Milk production and methane emissions from dairy cows fed a low or high proportion of red clover silage and an incremental level of rapeseed expeller

Livestock Science 197 (2017) 73–81

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Milk production and methane emissions from dairy cows fed a low or high proportion of red clover silage and an incremental level of rapeseed expeller Helena Gidlund, Mårten Hetta, Pekka Huhtanen

MARK

⁎

Swedish University of Agricultural Sciences, Department of Agricultural Research for Northern Sweden, SE-901 83 Umeå, Sweden

A R T I C L E I N F O

A BS T RAC T

Keywords: Legume silage Grass silage Crude protein Protein supplement Forage quality

This study evaluated the effects of including increasing levels of rapeseed expeller in dairy cow diets with a low or high proportion of red clover silage on milk production and methane emissions. A total of 32 lactating Swedish Red dairy cows were used in a cyclic change-over design with three periods of 21 days, in a 2×4 factorial arrangement of treatments. The total mixed ration consisted of 600 g/kg dry matter (DM) of forage and 400 g/kg DM of concentrate on a DM basis. The forage treatments consisted of a 30:70 or 70:30 ratio of grass to red clover silage (RC30 and RC70). A basal supplement consisted of crimped barley and premix, formulated to contain 130 g CP/kg DM. For the three additional concentrate supplements, crimped barley was gradually replaced with incremental levels of rapeseed expeller to reach 170, 210 or 250 g CP/kg DM. No differences in feed intake were found between RC30 and RC70, but a positive response was found to increased dietary CP concentration from rapeseed expeller. Increasing proportion of red clover silage did not have any effect on production, while increasing dietary CP concentration increased yield of milk, energy corrected milk (ECM) and milk protein. Nitrogen efficiency was higher with diet RC30 than with RC70 and decreased with increasing dietary CP concentration, while milk urea nitrogen increased. Methane (CH4) emissions per unit feed intake decreased with dietary CP concentration and tended to increase with increasing proportion of red clover silage in the diet. Increased CP intake from red clover silage in the diet of dairy cows had no positive effect on CH4 emissions.

1. Introduction Red clover (Trifolium pratense L) is the most common forage legume grown at northern latitudes in Europe, where grass or grass-red clover silage is the main ingredient in dairy cow diets. Due to its ability to fix atmospheric nitrogen (N), inclusion of red clover is of high value in leys in organic and low input dairy production systems. Future interest in using red clover within conventional dairy production may also increase with the rising cost of N fertiliser. When leys are composed of both grass and red clover, the regrowth has a higher proportion of red clover and a different quality of the grass, than the primary growth. On-farm, this results in forages of different qualities owing to the differences in feed characteristics between red clover and grasses. Red clover silage fed as a sole forage and in mixtures with grass silage has been reported to have better potential to increase dry matter intake (DMI) and milk yield than diets containing pure grass silage in the forage ration (Dewhurst et al., 2003b; Moorby et al., 2009). When revising their relative silage DMI (SDMI) index, Huhtanen et al. (2007)

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showed that when dairy cows were fed mixtures of grass and legumes, SDMI was higher than predicted from the mean of grass and legumes fed alone. Inclusion of rapeseed feedstuffs have increased milk production compared with soybean meal when fed as a protein supplement to dairy cows on a grass silage-based diet (Shingfield et al., 2003; Gidlund et al., 2015). Moreover, Martineau et al. (2013) found that feeding rapeseed feedstuffs increased the uptake of essential amino acids in the small intestine compared with soybean meal and other protein supplements. Furthermore, addition of rapeseed feedstuffs is reported to increase the omasal flow of non-ammonia N (NAN), with the positive production responses to the protein supplement being attributed to increased dietary NAN flow to the omasum (Ahvenjärvi et al., 1999). Red clover silage increases the flow of dietary and total NAN from the rumen compared with grass silage (Dewhurst et al., 2003a; Vanhatalo et al., 2009). The enzyme polyphenol oxidase (PPO) has been cited as the main cause of reduced degradation of dietary protein in the rumen and decreased N digestibility when red clover silage replaces grass silage in the diet (Dewhurst et al., 2003b; Merry et al.,

Corresponding author. E-mail address: [email protected] (P. Huhtanen).

http://dx.doi.org/10.1016/j.livsci.2017.01.009 Received 2 November 2016; Received in revised form 12 January 2017; Accepted 13 January 2017 1871-1413/ © 2017 Published by Elsevier B.V.

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2014; Halmemies et al., 2014). Huhtanen et al. (2014) suggested that the effect of PPO in red clover silage might decrease the post-rumen N digestibility compared with that of grass silage and concluded that the increased faecal N output when feeding red clover silage is not related to increased particle-associated crude protein (CP). An environmental concern with forage-based milk production is that high forage diets are known to increase ruminal methane (CH4) production compared with high concentrate diets (Johnson and Johnson, 1995). It has been suggested that legume silages could decrease CH4 emissions from ruminants (Beauchemin et al., 2008; Lüscher et al., 2014). However, there is currently a lack of data on CH4 emissions from ruminants fed legumes (Dewhurst, 2013). Due to the possible increase in dietary NAN with more red clover silage in the diet of dairy cows and the positive production response to rapeseed in the diet, our starting hypothesis was that increasing the proportion of red clover silage in the diet would have a protein supplement sparing effect. Our second hypothesis was that increasing red clover silage in the diet decreases CH4 emissions from dairy cows, by lowering the fibre content, and increases DMI. The overall aim of this study was to determine milk production responses and CH4 emissions in dairy cows fed diets based on grass silage and low or high inclusion of red clover, mimicking the composition of primary and regrowth grass-red clover ley. Incremental levels of rapeseed expeller were also included in the diets, to investigate the optimal supplemental CP levels and possible concentrate sparing effects when feeding low or high red clover silage to dairy cows.

Table 1 Proportions (g/kg DM) of different feed components in the experimental diets. Dietsa RC30

Grass silage Clover silage, primary growth Clover silage, regrowth Crimped barley Premixb Rapeseed expellerc

RC70

B

L

M

H

B

L

M

H

442 95 95 316 52 0

442 95 95 242 52 74

442 95 95 168 53 147

442 95 95 95 52 221

189 221 221 316 53 0

189 221 221 242 53 74

189 221 221 168 54 147

189 221 221 95 53 221

a Diets: RC30=30% red clover silage on DM basis in the forage ration and 70% grass silage, RC70=70% red clover silage in the forage ration and 30% grass silage, B = barley, no protein supplement, L = low level of rapeseed expeller, M = medium level of rapeseed expeller, H = high level of rapeseed expeller. b The premix (Fodercentralen, Umeå, Sweden) contained (g/kg feed) sugar beet pulp (250), sugar beet molasses (20), barley (166), oat (350), oat bran (50), NaCl (36), calcium carbonate (30), calcium-fat (40), mineral and vitamin concentrate (58). c Heat-moisture treated rapeseed expeller (Farmarin Öpex, Suomen Rehu, Hankkija Oy, Hyvinkää, Finland).

Four levels of concentrate CP were formulated. A basal concentrate (B; 130 g CP/kg DM) consisting of crimped barley and a premix (100 g/kg DM; Fodercentralen, Umeå, Sweden) was fed to fulfil mineral and vitamin requirements. Concentrate CP levels of 170, 210 and 250 g CP/kg DM (low (L), medium (M) and high (H), respectively) were achieved by replacing crimped barley with incremental levels of rapeseed expeller. All silages were harvested in 2012 with a mower conditioner and precision-chop forage harvester (theoretical chopping length of 16– 32 mm) and stored in separate bunker silos. The grass silage was a regrowth sward cut from a two-year-old ley of timothy and red clover (seed rate 80:20) fertilised with 40 kg N/ha (80 kg N/ha for the primary growth). The grass silage was harvested on 8 August and treated with an acid-based additive (Promyr TM XR 630, Perstorp, Sweden) at a rate of 3.5 L/tonne. The red clover silage came from a primary growth and a regrowth of a one-year-old pure red clover ley. It was fertilised with 30 kg N/ha for the primary growth, which was harvested on 6 July, while the regrowth was harvested on 15 August. Both primary growth and regrowth cuts were treated with the same acid-based additive as for the grass silage, at a rate of 6 L/tonne. The crimped barley (59.8% DM) was rolled using a mill (Murska 1400 S2×2, Murska, Ylivieska, Finland) adjusted to 0.3 mm between the rollers, treated with 3.5 L/tonne of propionic acid and stored in airtight bags (1.6 m×60 m, Ltd Rani Plast Oy, Terjärv, Finland). The treated rapeseed expeller was a commercial feed (Öpex, Mildola Ltd, Espoo, Finland), made by pressing double-zero rapeseeds (low in glucosinolates and erucic acid) under heating to extract the oil and then applying a heat-moisture treatment to the expeller.

2. Materials and methods The study was carried out with the permission of the Swedish Ethics Committee on Animal Research (Umeå, Sweden) and in accordance with Swedish laws and regulations and with the EU Directive 2010/63/ EU on animal research. 2.1. Experimental design, animals and management The study was conducted at the Röbäcksdalen research station, which is part of the Swedish Infrastructure for Ecosystem Science within the Swedish University of Agricultural Sciences in Umeå (63°45´N; 20 °17´E). A set of 32 lactating Swedish Red dairy cows (100 ± 34.4 days in milk; 32 ± 6.9 kg milk/day) were blocked according to parity and milk yield. Eight primiparous cows were blocked separately and older cows were blocked according to high, medium, or low yield. Within blocks, the cows were randomly assigned to treatments. The study was conducted as a cyclic change-over design (Davis and Hall, 1969), with eight treatments in a 2×4 factorial arrangement consisting of two grass-red clover silages and four levels of dietary CP fed to the cows in three experimental periods. Each period lasted 21 days and was divided into 14 d of adaptation and seven days of data recording and sampling. The cows were kept in an insulated, loose-housing barn and were milked twice a day, at 06:00 and 15:00. They were fed a total mixed ration ad libitum. A stationary feed mixer (Nolan A/S, Viborg, Denmark) processed the rations, which were then delivered with automatic feeder wagons into feed cribs three times per day, starting at 4:00, 10:00 and 17:00. During the trial, each cow had access to the same feed crib, shared with another experimental cow in pairs throughout the experiment.

2.3. Animal recordings Feed intake was recorded daily in Roughage Intake Control feeders (Insentec, B.V., Marknesse, the Netherlands), and data from day 15–21 in each period were used in the statistical analysis. Body weight was measured after morning milking on day 19–21 in each period. Milk yield was recorded daily with gravimetric milk recorders (SAC, S.A. Christensen and Co Ltd, Kolding, Denmark), and data from day 15–21 were used in the statistical calculations. Milk samples were collected at four consecutive milking, from the afternoon milking of day 19 until the morning milking of day 21 in each period. The morning samples were pooled together, as were the evening samples, and then stored at 8 °C until sent for analysis. The mass flux of CH4 and carbon dioxide (CO2) in exhaled air from individual animals was recorded by two portable open-circuit head

2.2. Diets The eight dietary treatments all had a forage-to-concentrate ratio of 60:40 on a DM basis (Table 1). The forage levels consisted of a mixture of either 70:30 (RC30) or 30:70 (RC70) grass silage and red clover silage on a DM basis. Furthermore, the red clover silage was a 50:50 (DM basis) mixture of a primary growth and regrowth ley from the same field. 74

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Table 2 Chemical composition (g/kg DM unless stated otherwise) of the experimental feeds.

Dry matter, g/kg feed Organic matter Crude protein MPa NDFb iNDFc Starch WSCd EEe In vitro digestibilityf MEg, MJ/kg DM Lactic acid Acetic acid Propionic acid Butyric acid Ammonia-N, g/kg N pH

Grass silage

Red clover silage, primary growth

Red clover silage, regrowth

Crimped barley

Premix

Rapeseed expeller

251 913 129 72 513 91

254 878 214 69 376 106

235 870 185 66 444 155

4 20 879 11.1 102.1 21.2 2.4 0.6 31.0 3.6

1 20 837 10.1 94.5 27.1 4.5 0.3 75.7 4.2

1 20 758 8.9 75.6 54.5 5.0 4.3 120.1 4.7

598 973 125 90 168 65 533 42 24

839 855 100 70 228 92 282 59 66

842 927 364 185 340 140 30 80 94

13.2 7.0 1.5 5.2 < 0.4 22.1 4.4

10.4

13.8

a

Metabolisable protein calculated according to Spörndly (2003). Neutral detergent fibre. c Indigestible neutral detergent fibre. d Water-soluble carbohydrates. e Ether extract for the silages are tabulated values (Spörndly, 2003), for concentrate ingredients ether extract was analysed. f In vitro digestibility was determined according to Lindgren (1979). g Metabolisable energy concentrations for the silages calculated according to Lindgren (1983), and for the concentrates according to Spörndly (2003). b

Espoo, Finland), with a 60:40 forage to concentrate ratio. All concentrate feedstuffs were analysed for starch (Larsson and Bengtsson, 1983). Silage samples were analysed for in vitro organic matter digestibility according to Lindgren (1979). The frozen silage samples were analysed for ammonium-N using direct distillation after adding MgO with Kjeltec 2100 Distillation Unit (Foss Analytical Ltd, Hillerød, Denmark). They were also analysed for volatile fatty acids (VFA) and lactic acid (Ericson and André, 2010). The frozen silage was thawed and pressed, and the pH in the liquid was measured with a pH-meter (Metrohm, Herisau, Switzerland). The milk samples were analysed for concentration of fat, protein, lactose and urea N, using a near infrared reflectance analyser (CombiFoss 6000, Foss Electric, Hillerød, Denmark).

chamber systems (GreenFeed system, C-lock Inc., Rapid City, SD). To capture gas emissions over the daily 24 h cycle, the cows were allowed a feed drop once every 5 h, delivered as six doses of 50 g concentrate with 40 s intervals. Airflow rates and gas concentrations were measured continuously in the system and the volumetric flux (L/min) of gases emitted by the animals was calculated as described by Huhtanen et al. (2015). The system recorded head position of the animal during the visit and data with inappropriate head positions were not used in the calculations. 2.4. Sampling of feeds and chemical analysis Feeds were sampled on day 16, 19 and 21 during each period and stored at −20 °C. Frozen silage samples were milled in a cutter mill (SM 2000, Retsch Ltd., Haan, Germany) to pass a 20 mm sieve and a part was kept in the freezer for analyses of silage fermentation quality. All feed samples were oven-dried at 60 °C for 48 h. The dried samples were milled by the same cutter mill as above to pass through a 2 mm or a 1 mm sieve for different analytical purposes. Concentration of DM was determined for feed samples by drying at 105 °C for 16 h. Oven DM concentration was corrected for volatile losses according to Huida et al. (1986). Ash concentration was determined by incinerating at 500 °C for 4 h. Feeds were analysed for CP (Nordic Committee of Food Analysis, 1979) using a 2020 Digestor and a 2400 Kjeltec Analyser Unit (FOSS Analytical A/S, Hillerød, Denmark), and water-soluble carbohydrates (Larsson and Bengtsson, 1983). Crude fat content was determined according to Method B in the Official Journal of the European Communities (1984), using a 1047 hydrolysing unit and a Sotex system HT 1043 extraction unit with petroleum ether (Foss Analytical A/S, Hillerød, Denmark). Neutral detergent fibre (NDF) concentration was analysed with stable ɑamylase and sodium sulphite (Mertens, 2002) using the Filter bag technique in an ANKOM200 Fibre Analyser (Ankom Technology Corp., Macedon, NY). The NDF values were expressed exclusive of residual ash. Concentration of indigestible NDF (iNDF) was determined following a 288-h rumen in situ incubation (Huhtanen et al., 1994) and the procedures by Krizsan et al. (2012). Two rumen-cannulated lactating cows were used for the incubation. They were fed a TMR based on grass silage, crimped barley and rapeseed expeller (Öpex, Mildola Ltd,

2.5. Calculations and statistical analysis Data from four cows were excluded from the whole dataset because of stealing from other experimental diets. No specific diet was more disposed to provoke stealing. Metabolisable energy (ME) concentration in the silages was calculated following the procedure of Lindgren (1979) and that in the concentrates following Spörndly (2003). Metabolisable protein (MP) was calculated according to Spörndly (2003). Energy corrected milk (ECM) production was calculated according to Sjaunja et al. (1990). The N efficiency was calculated as milk N / N intake. The SDMI index of the forage mixtures was calculated according to Huhtanen et al. (2007). The data were analysed with the MIXED procedure of SAS (SAS Inc. 2002–2003, release 9.3; SAS Inst., Inc., Cary, NC), using the statistical model:

Yijkl = μ + Bi +Cj (Bi )+Pk +Tl +ε ijkl where Yijkl is the dependent variable, µ is the mean of all observations, Bi is the effect of block i, Cj(Bi) is the effect of cow j within block i, Pk is the effect of period k, Tl is the effect of treatment l, and εijkl ~ N(0,σe2 ) is the random residual error. For treatment comparison, the following contrasts were used: comparison of low or high inclusion of red clover silage (F), linear (CP-Lin) and quadratic (CP-Quad) effects of dietary CP concentration, and interactions between the proportion red clover 75

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3.3. Milk production and composition

Table 3 Chemical composition (g/kg DM, unless stated otherwise) of the experimental diets.

There was no difference between forage treatments in terms of milk yield or ECM yield (Table 5). The milk protein concentration decreased (P < 0.01) with the RC70 diet. Milk urea N (MUN) concentration increased (P < 0.01) and N efficiency decreased (P < 0.01) with the RC70 diet. Manure N per kg ECM increased (P < 0.01) by about 20% with the RC70 diet. Increased dietary CP concentration quadratically increased (P≤0.01) the yield of milk, protein and lactose, with maximum yield observed with the M level of protein supplementation. Milk fat concentration (P≤0.05), and lactose concentration (P≤0.05) both decreased with increased dietary CP concentration. There was a interaction (P=0.02) between the forage treatment and quadratic effect of CP concentration; milk fat concentration decreased with RC30 and increased with RC70 when concentrate CP concentration increased from M to H. Increased dietary CP concentration also resulted in higher MUN concentration (P < 0.01) and lower N efficiency (P < 0.01). Furthermore, manure N per kg ECM increased linearly (P < 0.01) with increased dietary CP concentration.

Dietsa RC30

Dry matter, g/kg feed Organic matter Crude protein MPb NDFc pdNDFd iNDFe EEf MEg, MJ/kg DM SDMI indexh

RC70

B

L

M

H

B

L

M

H

369 921 145 79 380 288 92 26 11.4 103

373 918 157 84 390 294 96 29 11.4

376 915 172 90 402 301 101 34 11.5

380 912 186 95 411 306 105 38 11.5

357 913 160 79 352 253 99 26 11.1 103

361 909 174 83 361 257 105 30 11.1

365 906 189 90 371 262 109 35 11.2

369 903 205 95 385 270 115 39 11.2

a

Diets: See footnote 1 in Table 1 for explanation. Metabolisable protein calculated according to Spörndly (2003). Neutral detergent fibre. d Potentially digestible neutral detergent fibre. e Indigestible neutral detergent fibre. f Ether extract for the silages are tabulated values (Spörndly, 2003), for concentrate ingredients ether extract was analysed. g Metabolisable energy concentrations for the silages calculated according to Lindgren (1979), and for the concentrates according to Spörndly (2003). h Silage dry matter index according to Huhtanen et al. (2007). b c

3.4. Gas emissions Three of the experimental animals did not visit the GreenFeed units, which reduced the number of observations to 25 cows per period for gas recordings. Furthermore, in the last experimental period the CH4 sensor in one of the GreenFeed units malfunctioned, so CH4 emissions could only be measured in one unit. Therefore, in the last period, there were an additional nine missing observations of CH4 levels. The CO2 sensors functioned during all periods throughout the experiment. There were no differences in total gas emissions between diets RC30 and RC70 (Table 6), but the CH4 yield (g/kg DM intake) tended (P < 0.1) to be higher with RC70 compared with RC30. Increasing the dietary CP concentration, the emissions of CO2 (g/kg DMI) decreased (P < 0.01). With increasing dietary CP concentration the CH4 yield decreased (P < 0.05) and the decrease was greater with diet RC30 than with RC70 (forage × CP-Lin CP; P≤0.05). Furthermore, a tendency to an interaction (P < 0.10) between forage treatment and quadratic effect of increasing CP concentration was observed for CH4 yield. With diet RC30 the CH4 yield declined with increasing CP, whereas with RC70 the lowest CH4 yield was observed with the L level of protein. Methane intensity was lowest (15.3 g CH4/kg ECM) in cows fed the low level of dietary CP, but the differences between forage treatments did not reach statistical significance.

silage and linear (F × CP-Lin) and quadratic (F × CP-Quad) effects of dietary CP concentration.

3. Results 3.1. Experimental feeds The chemical composition of the experimental feeds is shown in Table 2 and that of the experimental diets in Table 3. The DM concentration was similar for all silages and the concentration of organic matter (OM) was lower for the red clover silages than the grass silage. The red clover silages had higher CP concentration than the grass silage, with the first cut red clover silage having the highest concentration (214 g/kg DM). The grass silage had the highest NDF concentration and the primary growth red clover silage the lowest. The regrowth red clover silage had a higher (155 g/kg DM) iNDF concentration than the other two silages. The ME concentration was lower for the clover silages and lowest for the regrowth red clover silage. The low pH and the concentrations of ammonia-N and VFA in the grass silage indicated good ensiling quality. The lactic acid concentration was high in the grass silage (102 g/kg DM) and in the primary growth red clover silage (95 g/kg DM). For the regrowth red clover silage, the concentrations of acetic acid and butyric acid were higher than preferred. The ammonia-N concentration was also high (120 g/kg N) compared with the primary growth red clover silage (76 g/kg N) and the grass silage (31 g/kg N).

4. Discussion 4.1. Silage composition This study evaluated the milk production responses and CH4 emissions from dairy cows to low or high inclusion of red clover in the diet, supplemented with barley and rapeseed expeller to four levels of dietary CP concentrations. Although the grass silage was made from a ley that was sown with timothy-red clover seed mixture, the low CP concentration and high digestibility indicate that the proportion of red clover was low in the grass silage. The higher ME and NDF concentrations and the lower CP and iNDF concentrations of the grass silage compared with the red clover silage confirmed the widely reported differences between the forage types (Bertilsson and Murphy, 2003; Halmemies et al., 2014). Comparing the primary growth and the regrowth red clover silages, the nutrient quality decreased in the same manner as with increasing maturity of the crop (Kuoppala et al., 2009). However, the relationship reported between nutrient quality of red clover silages and number of cuts and crop maturity varies between studies (Mela, 2003; Grabber,

3.2. Feed and nutrient intake Increasing the proportion of red clover silage in the diet of the dairy cows did not affect the total DMI, but significantly increased the intake of CP (P < 0.01). Furthermore, the RC70 diet decreased (P < 0.01) the intake of ME, NDF and pdNDF, while the intake of iNDF increased (P < 0.01) compared with the RC30 diet. The SDMI index was exactly the same (103) for both diets (Table 4). Increased dietary CP concentration increased (P < 0.01) total DMI, from 18.3 to 19.9 kg, and also increased the intake of all other nutrient parameters analysed (P < 0.01) (Table 4). 76

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Table 4 Effects of forage treatment and dietary CP concentration on dry matter and nutrient intake (kg/d unless stated otherwise). Dietsa RC30

c

n DMI OM CP MPd, g/d NDFe pdNDFf iNDFg Starch Crude fati MEj, MJ/d

Contrastsb

RC70

B

L

M

H

B

L

M

H

SEM

F

CP-Lin

CP-Quad

F ×CP-Lin

F ×CP-Quad

9 18.3 16.8 2.6 1443 7.0 5.3 1.7 3.0 0.5 209

11 19.2 17.6 3.0 1608 7.5 5.6 1.9 2.6 0.6 220

12 20.2 18.5 3.5 1819 8.1 6.1 2.0 2.1 0.7 233

12 20.2 18.4 3.8 1930 8.3 6.2 2.1 1.5 0.8 233

10 18.3 16.7 2.9 1430 6.4 4.6 1.8 3.2 0.5 203

9 19.1 17.4 3.3 1593 6.9 4.9 2.0 2.7 0.6 212

10 19.9 18.0 3.8 1782 7.3 5.2 2.2 2.2 0.7 222

11 19.6 17.7 4.0 1874 7.6 5.3 2.2 1.5 0.8 220

0.56 0.50 0.10 50.9 0.22 0.16 0.06 0.07 0.02 6.3

0.35 0.13 < 0.01 0.27 < 0.01 < 0.01 < 0.01 NDh ND 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

0.13 0.13 0.28 0.32 0.24 0.33 0.13 0.15 0.46 0.15

0.49 0.46 0.93 0.56 0.50 0.35 0.98 0.09 0.73 0.41

0.91 0.91 0.86 0.88 0.96 0.89 0.86 0.76 0.70 0.88

a

Diets: See footnote 1 in Table 1 for explanation. Probability of the dietary treatment effects: F =30 or 70% red clover silage in forage ration; CP-Lin = linear effect of dietary CP concentration; CP-Quad = quadratic effect of dietary CP concentration; F × CP-Lin = interaction between forage type and linear dietary CP concentrations F × CP-Quad == interaction between forage type and quadratic dietary CP concentration. c Number of observations. d Metabolisable protein calculated according to Spörndly (2003). e Neutral detergent fibre. f Potentially digestible NDF = NDF– iNDF intake. g Indigestible neutral detergent fibre. h Not determined. i Crude fat for the silages are tabulated values (Spörndly, 2003), for concentrate ingredients crude fat was analysed directly. j Metabolisable energy concentration of the silages calculated according to Lindgren (1979), calculations for the concentrate according to Spörndly (2003). b

Table 5 Effects of forage treatment and dietary CP concentration on milk production, N-efficiency, and body weight. Dietsa RC30

nc Yield Milk, kg/d ECMd, kg/d Protein, g/d Fat, g/d Lactose, g/d Concentration Protein, g/kg Fat, g/kg Lactose, g/kg MUNe, mg/dL N-efficiencyf, g/kg Manure N, g/kg ECMg Body weight, kg

Contrastsb

RC70

B

L

M

H

B

L

M

H

SEM

F

CP-Lin

CP-Quad

F ×CP-Lin

F ×CP-Quad

9

11

12

12

10

9

10

11

27.3 28.2 918 1152 1298

29.5 30.5 1016 1225 1431

30.5 31.0 1037 1248 1462

29.6 29.9 1012 1185 1424

27.0 28.9 901 1216 1304

29.2 29.8 989 1188 1392

30.1 30.0 1019 1189 1426

29.5 30.8 993 1289 1384

0.88 1.06 31.1 56.0 44.3

0.56 0.96 0.19 0.60 0.20

< 0.01 0.05 < 0.01 0.27 < 0.01

0.01 0.21 < 0.01 0.96 0.01

0.93 0.94 1.00 0.77 0.51

0.85 0.17 0.87 0.06 0.61

34.0 42.4 47.9 8.2 337 10.4 662

34.7 42.8 48.4 9.9 332 11.3 668

34.4 41.9 47.7 10.9 289 12.9 666

34.4 40.3 47.9 10.7 263 15.8 664

33.8 43.8 48.3 10.0 307 11.9 661

34.3 41.5 47.7 11.2 294 13.3 668

34.0 40.1 47.1 12.1 263 15.1 668

33.6 42.5 47.0 12.1 246 17.0 663

0.52 1.31 0.51 0.68 12.4 0.83 15.4

< 0.01 0.86 0.10 < 0.01 < 0.01 < 0.01 0.95

0.95 0.05 0.05 < 0.01 < 0.01 < 0.01 0.70

0.04 0.39 0.83 0.07 0.42 0.24 0.12

0.26 0.75 0.15 0.70 0.44 0.87 0.97

0.63 0.02 0.50 0.70 0.55 0.44 0.66

a

Diets: See footnote 1 in Table 1 for explanation. Probability of the dietary treatment effects: F =30 or 70% red clover silage in forage ration; CP-Lin = linear effect of dietary CP concentration; CP-Quad = quadratic effect of dietary CP concentration; F × CP-Lin = interaction between forage type and linear dietary CP concentrations was not significant (P≤0.15); F × CP-Quad = interaction between forage type and quadratic dietary CP concentration. c Number of observations. d Energy corrected milk calculated according to Sjaunja et al. (1990). e MUN = milk urea nitrogen. f Nitrogen efficiency = milk N/N intake. g Manure N output (g) per kg energy corrected milk. Calculated as ((CP intake /6.25) – (milk protein yield/6.38)) / ECM. b

that the buffer-soluble PPO could result in the lower proteolysis and hence explain the lower ammonia-N concentration in red clover silage compared with alfalfa silages. In the present study, the high ammoniaN and butyric acid concentrations in the regrowth red clover silage indicate that fermentation quality was lower for RC70 than for RC30 silage.

2009). Visual comparison of the primary growth and the regrowth red clover silages in the present study indicated that the regrowth contained much coarser stems than the primary growth, which is consistent with the decrease in nutrient quality. As reported previously by Bertilsson and Murphy (2003), the ammonia-N concentration was higher in red clover silage than in grass silage in the present study. However, others have found the opposite (Dewhurst et al., 2003b; Vanhatalo et al., 2009). Jones (1995) showed

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Table 6 Effects of forage treatment and dietary CP concentration on methane and carbon dioxide emissions. Dietsa RC30

c

n CH4 g/d g/kg DMI g/kg ECM CO2 kg/d g/kg DMI g/kg ECM CH4/CO2, g/kg

Contrastsb

RC70

B

L

M

H

B

L

M

H

SEM

F

CP-Lin

CP-Quad

F ×CP-Lin

F ×CP-Quad

8

9

8

8

8

8

9

7

455 24.6 16.7

459 23.9 15.6

457 22.9 15.2

441 21.8 14.9

466 25.3 17.8

434 23.0 15.0

478 24.1 16.4

471 24.6 16.3

15.3 0.67 0.83

0.32 0.07 0.25

0.87 0.03 0.13

0.86 0.18 0.17

0.20 0.05 0.58

0.22 0.08 0.40

11.4 618 419 39.9

11.6 613 399 38.9

11.8 592 397 38.4

11.5 564 389 37.5

11.7 637 424 39.5

11.5 610 404 37.5

11.8 596 406 40.6

11.7 593 417 39.6

0.25 16.1 19.5 0.88

0.38 0.31 0.36 0.33

0.53 0.01 0.36 0.40

0.45 0.98 0.43 0.70

0.80 0.72 0.53 0.07

0.25 0.31 0.69 0.71

a

Diets: See footnote 1 in Table 1 for explanation. Probability of the dietary treatment effects: F =30 or 70% red clover silage in forage ration; CP-Lin = linear effect of dietary CP concentration; CP-Quad = quadratic effect of dietary CP concentration was not significant (P≤0.17); F × CP-Lin = interaction between forage type and linear dietary CP concentrations was not significant; F×CP-Quad=interaction between forage type and quadratic dietary CP concentration. c Number of observations. b

milk production when cows are fed grass silage-based diets. Both Shingfield et al. (2003) and Gidlund et al. (2015) showed that rapeseed feeds have a positive effect on plasma histidine concentration and milk production compared with soybean meal. The effect of increased DMI with increased dietary CP concentration was linear in the present study, but seemed to level out above the M level of dietary CP concentration. This was also shown by Gidlund et al. (2015), who fed four incremental levels of dietary CP protein to dairy cows.

4.2. Feed intake The calculated SDMI index (according to Huhtanen et al. (2007)) was identical for diets RC30 and RC70, which is in agreement with the finding of similar DMI for the two forage treatments. Red clover silage in a mix with grass silage or fed as a pure crop is reported to increase DMI in comparison with pure grass silage (Dewhurst et al., 2003b; Moorby et al., 2009). Halmemies et al. (2014) found tendencies for a quadratic increase in DMI on increasing the red clover silage level in the forage from 0% to 33%, 67% and 100%. As found in the present study, there were no numerical differences in DMI between feeding 33 or 67% of the forage as red clover silage in that study. According to Huhtanen et al. (2007), red clover silage has positive effects on DMI when other nutritional factors such as D-value (concentration of digestible OM in DM) are equal. In the present study the RC70 forage had a lower D-value and higher concentration of fermentation acids than the RC30 forage. These factors, which are the two most important factors influencing silage DMI (Huhtanen et al., 2007), could have masked the positive DMI response expected from feeding a diet containing more red clover silage and less NDF. However, Kuoppala et al. (2009) found that delayed maturity of red clover silage increased DMI, even though the digestibility was decreased. Furthermore, they suggested that factors other than rumen fill limit DMI, when feeding early-harvested red clover silage in particular, since their red clover silage-based diets resulted in a smaller NDF rumen pool than grass silage-based diets. Diet digestibility was not measured in the present study, but the higher iNDF concentration in regrowth than primary growth red clover silage (155 compared with 106 g/kg DM) indicates lower digestibility of the former. Total DMI increased with increased dietary CP concentration. This is in agreement with Broderick (2003), who replaced rolled high moisture shelled corn with soybean meal to give a dietary CP concentration of 151, 167, and 184 g/kg DM and found that DMI increased from 21.2 to 22.6 kg/d. Gidlund et al. (2015) also increased the level of dietary CP in the concentrate ration, but found no increase in total DMI. Overall, however, increased CP concentration in the diet generally increases feed intake (Oldham, 1984). This effect is usually due to increased diet digestibility, but it is also suggested to be the result of better amino acid balance or increased nutrient requirement due to higher milk production (Oldham, 1984). In the present study, replacement of barley with rapeseed expeller increased the CP concentration at the expense of the starch concentration, which could have improved the amino acid balance for the lactating cows. Vanhatalo et al. (1999) suggested that histidine is the amino acid that first limits

4.3. Production responses Increasing the proportion of red clover silage in the diet of dairy cows did not increase milk yield or ECM yield, which is in line with the lack of DMI effect. The slightly lower (0.5 g/kg) milk protein concentration with the RC70 diets compared with RC30 is consistent with findings in other studies comparing increased proportion of red clover silage in the diet (Bertilsson and Murphy, 2003; Moorby et al., 2009), even though the decrease in those studies did not always reach statistical significance. According to Vanhatalo et al. (2009), the decrease in milk protein concentration with red clover silage could be due to an imbalanced amino acid profile of the digesta entering the lower tract. The lack of decrease in milk fat concentration in the present study with diet RC70 has been reported previously (Dewhurst et al., 2003b (Experiment 1); Vanhatalo et al., 2006; Halmemies et al., 2014). However, milk fat concentration usually decreases when red clover silage replaces grass silage in the diet (Dewhurst et al., 2003a, 2003b (Experiment 2); Vanhatalo et al., 2009). Steinshamn (2010) concluded that the mechanisms behind the decrease in milk fat concentration with red clover silage need to be further investigated. In a meta-analysis, dietary CP concentration was found to be the best predictor of N efficiency in dairy production (Huhtanen and Hristov, 2009). Hence, the decreased N efficiency with diet RC70 due to the greater CP intake was expected and in accordance with previous studies (Bertilsson and Murphy, 2003; Moorby et al., 2009; Halmemies et al., 2014). The higher N intake with diet RC70 would have increased N excretion via urine and faeces (Moorby et al., 2009; Vanhatalo et al., 2009), thereby resulting in greater losses to the environment. The increases in milk, ECM and milk protein yields with increased dietary CP concentration confirm previous findings (Broderick, 2003). Furthermore, a decrease in N efficiency and increase in MUN has been reported by Olmos Colmenero and Broderick (2006). Shingfield et al. (2003) found a similar effect when feeding an increasing amount of rapeseed expeller to dairy cows. In the meta-analysis by Martineau et al. (2013), rapeseed feeds were concluded to be an excellent protein 78

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clover silage was due to inadequate supply of methionine to the mammary gland. The additional CP intake from red clover inclusion was utilised efficiently in the rumen as increased NAN flow to the lower tract (Dewhurst et al., 2003a; Vanhatalo et al., 2009), but increased NAN flow did not increase milk protein yield. This is consistent with the lack of protein supplement sparing effects in the present study. Further studies are needed to determine why cows do not display intake or production responses with incremental CP intake from red clover, despite being responsive to supplementary protein. The N fractions from the red clover silage are not used efficiently in the animal for some reason, and more N excretion via the manure would be expected.

supplement to dairy cows. The quadratic effect on milk yield with increasing dietary CP concentration in the present study resulted in maximum yield with the M level of protein supplementation (M) diets. With the H diets, the milk yield declined, as found by Gidlund et al. (2015). The decline in milk yield would make the highest dietary CP concentration unnecessary in the ration formulation to dairy cows, since there would be no increased production, only increased loss of N via the manure. Red clover silage inclusion in the diet of dairy cows increases both CP intake and the flow of NAN into the small intestine (Dewhurst et al., 2003a; Vanhatalo et al., 2009; Halmemies et al., 2014). This is mainly due to increased flow of dietary NAN, which is probably explained by the effects of the enzyme PPO (Dewhurst et al., 2003a). Due to the increased flow of NAN to the lower digestive tract with red clover silage, it can be speculated that including red clover silage in the diet could save on the use of protein supplements. However, the lack of production response to incremental CP from red clover silage in the present study indicates that protein supplementation cannot be reduced by feeding more red clover, without compromising animal performance. The marginal milk protein yield was the same for diets RC30 and RC70 (128 g milk protein/kg CP intake) calculated between the basal diet and M protein diet, which was similar to the responses with heattreated rapeseed feeds (meal and expeller; 133 g milk protein/kg CP intake) in the meta-analysis by Huhtanen et al. (2011), but higher than the observed response for soybean meal (98 g milk protein/kg CP intake). Furthermore, the marginal milk protein yield response was greater in the present study than observed by Gidlund et al. (2015), who fed cows grass silage-based diets supplemented with heat-treated rapeseed meal or soybean meal. The lack of production response with increased red clover inclusion in this study was not because the cows had reached their maximum production level, since increased level of rapeseed expeller in the diets increased production. The increase in MUN with an increase in CP intake was higher for incremental red clover silage inclusion (5.2 mg/ dL per kg CP intake) than for incremental rapeseed expeller inclusion (3.0 mg/dL per kg CP intake). This difference also indicates that there are some other factors limiting the use of the additional CP provided from red clover silage for increased milk protein synthesis. The reduced ME intake with the RC70 diets compared with RC30 could explain the lack of production response to increased feed protein from red clover. The CP concentration of the RC30 diets was consistently similar to the lower level of rapeseed meal of the RC70 diets. For example, RC30L contained 157 g CP/kg DM and RC70-B contained 160 g CP/kg DM, but the production was markedly greater with RC30-L than with RC70B (30.5 and 28.9 kg ECM, respectively). The N efficiency followed the same pattern, which further indicates that the incremental increase in CP intake from red clover silage in the diet had no beneficial effects on milk production. Similarly, when other strategies were used to manipulate forage CP concentration, the responses to supplementary protein were not related to forage CP concentration. Rinne et al. (1999) harvested four grass silages at one-week intervals and found that the silage CP concentration decreased from 172 to 113 g/kg DM, but the production responses to rapeseed meal supplementation were similar irrespective of silage CP concentration. Shingfield et al. (2001) found no effect of incremental CP from grass silage fertilised to supply 120 or 150 g CP/kg DM, but a great production response when rapeseed expeller was used as the protein supplement. In addition, Jaakkola et al. (2009) replaced grass silage with whole crop barley silage from 0 to 600 g/kg DM and found that, although the forage CP concentration decreased from 138 to 115 g/kg DM, the production responses to rapeseed meal inclusion were similar between the forage treatments. Vanhatalo et al. (2009) showed generally greater omasal canal flow and higher plasma concentration of all amino acids, except methionine, with red clover silage diets compared with grass silage diets. They suggested that the lack of further increase in milk production with red

4.4. Gas emissions Beauchemin et al. (2008) suggested that legumes could possibly reduce CH4 mitigation due to the lower fibre content, higher DMI and faster ruminal passage rate compared with grasses. However, a higher proportion of red clover in the diet in the present study did not decrease CH4 production and in fact tended to increase CH4 emissions. Similarly, van Dorland et al. (2007) concluded that white and red clovers had similar effects on CH4 emissions. Hammond et al. (2014) found no differences between red clover and perennial ryegrass fed as haylage or pasture grass in their effects on CH4 yield. Red clover had a lower CH4 output per g DM in vitro than perennial ryegrass, but this effect was reversed when expressed per g digested DM (Navarro-Villa et al., 2011). Despite the lower NDF concentration in red clover compared with grass, differences in rumen fermentation pattern do not explain the lower CH4 emissions from red clover diets. Greater molar proportions of acetate and lower proportions of propionate in VFA when comparing red clover and grass was shown both in vivo (Vanhatalo et al., 2009) and in vitro (Navarro Villa et al., 2011). However, unchanged rumen fermentation pattern between these forage types was reported by Bertilsson and Murphy (2003) and Dewhurst et al. (2003a). Overall, the relationships between dietary NDF concentration and fermentation pattern are reported to be not very strong in animals fed up to 40–50% concentrates in total DMI, and the differences in CH4 yield between non-fibre carbohydrates and NDF are also relatively small (Ramin and Huhtanen, 2013). Consistently with our previous study (Gidlund et al., 2015), increased protein supplementation decreased CH4 yield. One obvious reason for reduced CH4 yield with increased dietary CP concentration is that ruminal fermentation of protein produces less CH4 than ruminal fermentation of carbohydrates. According to the stoichiometric equations developed by Bannink et al. (2006) and Sveinbjörnsson et al. (2006), protein fermentation produces approximately 30 to 50% less CH4 than fermentation of carbohydrates. Increased DMI can also contribute to reduced CH4 yield as a function of increased protein supplementation (Yan et al., 2000; Ramin and Huhtanen, 2013). According to an equation by Ramin and Huhtanen (2013), the increase in DMI per kg body weight from the basal (B) to M level of protein would reduce the CH4 yield by 0.7 g/kg DMI. The rapeseed expeller used in the present study had a high fat concentration (94 g/kg DM), increasing the dietary fat concentration from 26 to 39 g/kg DM. According to the equation of Ramin and Huhtanen (2013), this would decrease CH4 yield by 0.4 g/kg DMI. An equation by Grainger and Beauchemin (2011), derived from fat supplementation studies predicted a difference of about 1.0 g/kg DMI decrease in CH4 yield between the B and H levels of protein supplementation. The reasons for the greater decline in CH4 yield with RC30 compared with RC70 remain unclear. Methane intensity (g CH4/kg ECM) decreased numerically with protein supplementation, but the linear trend did not reach significance because no further decreases were observed above the L level of protein supplementation. The effects of protein supplementation on CH4 intensity were similar to those observed in our previous study 79

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sacco evaluations of rumen function. J. Dairy Sci. 86, 2612–2621. Dewhurst, R.J., Fischer, W.J., Tweed, J.K.S., Wilkins, R.J., 2003b. Comparison of grass and legume silage for milk production. 1. Production responses with different levels of concentrate. J. Dairy Sci. 86, 2598–2611. Ericson, B., André, J., 2010. HPLC – applications for agricultural and animal science. In: Proceedings of the 1st Nordic Feed Science Conference, the Swedish University of Agricultural Sciences, Uppsala, Sweden, 23–26. Gidlund, H., Hetta, M., Krizsan, S.J., Lemosquet, S., Huhtanen, P., 2015. Effects of soybean meal or canola meal on milk production and methane emissions in lactating dairy cows fed grass silage-based diets. J. Dairy Sci. 98, 8093–8106. Grabber, J.H., 2009. Forage management effects on protein and fiber fractions, protein degradability, and dry matter yield of red clover conserved as silage. Anim. Feed Sci. Technol. 154, 284–291. Grainger, C., Beauchemin, K.A., 2011. Can enteric methane emissions from ruminants be lowered without lowering their production. Anim. Feed Sci. Technol. 166–67, 308–320. Halmemies-Beauchet-Filleau, A., Vanhatalo, A., Toivonen, V., Heikkilä, T., Lee, M.R.F., Shingfield, K.J., 2014. Effect of replacing grass silage with red clover silage in nutrient digestion, nitrogen metabolism, and milk fat composition in lactating cows fed diets containing a 60:40 forage-to-concentrate ratio. J. Dairy Sci. 97, 3761–3776. Hammond, K.J., Humphries, D.J., Westbury, D.B., Thompson, A., Crompton, L.A., Kirton, P., Green, C., Reynolds, C.K., 2014. The inclusion of forage mixtures in the diet of growing dairy heifers: impacts on digestion, energy utilisation, and methane emissions. Agric. Ecosyst. Environ. 197, 88–95. Huhtanen, P., Hristov, A.N., 2009. A meta-analysis of the effects of dietary protein concentration and degradability on milk protein yield and milk N efficiency in dairy cows. J. Dairy Sci. 92, 3222–3232. Huhtanen, P., Kaustell, K., Jaakkola, S., 1994. The use of internal markers to predict total digestibility and duodenal flow of nutrients in cattle given 6 different diets. Anim. Feed Sci. Technol. 48, 211–227. Huhtanen, P., Rinne, M., Nousiainen, J., 2007. Evaluation of the factor affecting silage intake of dairy cows: a revision of the relative silage dry-matter intake index. Animal 1, 758–770. Huhtanen, P., Nousiainen, J.I., Rinne, M., Kytölä, K., Khalili, H., 2008. Utilization and partition of dietary nitrogen in dairy cows fed grass silage-based diets. J. Dairy Sci. 91, 3589–3599. Huhtanen, P., Hetta, M., Swensson, C., 2011. Evaluation of canola meal as a protein supplement for dairy cows: a review and a meta-analysis. Can. J. Anim. Sci. 91, 529–543. Huhtanen, P., Bayat, A., Krizsan, S.J., Vanhatalo, A., 2014. Compartmental flux and in situ methods underestimate total feed nitrogen as judged by the omasal sampling method due to ignoring soluble feed nitrogen flow. Br. J. Nutr. 111, 535–546. Huhtanen, P., Cabezas-Garcia, E.H., Utsumi, S., Zimmerman, S., 2015. Comparison of methods to determine methane emissions from dairy cows in farm conditions. J. Dairy Sci. 98, 3394–3409. Huida, L., Väätäinen, H., Lampila, M., 1986. Comparison of dry matter contents in grass silages as determined by oven drying and gas chromatography water analysis. Ann. Agric. Fenn. 25, 215–230. Jaakkola, S., Saarisalo, E., Heikkilä, T., 2009. Formic acid treated whole crop barley and wheat silages in dairy cow diets: effect of crop maturity, proportion in the diet, and level and type of concentrate supplementation. Agric. Food Sci. 18, 234–256. Johnson, K.A., Johnson, D.E., 1995. Methane emissions form cattle. J. Anim. Sci. 73, 2483–2492. Jones, B.A., 1995. Red clover extracts inhibit legume proteolysis. J. Sci. Food Agric. 67, 329–333. Krizsan, S.J., Nyholm, L., Nousinainen, J., Südekum, K.H., Huhtanen, P., 2012. Comparison of in vitro and in situ methods in evaluation of forage digestibility in ruminants. J. Anim. Sci. 90, 3162–3173. Kuoppala, K., Ahvenjärvi, S., Rinne, M., Vanhatalo, A., 2009. Effects of feeding grass or red clover silage cut at two maturity stages in dairy cows. 2. Dry matter intake and cell wall digestion kinetics. J. Dairy Sci. 92, 5634–5644. Larsson, K., Bengtsson, S., 1983. Bestämning av lätt tillgängliga kolhydrater i växtmaterial. Method no. 22. Metodbeskrivning – Statens Lantbrukskemiska Laboratorium. Uppsala, Sweden. Lindgren, E., 1979. The nutritional value of roughages determined in vivo and by laboratory methodsReport no. 45. The Swedish University of Agricultural Sciences, Uppsala, Sweden (in Swedish). Lindgren, E., 1983. Nykalibrering av VOS-metoden för bestämmning av energivärde hos vallfoder. The department of animal nutrition and management. Stencil. The Swedish University of Agricultural Sciences, Uppsala, Sweden (in Swedish). Lüscher, A., Mueller-Harvey, I., Soussana, J.F., Rees, R.M., Peyraud, J.L., 2014. Potential of legume-based grassland-livestock systems in Europe: a review. Grass Forage Sci. 69, 206–228. Martineau, R., Ouellet, D.R., Lapierre, H., 2013. Feeding canola meal to dairy cows: a meta-analysis on lactational responses. J. Dairy Sci. 96, 1701–1714. Mela, T., 2003. Red clover grown in a mixture with grasses: yield, persistence and dynamics of quality characteristics. Agr. Food Sci. Finl. 12, 195–212. Merry, R.J., Lee, M.R.F., Davies, D.R., Dewhurst, R.J., Moorby, J.M., Scollan, N.D., Theodorou, M.K., 2014. Effects of high-sugar ryegrass silage and mixtures with red clover silage in ruminant digestion. 1. In vitro and in vivo studies of nitrogen utilization. J. Anim. Sci. 84, 3049–3060. Mertens, D.R., 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beakers or crucibles: collaborative study. J. AOAC Int. 85, 1217–1240. 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(Gidlund et al., 2015). In that study rapeseed meal improved CH4 intensity more than soybean meal, mainly because of the greater production response with diets containing rapeseed meal. Improved CH4 intensity resulted partly from increased ECM yield and partly from reduced CH4 yield. However, the positive effects of protein supplementation on CH4 intensity are counterbalanced by the increased manure N output. With increased dietary protein concentration a large proportion of incremental manure output is urinary N, which is more susceptible to leaching and evaporative losses than N in faeces (Castillo et al., 2000; Huhtanen et al., 2008). In terms of environmental effects, the L level of protein in the present study was optimal, as it decreased CH4 intensity by 11% without markedly increasing N losses. This level of protein also produced 70% and 78% of maximum milk and protein yield responses, respectively. This suggests that the L level of protein supplementation was optimal, both in terms of concern for the environment and farm profitability. Between the L and M protein levels, the manure N output per kg ECM was 20% greater for the M level, without any improvements in CH4 intensity. 5. Conclusions The suggested protein supplement sparing effect of including more red clover silage in the diet of dairy cows was rejected in this study. Increasing the CP intake from red clover silage did not show the same positive effect on milk production as increasing the CP intake from rapeseed expeller, although ME intake could have been a limiting factor. The lower fermentation quality with increased proportion of red clover silage could have been a confounding factor explaining the lack DMI response to increased silage proportion of red clover. Moreover, increased red clover silage inclusion did not decrease CH4 emissions, contradicting expectations. The optimal diet in environmental terms had low inclusion of red clover silage and low inclusion of rapeseed expeller. It produced about 70% of maximum milk yield and decreased CH4 intensity, without a major increase in N losses compared with the other diets. Funding This study was supported by the programme SLU Ekoforsk for field research projects within organic agriculture and horticulture, which is coordinated by the Swedish University of Agricultural Sciences (grant number: SLU ua Fe 20011.5.9-159). Conflicts of interest We confirm that there is no conflict of interest. References Ahvenjärvi, S.A., Vanhatalo, A., Huhtanen, P., Varvikko, T., 1999. Effects of supplementation of a grass silage and barley diet with urea, rapeseed meal and heatmoisture-treated rapeseed cake on omasal digesta flow and milk production in lactating dairy cows. Acta Agric. Scand. Sect. A Anim. Sci. 49, 179–189. Bannink, A., Kogut, J., Djikstra, J., France, J., Kebreab, E., Van Vuuren, A.M., Tamminga, S., 2006. Estimation of the stoichiometry of volatile fatty acid production in the rumen of lactating cows. J. Theor. Biol. 238, 36–51. Beauchemin, K.A., Kreuzer, M., O´Mara, F., McAllister, T.A., 2008. Nutritional management for enteric methane abatement: a review. Aust. J. Exp. Agric. 48, 21–27. Bertilsson, J., Murphy, M., 2003. Effects of feeding clove silages on feed intake, milk production and digestion in dairy cows. Grass Forage Sci. 58, 309–322. Broderick, G.A., 2003. Effects of varying dietary protein and energy levels on the production of dairy cows. J. Dairy Sci. 86, 1370–1381. Castillo, A.R., Kebreab, E., Beever, D.E., France, J., 2000. A review of efficiency of nitrogen utilisation in lactating dairy cows and its relationship with environmental pollution. J. Anim. Feed Sci. 9, 1–32. Davis, A.W., Hall, W.B., 1969. Cyclic change-over designs. Biometrika 56, 283–293. Dewhurst, R.J., 2013. Milk production from silage: comparison of grass, legume and maize silage and their mixtures. Agric. Food Sci. 22, 57–69. Dewhurst, R.J., Evans, R.T., Scollan, N.D., Moorby, J.M., Merry, R.J., Wilkins, R.J., 2003a. Comparison of grass and legume silages for milk production. 2. In vivo and in

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production and milk quality in dairy cows. J. Dairy Sci. 92, 1148–1160. Navarro-Villa, A., O´Brian, M., López, S., Boland, T.M., O´Kiely, P., 2011. In vitro rumen methane output of red clover and perennial ryegrass assayed using the gas production technique (GPT). Anim. Feed Sci. Technol. 168, 152–164. Nordic Committee on Food Analysis, 1979. Nitrogen, Determination in food and feed according to Kjeldahl, Report no. 6, Uppsala, Sweden. Oldham, J.D., 1984. Protein-Energy interrelationships in dairy cows. J. Dairy Sci. 67, 1090–1114. Olmos Colmenero, J.J., Broderick, G.A., 2006. Effect of dietary crude protein concentration on milk production and nitrogen utilization in lactating dairy cows. J. Dairy Sci. 89, 1704–1712. Ramin, M., Huhtanen, P., 2013. Development of equations for predicting methane emissions from ruminants. J. Dairy Sci. 96, 2476–2493. Rinne, M., Jaakkola, S., Kaustell, K., Heikkilä, T., Huhtanen, P., 1999. Silages harvested at different stages of grass growth v. concentrate foods as energy and protein sources in milk production. Anim. Sci. 69, 251–263. Shingfield, K., Vanhatalo, A., Huhtanen, P., 2003. Comparison of heat-treated rapeseed expeller and solvent-extracted soya-bean meal as protein supplement for dairy cows given grass silage based diets. Anim. Sci. 77, 305–317. Shingfield, K.J., Jaakkola, S., Huhtanen, P., 2001. Effect of level of nitrogen fertilizer application and various nitrogenous supplements on milk production and nitrogen utilization of dairy cows given grass silage-based diets. Anim. Sci. 73, 541–554. Sjaunja, L.O., Baevre, L., Junkkarinen, L., Pedersen, J., Setälä, J., 1990. A Nordic proposal for an energy corrected milk (ECM) formula. In: Gallion, P., Chabert, Y. (Ed.), Proc. 27th Session Intl. Committee of Recording and Productivity of Milk

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