., 1985; Morris et al., 2020).
In our study, although the efficiency of converting ME into milk energy was not different between diets, the ratio of ME intake to GE
intake was greater for the cows fed LF diets compared with HF diets, which agrees with our previous observations (Bayat et al., 2017).
The greater proportion of concentrate in the diets and the inclusion of RO supplement did not change energy partitioning toward milk
synthesis as evaluated by milk energy as a proportion of ME intake, while cows fed LF diets (especially LFO) showed a greater milk
yield. The high quality of grass silage and consequently less difference with the supplemental concentrates might have contributed to
the lack of effect on energy partitioning.
Nitrogen balance was not different among treatments, whereas previously we observed a positive effect of greater proportion of
concentrates or dietary starch on N balance (Aguerre et al., 2011; Morris et al., 2020). In this study, regardless of RO supplementation,
cows fed LF diets had lower daily N excretion in feces as a proportion of N intake while they had greater N intake compared with HF
diets (688 vs. 558 g/d). This caused improved efficiency of N utilization in LF than HF cows. Even though high proportion of dietary
concentrates may stimulate microbial protein synthesis in the rumen and increase intestinal absorption of essential amino acids
leading to higher milk protein synthesis, in the current study, FC ratio did not change milk N secretion as a proportion of N intake
despite the difference in dietary CP concentration (on average 148 vs. 168 g/kg for HF and LF diets, respectively). However, feeding
RO increased efficiency of dietary N utilization, defined as the ratio of milk N output to N intake. Our result is consistent with Hassanat
and Benchaar (2021) who demonstrated that supplementing linseed oil (20–40 g/kg DM) led to improved efficiency of dietary N
utilization due to simultaneous decreases in N intake and increases in milk N secretion as proportion of N intake. Based on these
observations, the increase in efficiency of N utilization may also be explained by the decrease in dietary CP concentration when RO was
added to the diets. Further, the partitioning of N towards milk in cows fed RO-supplemented diets was likely related to an increase in
dietary energy content which is consistent with the observations of Rius et al. (2010).

4.4. Feed and milk fatty acid compositions

Dietary addition of RO increased the intakes of the most dietary FA (including 18:0, cis-9 18:1, cis-9, cis-12 18:2, and cis-9, cis-12,
cis-15 18:3) with a different magnitude when used in LF and HF diets. These variations were reflected in milk fat where concentrations
of the most of these FA were increased when cows received RO. Similarly, feeding LF diets increased the intake of cis-9, cis-12 18:2
which explains the higher concentration of this essential FA in milk fat as compared with HF diets.
The main factor in the variation of BH in the rumen is the FC ratio of the diet, and basal diet appears to have a profound effect on
ruminal metabolism of FA when diet is supplemented with oil sources (Shingfield et al., 2005). Feeding a low FC ratio diet markedly
affects the ratio of cellulolytic to propionigenic, lactogenic, and amylolytic bacteria, which in turn affects ruminal BH (Chilliard et al.,
2007). Consequently, interactions between level of oil supplementation and other dietary changes are likely to occur. In the present
study, the concentration of SFA in milk fat was negatively affected by RO supplementation but a significant interaction effect with FC
ratio was observed; with more pronounced decrease in SFA from cows fed LFO than HFO diet. In particular, this trend was observed for
8:0, 10:0, and 12:0 which are the SFA in milk, and, to a minor extent, for 4:0 and 14:0. Previous research reported similar effects of
dietary RO on milk FA composition using similar amount of RO in the diet with 60:40 FC ratio, and the effects were of similar
magnitude (Bayat et al., 2018). Stearic acid (18:0) increased in the milk fat by feeding RO due to the abundance of 18-carbon UFA in
RO diet which is hydrogenated to 18:0 in the rumen. Likewise, Kliem et al. (2019) reported a reduction in milk fat SFA concentration
with 59 g/kg DM of milled rapeseed (500 g/d oil) added to a maize silage/grass silage-based diet with 50:50 FC ratio, but the reduction
was mainly due to a significant reduction of 16:0 in milk fat. In a previous experiment (Leskinen et al., 2019), sunflower oil sup­
plementation (50 g/kg DM) tended to decrease SFA in milk fat more when supplemented in low-forage diet (35:65) compared with
high-forage diet (65:35), which is consistent with the present experiment, although the magnitude of the changes were different. Milk

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SFA were mainly replaced with cis-MUFA following oil supplementation, the most predominant being cis-9 18:1. Intake of cis-9 18:1
was high for the HFO and LFO, and the appearance of cis-9 18:1 in milk is associated with both increased intake and increased rumen
outflow of 18:0 that is subsequently desaturated to cis-9 18:1 by mammary Δ− 9 desaturase (Bauman and Griinari, 2003). Kliem et al.
(2019) reported a smaller increase in cis-9 18:1 in milk fat than in the current study, but the amount of oil from rapeseed in the diet was
also lower, whereas in the study of Bayat et al. (2018) the increase in milk cis-9 18:1 was greater with similar amount of RO in the diet.
The effect of FC ratio on milk 18:1 FA composition differed, depending on the presence or absence of RO in the basal diet which was
expected. Among the intermediates of ruminal BH, the level of trans-11 18:1 increased more by feeding HFO compared with LFO diet,
but an inverse interaction was observed on the concentration of trans-10 18:1. Feeding lower proportion of forage promotes the
alternate (trans-10) BH pathway instead of the normal pathway (trans-11; Shingfield et al., 2010) as reported in the current trial as well
as in previous experiments (Sterk et al., 2011; Saliba et al., 2014). In many cases, reductions in milk fat secretion have consistently
been associated with increases in milk trans-10, cis-12 CLA and, in some cases, in milk trans-10 18:1 (Shingfield et al., 2010). In the
present experiment, an interaction effect between FC ratio and RO addition was observed on milk fat concentration but not on milk fat
yield. Compared with other diets, the milk fat concentration reduced in cows fed LFO by 19% which may be explained by the inhibitory
effect of trans FA biohydrogenation intermediates on milk fat synthesis. The concentrations of trans-10 18:1 and trans-10, cis-12 CLA in
milk fat increased by 89% and 80% of total FA, respectively, in cows fed LFO compared with other diets. The greater concentration of
alternate BH intermediates (i.e., 3.25% trans-10 18:1) in milk from cows fed LFO originated from enhanced diet fermentability and
PUFA content.
Feeding RO increased cis-9, trans-11 CLA concentration in milk fat. The cis-9, trans-11 CLA is produced mainly endogenously by the
mammary desaturation of trans-11 18:1 by stearoyl-CoA desaturase enzyme but also as an intermediate in rumen BH (Bauman and
Griinari, 2003; Chilliard et al., 2007). The concentration of cis-9, trans-11 CLA, in fact, more than doubled in milk from cows fed HFO
and LFO diets compared with HF and LF diets. However, the level of CLA enrichment was greater in the present study than in previous
trials (Altenhofer et al., 2014; Kliem et al., 2019) where dairy cow diets were supplemented with vegetable oil. This result might be
attributed to the greater amount of RO supplemented in the present trial.

5. Conclusions

Low-forage diet and rapeseed oil supplement fed together increased the yields of milk, milk protein and lactose compared with
other diets, while it decreased milk fat concentration. Energy intake lost as methane was lower whereas ME intake was greater for the
low- compared with high-forage diets. Rapeseed oil decreased the excretion of methane energy as a proportion of energy intake in both
low- and high-forage diets which was explained, at least in part, by decreases in feed intake. Further, dietary rapeseed oil supple­
mentation increased the partitioning of N towards milk protein synthesis on low-forage diets in this study. Diets did not influence
energy and N balances. The low-forage diet combined with rapeseed oil supplement increased milk trans-10 18:1 and trans-10, cis-12
18:2, resulting in the lowest milk fat concentration without influencing milk fat yield compared with other diets. Rapeseed oil
increased cis monounsaturated FA, while feeding low-forage diet with rapeseed oil decreased saturated FA and increased total trans FA
in milk fat. Overall, higher dietary concentrate ratio was effective in improving energy partitioning towards milk, whereas rapeseed oil
supplement improved N partitioning towards milk when supplemented to both high- and low-forage diets.

CRediT authorship contribution statement

Ali Razzaghi: Formal analysis, Writing – original draft, Writing – review & editing. Heidi Leskinen: Writing – review & editing.
Seppo Ahvenjärvi: Writing – review & editing. Heikki Aro: Writing – review & editing. Ali Reza Bayat: Conceptualization, Meth­
odology, Supervision, Writing – review & editing.

Conflicts of interest

The authors confirm that there are no recognized conflicts of interest associated with this publication and there has been no
financial support for this work that could have influenced its outcome.

Acknowledgements

The authors express their appreciation to the staff of Natural Resources Institute Finland (Luke) in Jokioinen Research Barn for
technical support, care of experimental animals, and assistance in sample collection. The laboratory staff of Luke is acknowledged for
the chemical analysis of samples. The financial aid by CEDERS project under the ERA-NET Co-fund scheme FACCE ERA-GAS for
Monitoring & Mitigation of Greenhouse Gases from Agri- and Silvi-Culture is acknowledged. The financial support (#1323/03.01.01/
2017) from Ministry of Agriculture and Forestry of Finland (MMM) is highly appreciated. The authors confirm that there are no
recognized conflicts of interest associated with this publication.

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