White or red clover-grass silage in organic dairy milk production: Grassland productivity and milk production responses with different levels of concentrate

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Livestock Science 119 (2008) 202 – 215

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White or red clover-grass silage in organic dairy milk production: Grassland productivity and milk production responses with different levels of concentrate Håvard Steinshamn a,⁎, Erling Thuen b a

Organic Food and Farming Division, Norwegian Institute for Agricultural and Environmental Research, Tingvoll gard, N-6630 Tingvoll, Norway b Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 A°s, Norway Received 27 November 2007; received in revised form 8 April 2008; accepted 9 April 2008

Abstract Red (RC) or white (WC) clover were grown in mixture with grasses, ensiled and offered to dairy cows in early lactation over two successive years (48 cows per year) to compare grassland yield, feed intake, milk production and milk quality. The crops were ensiled in round bales and proportional mixtures of the second and third cut prepared each year were used to ensure that the silage treatments were representative of the crop. In addition to silage type, concentrate supplementation, without and with (10 kg/day), was included as a factor in a 2 × 2 factorial, continuous experiment. Total dry matter (DM) yield, silage chemical composition and total DM intake was hardly affected by silage type. There was no effect of silage type on milk yield and milk constituents either, except for higher milk protein content (P b 0.05) on WC and higher milk fat content of C18:3n-3 (P b 0.001), C18:2n-6 (P b 0.05) fatty acids (FAs) and sum of polyunsaturated FA (P b 0.001) and lower n-6/n-3 FA ratio (P b 0.01) on RC. Concentrate supplementation increased total DM, N and net energy intakes (P b 0.001), milk yield (P b 0.001), milk fat (P b 0.01) and protein (P b 0.001) content, decreased the milk urea content (P b 0.001), and increased the milk fat content of short- and medium-chained FAs (b C16, P b 0.001), C18:0 (P b 0.01) and C18:2n-6 (P b 0.001), decreased the content of C16:0 (P b 0.05), C18:1t11 (P b 0.001) and C18:3n-3 (P b 0.001), and increased the n6/n-3 FA ratio (P b 0.001). The effect of concentrate supplementation was not affected by silage type, except for milk protein content where the positive effect of supplementation was stronger on WC than on RC diets (P b 0.05). This study illustrates that the white- and red clover-grass mixtures investigated were widely similar with regard to their effects on grassland yield, silage intake and milk production and milk constituents, except for a higher milk fat content of C18:3n-3 and C18:2n-6 and lower n-6/n-3 FA ratio on red clover diets. Our findings also show that N conversion efficiency from feed to milk on pure forage diets is more sensitive to changes in dietary protein intake than silage diets containing cereal based concentrates. © 2008 Elsevier B.V. All rights reserved. Keywords: Organic farming; Legumes; Silage; Concentrate supplementation; Milk production; Fatty acids

1. Introduction

⁎ Corresponding author. Tel.: +47 404 80 314; fax: +47 71534405. E-mail address: [email protected] (H. Steinshamn). 1871-1413/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2008.04.004

In cool temperate regions, like in northern parts of Scandinavia and Finland, red clover is the dominant grassland legume, while white clover has been regarded suitable for grazing and not for cutting. In other

H. Steinshamn, E. Thuen / Livestock Science 119 (2008) 202–215

temperate regions, large leaf cultivars of white clover are also used for conservation (Abberton and Marshall, 2005). Red clover outyields white clover, but under less favourable growth conditions red clover and white clover have a more similar yield potential than on sites with more favourable conditions, and white clover grass tends to improve in yield over time compared to red clover grass (Halling et al., 2004). Previous investigations have shown that it is harder to maintain the breeding efficiency in dairy cows bred during winter in organic than in conventional husbandry (Reksen et al., 1999), which is likely due to both lower concentrate levels and forage energy concentrations and thereby lower dietary energy supply for the organically than for the conventionally managed cows (Kristensen and Kristensen, 1998; Olesen et al., 1999; Sehested et al., 2003; Haas et al., 2007). More frequent cuts may be a strategy to improve the quality of farm-grown forages for winter feeding. As white clover is more persistent and better adapted to frequent defoliation than red clover, it is worth considering white clover in organic ley farming in cool temperate regions. However, knowledge and research is limited on the cultivation and use of white clover in leys for cutting and silage preservation and its feeding value under cool temperate conditions. Earlier studies have showed similar feed intake and milk yield and composition to clover species (Bertilsson and Murphy, 2003; Dewhurst et al., 2003; Van Dorland et al., 2006). Red and white clovers are therefore to a large extent equivalent in usable energy and protein supply (Dewhurst et al., 2003; Van Dorland et al., 2006), or that differences between them depend on year (Bertilsson and Murphy, 2003). Intake of both red clover and white clover silages has shown to yield higher levels of polyunsaturated fatty acids (FA) in dairy cow milk, particularly of α-linolenic acid, than intake of grass silage (Dewhurst et al., 2003; Al Mabruk et al., 2004; Van Dorland et al., 2008; Vanhatalo et al., 2007). Comparisons of white and red clover silages have revealed only small differences in milk FA profiles (Dewhurst et al., 2003; Van Dorland et al., 2008). However, previous comparisons between red and white clover silages were made with clovers grown and fed as pure clovers or mixed at feeding with ryegrass (Lolium perenne L.) also cultivated in monoculture and not with silages prepared from mixed stands of grasses and clovers that is usually done in practice. The objective of the present study was to evaluate diets based on clover-grass silages prepared from either white or red clover, grown in mixture with grasses, without and with concentrate supplementation, representing the extremity in the adaptation to the standards

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for organic production (Council regulation (EC) No 1804/1999, 1999). The work tested the hypothesis that there are different effects of white- and red clover-grass leys and silages on grassland yield, feed intake, milk production and milk quality, including fatty acid composition, and N utilisation by organically managed dairy cows in early lactation. Additionally, a difference between white- and red clover-grass silage could depend on the concentrate supplementation level. 2. Materials and methods 2.1. Forage establishment Organically managed mixed stands of grass and clover were established in the spring of 2003 with barley (Hordeum vulgare cv. ‘Sunnita’, 195 kg/ha seeding rate) as a cover crop on a silty clay loam at the experimental farm of the Norwegian University of Life Sciences, Ås (59°66′N, 10°78′E. 95 m above sea level). The dominating soil texture classes are silty clay loam and loam, and the soil is well-drained, limed and in good fertility condition with respect to the content of P and K. A mixture of the grasses timothy (Phleum pratense cv. ‘Grindstad’, 8 kg/ha), meadow fescue (Festuca pratensis cv. ‘Fure’, 8 kg/ha), and perennial ryegrass (L. perenne cv. ‘Napoleon’, 4 kg/ha) was sown either with white clover (Trifolium repens cvs. ‘Milkanova’ and ‘Sonja’, 2.5 + 2.5 kg/ ha) or red clover (Trifolium pratense cvs. ‘Bjursele’ and ‘Nordi’, 2.5 + 2.5 kg/ha) in three replicates (average field size 2.3 ha). The forages were grown and harvested in the two following production years (2004 and 2005). Just before sowing, cattle slurry, on average 25 Mg/ha, was spread and incorporated into the soil within 24 h after application. Thereafter, no fertilizer or manure was applied to the crops. 2.2. Experimental silages The crops were cut for silage three times both years. The first cut was taken at booting stage of timothy and the two other cuts at approximately 7 week intervals thereafter. The first cut was ensiled in tower silos, while the second and third cuts, used as experimental feed, were ensiled in round bales. The leys were mowed using a disc-mower with conditioner. The crops were wilted in the mower swaths without any spreading and lifted with a precision chop harvester (1. cut) or by a round baler with a chopper (2. and 3. cuts) within 48 h of cutting. The target DM content was 30%. A potassium formate based additive (GrasAAT Eco; Yara Formates AS, Porsgrunn, Norway) was used at the average rate of 3.9 L/Mg of crop (recommended rate is 4–6 L/Mg). Round bales from the second and third cuts over the three replicates from each crop were mixed and used in the feeding experiment to ensure that the two silages were representative for the corresponding crops. First cut silage was not used primarily because of relative low clover proportion. The bales were unwinded, chopped and mixed just prior to feeding.

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2.3. Animals and dietary treatments

Table 1 Ingredient composition of concentrate supplements used

Each year 48 Norwegian Red dairy cows were used; 8 cows were primiparous in 2004 and 20 in 2005. The cows were housed in a free stall barn during an experimental period of 100 days in 2004 and 91 days in 2005. Before calving each year, the cows were allocated to 12 blocks according to expected calving date and lactation and then randomly allocated to four dietary treatments arranged according to a 2 × 2 factorial design: white clover-grass silage (WCS0), white clovergrass silage with concentrate supplementation (WCS1), red clover-grass silage (RCS0) and red clover with concentrate supplementation (RCS1). Forages and water were accessible ad libitum throughout the experiment. The cows with concentrate supplementation were given a constant amount of 10 kg/day of a standard commercial, certified organic concentrate mixture (Natura Drøv 10, Felleskjøpet Agri, Oslo, Norway) both years (Table 1). The concentrate levels were chosen to represent the extremes of the adjustment to the organic standards; i) whole ration based entirely on on-farm produced forages or ii) up to 40% of the daily DM intake as concentrated feed. As some cows calved during the experimental period the average number of days in experiment and in lactation was 72 (standard deviation (SD) = 32) and 70 (SD = 31) in 2004 and 71 (SD = 19) and 77 (SD = 21) in 2005, respectively. Before and after the experiment all cows were offered the same type of forage; organically produced third cut round bale silage (2004) or first cut silage (2005) before and first cut silage (2004) or second cut silage (2005) after the experiment.

Ingredients

Year 1

Year 2

g/kg fresh weight Oats Barley Wheat Cane molasses Fishmeal a Mineral and vitamin premix

550 240 50 60 70 30

600 260 – 40 70 30

Mineral/vitamin premix Vitamins A, IU/kg Vitamins D3, IU/kg Vitamins E, mg/kg Cu, mg/kg Se, mg/kg Zn, mg/kg Mn, mg/kg I, mg/kg Co, mg/kg

5000 2000 50 10 0.25 65 30 2 0.2

5000 2000 50 10 0.25 65 – 2 0.2

2.4. Data collection, sampling and chemical analysis Dry matter yield and botanical composition were determined by harvesting 6 plots of 0.5 m2 distributed evenly on a diagonal through each field (replica) the day before or the same day as the fields were cut for silage making. The plots were cut to a stubble height of 6 cm and the material was split into two sub samples, one for botanical analysis by hand separation and the other for DM determination by freeze drying. Individual feed intake was monitored by electronic balances registering intake at each access. Silage samples were taken from each bale before feeding and stored frozen and thereafter mixed to a monthly sample for each silage type, cut and replicate. Samples of concentrate (for all analyses) and silage (for DM, total N, pH, ammonia, lactic acid) were submitted wet (frozen). All other analyses were conducted using freeze-dried material after milling through 1.0 mm or 1.5 mm (for indigestible NDF determination) screens. The DM content of grass silage was determined by freeze drying and oven drying (Commission Directive 71/393/EEC, 1971). Silage samples were diluted with distilled water and stored frozen, then thawed and filtrated before analysing organic acids and ethanol by HPLC (Nucleogel Ion 300 OA, 300 × 7.7 mm, column). Detection of lactic acid was performed with an UV

a

Fishmeal is allowed in organic milk production in Norway.

detector, while the detection of other volatile acids and ethanol was performed with an IR detector. Ammonia N in silage was determined according to the Standard Methods for the Examination of Water and Wastewater (1995). Ash content was determined by combustion at 550 °C (Commission Directive 71/250/EEC, 1971). Crude protein (N × 6.25) was determined using a Kjeldahl procedure (Kjeltec1035 analysator) according to Commision Directive 93/28/EEC (1993) and crude fat according to Commission Directive 98/64/EC (1998). Neutral detergent fibre was determined using an ANKOM220 fibre analyser (ANKOM Technology, Fairport, NY) with sodium sulphite and α-amylase in the neutral detergent and in vitro dry matter digestibility (IVDMD) according to Tilley and Terry (1963) using the DAISYII Incubator (ANKOM Technology, Fairport, NY). Fatty acid methyl esters (FAME) were prepared in a one-step extraction–transesterification procedure (Sukhija and Palmquist, 1988) using 2% (vol/vol) methanolic sulphuric acid as a catalyst and C19 as an internal standard. Methyl esters were quantified by gas–liquid chromatography (GC) using a gas chromatograph fitted with a 100 m fused-silica capillary column (Restek Rt-2560) and He as the carrier gas. The total FAME profile in a 2 µL sample volume at a split ratio of 1:70 was determined using a temperature gradient programme. Peaks were identified using authentic standards (Supelco TM 37 Component FAME MIX). Rumen indigestible NDF (iNDF) content of the experimental silages was determined in sacco according to the NorFor standard 070910 (NorFor http://www.norfor.info/). The content of net energy lactation (NEL) in the feed was calculated from feed chemical composition and digestibility values according to Van Es (1975, 1978). The cows were milked twice a day. Milk yield was recorded for each milking, and live weight of the cows was recorded after each milking by an electronic balance. Milk

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samples were collected from 6 consecutive milkings every second week, conserved with Bronopol (D&F Inc., Dublin, CA, USA) and analysed for fat, protein, lactose, urea and free fatty acids with an infrared spectrophotometer (MilcoScan(tm) FT6000, Foss Electric, Hillerød, Denmark). Energy corrected milk yield (ECM) was calculated from chemical composition (Sjaunja et al., 1990). Additional milk samples were collected from 28 cows (7 in each treatment, selecting the once that calved first within each treatment but balancing for parity and lactation stage) at 4 week intervals at 2 occasions the first year and 3 occasions the second year. These samples were stored at −20 °C without preservation, prior to analysis of fatty acids by GC as FAME according to Feng et al. (2004) at AS Vitas, Oslo. Analyses were performed with a 6890N GC with a split/ splitless injector, a 7683B automatic liquid sampler, and flame ionization detection (Agilent Technologies, Palo Alto, CA). Separation was performed with a CP-Select CB for FAME (200 m × 0.25 mm i.d. × 0.25 µm film thickness) fused-silica capillary column (Varian Inc.). Fatty acid analysis was performed by autoinjection of 1 µL of each sample at a split ratio of 70:1. Carrier gas was H2 with a pressure of 314 kPa, with a H2 flow of 151 mL/min and a temperature of 280 °C. The flame ionization detector temperature was 290 °C with H2, air and N2 make-up gas flow rates of 40, 450 and 45 mL/min, respectively. A temperature gradient (starting at 70 °C with 4 min hold, 20 °C/min rise to 160 °C, hold for 80 min, 3 °C/ min rise to 220 °C, hold for 28 min) was used to separate the FAME. The sampling frequency was 10 Hz. The run time for a single sample was 136 min. 2.5. Statistical analysis Weighted mean values of each year were used in the statistical analysis of feed intake, milk production, and milk composition. Statistical analysis was performed using the Restricted Maximum Likelihood method of the MIXED procedure in (SAS, 1999). The following statistical model was used: Yijklm ¼ μ þ ai þ bj þ ck þ em þ D þ Am ðai bj ck Þ þ εijklm where: Y = dependent variable, μ = overall mean, a = fixed effect of year (i = 2004 or 2005), b = fixed effect of silage type j ( j = white clover or red clover-grass silage); c = fixed effect of concentrate supplementation k (k = 0 (without) or 1 (with)); e = fixed effect of lactation number (m = 1 (primiparous) or 2 (multiparous)); D= fixed effect of average days in lactation during the experiment (continuous); Am(aibjck) = random effect of cow m within treatment i and j and k; random residual variation εijklm ∼ N(0,σ2). All fixed effect interactions were also included. As some cows were present in both years (22 out of 71 cows used) the covariation within animal was accounted for in an analysis of repeated measure. The optimal covariance structure was assessed for each variable with attention to Akaike's Information Criterion and Schwartz's Bayesian Criterion (Littell et al., 1998). The tables give treatment LSmeans and the average group standard errors of the means (SEM) of the LSmeans across years. One cow in

205

Table 2 Effect of ley type (WCL, white clover-grass ley; RCL, red clover-grass ley) and cut on dry matter yield (g/m2) and herbage species composition (% of DM yield) averaged across years (n = 6) Item

Cut

WCL RCL SEM a Significance b

Dry matter yield, g/m2

1 2 3 Total

275 340 289 904

261 369 283 913

17 32 33 29 21 15 35 36 48 4.3 2.1 0.6

26 46 52 29 17 8 28 27 35 3.0 2.8 1.4

Species, % of DM yield Clover 1 2 3 Timothy 1 2 3 Other sown grasses c 1 2 3 Herbs not clover 1 2 3

S

Cut S × Cut

20.5

ns

*** *

60.8

ns

–

–

4.4

*** *** ns

3.8

ns

*** ns

4.4

(*)

**

ns

1.68

ns

*

ns

a

SEM = standard error of the means. Statistical significance; (*), P b 0.1; *, P b 0.05; **, P b 0.01; ***, P b 0.001; S = effect of clover species; Cut = effect of cut; S × Cut = effect of clover species × cut interaction. c Perennial ryegrass and Meadow fescue. b

2004 and three cows in 2005 were omitted due to failure with electronic responder (1 cow), missing forage intake data (2 cows) and teat injury (1 cow).

3. Results 3.1. Crop yield and clover proportion Total annual forage yields were about 9 t DM/ha, and the yields were similar between the white clover-grass ley (WCL) and the red clover-grass ley (RCL) (Table 2). Second cut yielded higher (P b 0.001) than the two other cuts in both crops. The yield difference between the second and the two other cuts was greater in RCL than in WCL as indicated by the significant interaction between cut and clover species (P b 0.05). The mean weighted clover content (%) in DM yield was higher (P b 0.001) in RCL than WCL in all three cuts (Table 2). In both crops, the clover content increased (P b 0.001) from the first to the two subsequent cuts. This resulted in a higher clover proportion in the experimental feed than in total yield. The yield proportion of timothy decreased (P b 0.001) while the sum of other sown grasses increased (P b 0.01) from the first to the third cut.

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3.2. Feed composition Chemical composition of the concentrates and the experimental silages are shown in Tables 3 and 4, respectively. Concentrate in vitro DM digestibility (IVDMD) and net energy (NEL) were higher in Year 1 than 2 (Table 3). There were only small differences in silage DM composition, IVDMD and NEL between white clover-grass silage (WCS) and red clover-grass silage (RCS) within a year. DM content in WCS was higher (P b 0.001) than in RCS the first year, but lower (P b 0.05) than in RCS the second year. Crude protein content in both silages was higher the first than the second year, reflecting a reduction in herbage clover content in both leys. The NDF content in the two silages was low both years, with a numerically small but significant higher (P b 0.05) NDF content in WCS than in RCS the second year. The major fatty acids in the silage were C18:3, followed by C16:0 and C18:2 (Table 4). Fermentation measurements indicate that all silages were well-preserved (Table 4). In the first year, the total diet levels of crude protein and net energy were higher and of NDF and fat lower than in the second year (Table 5). 3.3. Feed intake Cows offered WCS had on average 0.6 kg DM higher (P b 0.1) silage DM intake (DMI) than cows offered Table 3 Chemical composition and feed values of concentrate supplements and hay (g/kg) DM, unless stated otherwise Year 1

n Dry matter (g/kg) Ash Crude protein NDF a Crude fat Starch IVDMD b (%) NEL c (MJ/kg DM) Fatty acids Total C16:0 C18:0 C18:1 cis-9 C18:2n-6 C18:3n-3 a b c

Year 2

Concentrate

Hay

Concentrate

Hay

5 876 71 161 213 48 429 82.2 7.00

2 893 51 114 398 30 – 76.8 6.61

3 886 68 159 215 46 414 79.1 6.70

4 919 53 87 660 13 – 62.9 5.20

33.86 10.27 1.04 9.42 4.46 0.90

17.68 4.90 0.39 1.71 2.02 2.36

37.62 9.30 0.95 12.79 10.46 1.41

10.50 2.72 0.21 0.75 1.90 2.68

NDF, neutral detergent fibre with ash Year 1 and ash free in Year 2. IVDMD, in vitro dry matter digestibility. NEL, calculated according to Van Es (1975, 1978).

RSC (Table 6). Concentrate supplementation reduced (P b 0.001), as anticipated, silage DMI of both silages. The reduction in silage DMI per kg DM of concentrate was slightly stronger in WCS (0.32 kg/kg) than in RCS (0.26 kg) and resulted in only small differences in total DMI between the two types of silages supplemented with concentrates. Intake differences between silage types and concentrate supplementation level of individual FA mostly reflected differences in feed concentration and DM intake (Table 6). The intake of C18:3n-3 was higher (P b 0.1) in cows offered WSC than RCS, otherwise there was no significant effect of silage type on FA intake. On average, concentrate supplementation doubled the intake of C16:0, C18:0 and C18:2n-6 (P b 0.001), increased the intake of C18:1c-9 with a factor of ten (P b 0.001), but reduced the intake of C18:3n-3 (P b 0.01). The N intake was significantly influenced by clover species (P b 0.01) and concentrate level (P b 0.001) (Table 6). The daily intake of N was higher for the cows offered WCS than RCS diets the first year (figures not shown), but similar for both silages the second year with an overall daily mean difference in N intake of 53 g in favour of WCS for both years. Concentrate supplementation increased on average daily N intake by 131 g in WCS and 150 g in RCS. 3.4. Milk production and composition There were only small effects of clover species on most of the production parameters (Table 7). On average, intake of WCS diets resulted in higher (P b 0.05) protein content in milk than RCS. However, there were both clover species with year interaction (P b 0.05, figures not shown) and clover species with concentrate supplementation interaction (P b 0.05) effects on milk protein content. The higher milk protein content on WCS occurred only in the first year and in cows offered concentrate. There was a tendency (P b 0.1) that cows on WCS produced milk with a higher content of fat than cows fed RCS. Concentrate supplementation had a significant elevating effect on most production parameters (Table 7). The overall response across years in daily milk and ECM production per kg concentrate DM was similar for the two silages; 0.76 kg and 0.78 kg milk and 0.96 kg and 0.87 kg ECM for cows offered WCS and RCS, respectively. The milk yield response to extra energy supply with supplementation was on average + 0.16 and +0.15 kg milk/MJ NEL and + 0.20 and + 0.17 kg ECM/ MJ NEL for WCS and RCS, respectively. The concentrate effect on milk and ECM production was more

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207

Table 4 Chemical composition and feed values of the experimental silages (g/kg DM, unless stated otherwise); grass-white clover (WCS) and grass-red clover (RCS) weighted according to the proportion represented by each sample of the crop yield Year 1

Year 2 WCS

RCS

SEM

Sign a

** * ns ns ns ns ns ns

3 268 92 150 447 97 42 74.9 6.07

3 284 95 147 441 103 40 72.7 5.84

2.2 0.9 2.0 1.0 5.9 0.3 0.72 0.075

* ns ns * ns (*) ns ns

0.982 0.155 0.014 0.036 0.160 0.502 0.025 2.13 1.63

ns ns ns ns ns ns ns * ns

25.78 3.70 0.43 0.65 3.51 11.32 4.64 67.2 7.48

27.37 3.93 0.45 0.61 3.64 11.58 4.76 66.5 6.34

0.534 0.080 0.007 0.022 0.022 0.100 0.044 1.40 0.779

ns ns ns ns (*) ns ns ns ns

2.22 0.36 0.024 0.002

ns * (*) ns

59.4 13.7 1.52 0.04

58.9 13.6 1.36 0.00

1.48 0.26 0.168 0.029

ns ns ns ns

WCS

RCS

SEM

Sign

4 313 94 171 419 101 37 75.2 6.10

4 285 99 163 426 96 36 74.7 6.00

1.6 0.7 2.8 3.6 8.0 2.0 0.58 0.059

Fatty acids (g/ kg DM) Total C16:0 C18:0 C18:1 cis-9 C18:2n-6 C18:3n-3 pH Ammonia N (g/kg total N) Ethanol (g/kg DM)

26.12 3.91 0.43 0.58 3.66 9.80 4.64 78.3 13.2

22.72 3.57 0.38 0.47 3.46 9.22 4.57 67.4 9.1

Fermentation acids (g/kg DM) Lactate Acetate Propionate Butyrate

40.1 10.8 2.74 0.05

43.5 13.5 3.58 0.01

n Dry matter (g/kg) Ash Crude protein NDF b iNDF c Crude fat IVDMD d (%) NEL e, (MJ/kg DM)

a b c d e

a

Statistical significance; (*), P b 0.1; *, P b 0.05; **, P b 0.01; ***, P b 0.001. NDF, neutral detergent fibre with ash Year 1 and ash free in Year 2. iNDF, indigestible neutral detergent fibre. IVDMD, in vitro dry matter digestibility. NEL, net energy lactation calculated according to Van Es (1975, 1978).

pronounced the first than the second year (P b 0.01, figures not shown). Supplementation also increased the milk fat (P b 0.01) and protein (P b 0.001) content. Milk urea decreased (P b 0.001) with concentrate supplementation, and there was a tendency (P b 0.1) that supple-

mentation also reduced the milk content of free fatty acids. Cows offered concentrate maintained on average their live weight, while those who were not supplemented lost on average 370 and 500 g daily on WCS and RCS, respectively (P b 0.001).

Table 5 Chemical analysis of the total diet (g/kg DM, unless stated otherwise); grass-white clover (WCS) and grass-red clover (RCS) without (0) and with (1) concentrate supplementation Year 1

DM, g/kg Crude protein NDF a Fat NEL b (MJ/kg DM) Total fatty acids a b

Year 2

WCS0

WCS1

RCS0

RCS1

WCS0

WCS1

RSC0

RCS1

321 168 422 37 6.2 22.7

412 166 344 41 6.6 26.5

291 162 429 35 6.2 22.4

394 162 344 40 6.5 26.9

270 150 449 42 6.1 27.0

377 153 356 44 6.3 31.3

288 145 442 40 5.9 27.1

398 150 352 42 6.2 31.5

NDF, neutral detergent fibre with ash Year 1 and ash free in Year 2. NEL, net energy lactation calculated according to Van Es (1975, 1978).

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Table 6 Effects of clover silages (WCS, grass-white clover; RCS grass-red clover) and concentrate supplementation (0, without; 1, with 10 kg per day and cow) on intake of DM, N, crude fat, NDF, net energy (NEL) and major fatty acids Item

WCS 0

n

SEM a

RCS 1

0

1

22

24

23

23

Feed DM intake (kg/day) Silage Hay Total N intake (g/day) Fat intake (g/day) NDF intake (kg/day) NDF intake (g/kg LW) NEL (MJ/day)

14.4 0.5 14.8 373 581 6.4 11.8 90

12.0 0.3 19.9 504 839 6.9 11.9 127

13.6 0.4 13.9 337 522 6.0 11.1 83

11.6 0.4 19.7 487 805 6.8 11.6 124

Fatty acids intake (g/day) C16:0 C18:0 C18:1 cis-9 C18:2n-6 C18:3n-3

58 6 9 53 154

123 13 94 102 134

51 6 8 47 139

123 13 97 101 128

Significance b S

C

S×C

0.35 0.04 0.35 8.6 13.1 0.15 0.35 2.1

(*) ns ns ** *** ns (*) *

*** * *** *** *** *** ns ***

ns * ns ns ns ns ns ns

2.1 0.2 0.9 1.9 5.6

ns ns ns ns (*)

*** *** *** *** **

ns ns * ns ns

a

SEM = standard error of the means. Statistical significance; (*), P b 0.1; *, P b 0.05; **, P b 0.01; ***, P b 0.001; S = effect of clover species; C = effect of concentrate supplementation; S × C = effect of clover species × supplementation interaction. b

(P b 0.05), C18:3n-3 (P b 0.001), C20:5n-3 (P b 0.01) and total PUFA (P b 0.001), had a higher (P b 0.01) ratio between unsaturated and saturated FA and lower (P b 0.01) ratio between n-6 and n-3 FA (Table 8). The

3.5. Milk fatty acid profile Milk from cows offered RCS contained higher levels of the polyunsaturated fatty acids (PUFA) C18:2n-6

Table 7 Effects of clover silages (WCS, grass-white clover; RCS grass-red clover) and concentrate supplementation (0, without; 1, with 10 kg per day and cow) on milk production performance, excretion of milk fat, lactose, protein and milk free fatty acids, live weight change, nitrogen utilisation, feed conversion and energy balance Item

WCS 0

1

n

22

24

Milk production and content Milk yield (kg/day) ECM (kg/day) Milk fat (g/kg) Milk protein (g/kg) Milk lactose (g/kg) Urea (mmol/L) Free fatty acids (mEq/L) Live weight change (kg/day) N efficiency (milk N/feed N, %) Feed conversion (EKM/ kg DM) Energy balance (MJ/day) c

22.1 20.8 37.0 30.3 46.4 4.55 0.60 − 0.50 28.5 1.34 − 8.2

27.9 28.1 39.6 33.6 47.1 3.71 0.50 0.11 28.9 1.37 2.5

a

SEM a

RCS 0

1 23

22.0 20.6 36.2 30.2 46.8 4.55 0.62 − 0.37 30.9 1.46 − 14.6

Significance b S

C

S×C

ns ns (*) * ns ns ns ns ns ns ns

*** *** ** *** ns *** (*) *** ns ns ***

ns ns ns * ns ns ns ns ns ns ns

23

28.1 27.4 38.2 32.4 47.1 3.85 0.50 0.01 29.2 1.35 2.0

0.65 0.65 0.72 0.37 0.32 0.083 0.050 0.084 0.96 0.069 3.05

SEM = standard error of the means. Statistical significance; (*), P b 0.1; *, P b 0.05; **, P b 0.01; ***, P b 0.001; S = effect of clover species; C = effect of concentrate supplementation; S × C = effect of clover species × supplementation interaction. c Energy balance was calculated as: Net energy intake – net energy required for maintenance – net energy secreted in milk. b

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Table 8 Effects of clover silages (WCS, grass-white clover; RCS grass-red clover) and concentrate supplementation (0, without; 1, with 10 kg per day and cow) on milk fatty acid composition (g/100 g FAME), groups of fatty acids (g/100 g FAME), fatty acid ratios (g/g), and apparent recoveries of dietary C18:2 and C18:3 in milk fat from dairy cows fed the experimental diets (%) Fatty acids

WCS

SEM a

RCS

0

1

0

1

14

14

14

14

Individual fatty acid (g/100 g FAME) C4:0 4.36 C6:0+C12:0 3.67 C10:0+C12:0 5.63 C14:0 10.61 C16:0 28.54 C18:0 9.71 C18:1 cis-9 18.93 C18:1 trans-11 1.99 C18:2n-6 0.86 C18:3n-3 0.87 C18:2 t-9, c-11 + C20:0 1.02 C20:4 (ARA) 0.08 C20:5 (EPA) 0.11 C22:6 (DHA) 0.01

4.54 4.06 6.43 11.09 27.20 10.55 19.24 1.47 1.26 0.58 0.91 0.09 0.10 0.10

4.27 4.17 5.29 10.23 27.85 9.80 19.93 2.01 0.89 1.04 1.02 0.08 0.12 0.01

4.92 5.29 6.79 11.03 26.15 11.08 18.68 1.60 1.35 0.69 0.92 0.09 0.11 0.12

Fatty acid groups (g/100 g FAME) c SFA 65.00 MUFA 23.98 PUFA 2.84 bC16 20.24 C16:0+C16:1 29.93 NC16 35.32

65.65 23.84 2.92 20.97 28.48 36.00

63.43 25.12 3.09 19.78 29.29 36.71

65.57 23.28 3.17 21.14 27.28 36.39

n

Fatty acid ratios (g/g) c (PUFA+MUFA)/SFA n-6/n-3

0.044 0.94

Apparent recovery from feed to milk (%) C18:2n-6 11.0 C18:3n-3 4.08

0.045 1.77

13.6 4.44

0.049 0.86

14.0 6.02

0.049 1.61

14.3 5.40

Significance b S

C

S×C

0.123 0.088 0.230 0.230 0.664 0.323 0.615 0.078 0.037 0.028 0.034 0.003 0.003 0.005

ns ns ns ns ns ns ns ns * *** ns ns ** (*)

(*) *** *** *** * ** ns *** *** *** ** ** *** ***

ns ns ns ns ns ns ns ns ns ns ns ns ns ns

0.777 0.691 0.072 0.312 0.667 0.858

ns ns *** ns ns ns

(*) ns ns ** ** ns

ns ns ns ns ns ns

0.002 0.044

** **

ns ***

ns ns

0.91 0.393

* ***

ns ns

ns ns

a

SEM = standard error of the means. Statistical significance; (*), P b 0.1; *, P b 0.05; **, P b 0.01; ***, P b 0.001; S = effect of clover species; C = effect of concentrate supplementation; S × C = effect of clover species × supplementation interaction. c SFA, saturated fatty acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; bC16, fatty acids with chain length less than 16 C atoms; NC16, fatty acids with chain length containing more than 16 C atoms; n-6, n-6 fatty acids; n-3, n-3 fatty acids. b

effect of RCS was consistent and independent of concentrate level as there was no significant interaction of silage type with supplementation. Concentrate supplementation reduced the content of the FAs C16:0 (P b 0.05), C18:1t11 (P b 0.001), C18:3n-3 (P b 0.001) and C20:5n-3 (P b 0.01), and increased the content of short (C6:0–C12:0, P b 0.001) and medium (C14:0, P b 0.01) chain FA, C18:0 (P b 0.01), C18:2n-6 (P b 0.001) and C22:6n-3 (P b 0.001) (Table 8). There was a tendency that concentrate supplementation increased (P b 0.1) the content of saturated FA but had no significant effect on the sum of monounsaturated

and polyunsaturated FA (Table 8). Supplementation increased (P b 0.001) the n-6/n-3 FA ratio. 3.6. Feed and nitrogen conversion Feed conversion, defined as kg ECM per kg DM intake, and N utilisation, defined as the ratio between milk and feed N, was similar between treatments (Table 7). The two conversion indicators were, however, significantly influenced by the interaction between year and clover species in that the both values were highest in RCS the first year but highest in WCS the second (figures not shown).

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3.7. Reproduction and health The number of cows was limited, and only treatment sums and average values of reproductive performance and clinical treatments are presented (Table 9). There were no differences in reproduction performance measured by the number of services and calving interval between cows with and without concentrate. The incidents of metabolic diseases, milk fever and ketosis were low with no apparent differences between treatments (Table 9). 4. Discussion The purpose of the present study was to compare the effects of white and red clover grown in mixture with grasses on forage yields and on forage intake, milk production and quality when offered as silage. Previous comparisons between red and white clover silages were made with clovers grown and fed as pure clovers or mixed at feeding with ryegrass (L. perenne), also cultivated in monoculture (Bertilsson and Murphy, 2003; Dewhurst et al., 2003; Van Dorland et al., 2006). Experimentally, there are good reasons for separate species cultivation and feeding. This makes it possible to administrate the clover in a desired proportion and the interpretation of the results is simpler since the effect of legume species may be confounded with the legume proportion when grown in mixtures with grasses. However, cultivation and feeding of pure clover may have undesirable consequences such as a risk of N leaching losses in field (Loiseau et al., 2001), high forage protein content and thereby poor N utilisation in the animal (Steg et al., 1994; Van Dorland et al., 2006) and bloat (Kuusela et al., 2004). Furthermore, nutrient content and forage quality of grasses and clovers, particular grasses, may differ when grown in pure or mixed grass-clover

stands (Lehmann et al., 1978). The effect of a plant species grown in pure stands on milk production and quality is thus not necessarily the same as when those species are cultivated in mixed stands. The level of clover in the complete diets of the present study ranged from 17 to 37% on WC containing diets and from 23 to 53% on RC containing diets. Therefore, effects of other diet ingredients and of clover proportion cannot be excluded. However, the clover proportion level and the difference between the two grassland types in clover proportion are what may realistically be achieved in mixed grass-clover leys and in organic ley farming (Frame and Harkess, 1987; Lunnan, 1989; NilsdotterLinde et al., 2002; Kunelius et al., 2006). Our findings thus have considerable applied relevance. 4.1. Forage yields The observed similarity in forage yield differs from what is usually found when red and white clover are managed under the same cutting regime, whereas the lower clover proportion in the white clover swards is in agreement with other studies (Frame and Harkess, 1987; Lunnan, 1989; Mallarino and Wedin, 1990; Kunelius et al., 2006). However, similarity in total yields between red clover and white clover-grass mixtures is also reported by others even when managed under the same cutting regime during the two first production years as in the present study (Svanäng and Frankow-Lindberg, 1994; Nilsdotter-Linde et al., 2002). In these studies, lower clover proportion in the white clover swards was also observed, but beyond the second production year the DM yield and clover proportion was higher in the white clover than in the red clover swards. The present study was run on a soil of high nutrient status, and the companion grasses are high yielding species that compete strongly with white clover and presumably

Table 9 Number of cows clinically treated for hypocalcemia and ketosis, number cows culled because of infertility, average number of services per cow and calving intervals in days in each clover silage treatment (WCS, grass-white clover; RCS grass-red clover) and concentrate supplementation (0, without; 1, with 10 kg per day and cow) across both experimental years Item

WCS

RCS

0 Mean/sum Clinical treatments (no. of cows treated) Hypocalcemia 3 Ketosis 1 No. culled due to infertility 1 No. of services per cow 1.92 Calving interval (days) 386

1 SD

Mean/sum

1.06 123

4 0 2 1.96 376

0 SD

Mean/sum

1.50 163

3 2 0 1.42 371

1 SD

Mean/sum

SD

0.58 35

3 0 2 2.29 396

1.88 164

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compensated for the lower yield potential of white clover. Nevertheless, the results indicate that white clover grown in mixtures with high yielding cool temperate grasses may give similar yields as red clover grass, even in short term rotational leys. 4.2. Feed nutritive value The differences in DM found between the silage types were likely due to differences in forage clover proportion (high moisture content in RC at harvest). The dietary crude protein content was lower but the NDF content similar to the observations in other studies (Dewhurst et al., 2003; Bertilsson and Murphy, 2003; Van Dorland et al., 2006). Pure white clover silage has usually higher CP content and lower NDF content than pure red clover silage (Dewhurst et al., 2003; Bertilsson and Murphy, 2003; Van Dorland et al., 2006). The small difference in silage digestibility and content of CP and NDF found in the present study is likely a result of difference in clover proportion, being lower in the white clover-grass silage. Due to their high buffering capacity and relatively low water-soluble carbohydrate content, legumes are regarded as difficult to ensile, but the fermentation characteristics indicate a moderate fermentation and high fermentation quality. The C18:3n-3, C18:2n-6 and C16:0 contents of experimental silages were mainly as observed previously in wilted clover silages (Dewhurst et al., 2003; Van Dorland et al., 2008; Vanhatalo et al., 2007). Differences in C18:3n-3 among studies could be due to differences in sward composition, and management practices like pre wilting and the use of silage additives (Dewhurst et al., 2006). The differences in concentrate composition (Table 1) explains the higher content of polyunsaturated fatty acid, particular linoleic acid, lower content of starch and lower digestibility and energy concentration in the second than in the first year (Table 3). 4.3. Feed intake Higher silage DMI of WCS than of RCS can hardly be explained by the chemical characteristics of the silage as important variables known to influence DMI, such as the NDF content and DM digestibility were similar (Mertens, 1994). The extent of fermentation and DM content in the present study was significantly higher on WSC. However, the difference in fermentation was small, and using the silage DMI index equation of Huhtanen et al. (2007), which includes both effect of digestibility, DM content, fermentation acid content and clover proportion, revealed

211

that the silages had the same index value. Thus, the chemical analysis of the silages could not explain the slightly lower intake of RCS than of WCS. The substitution effect of concentrate for silage was moderate (average − 0.32 and − 0.26 kg/kg for WCS and for RCS, respectively) and agreed broadly with results of Dewhurst et al. (2003) who found substitution rates between − 0.29 and − 0.41 kg/kg and with − 0.41 found in an analysis across different silage feeding experiments by Rook et al. (1991). 4.4. Milk production and composition The higher nutritive value of the first year concentrate is the likely reason for the greater milk production response to supplementation in the first than in the second year. On average, the milk yield response to increased energy supply was similar for WCS (0.16 kg/MJ NEL) and RCS (0.15 kg/MJ NEL) and comparable to the 0.13 kg/MJ NEL found in cows in early lactation by Coulon and Remond (1991). The positive response of milk protein content to concentrate supplementation was likely related to higher intake of all nutrients. Extra energy intake in the supplemented cows stimulated the production of glucogenic nutrients in the rumen, which in turn increased rumen microbial protein synthesis (Rigout et al., 2003). Previous clover silage studies and studies with organically managed cows have shown that grain based supplementation increases milk protein content (Sehested et al., 2003; Cohen et al., 2006). The average response to increased energy supply on WCS diets of 87 mg protein per kg milk/MJ NEL was higher than on RCS diets (58), but similar to the response (85 mg protein per kg milk/ MJ NEL) found in early lactating multiparous cows by Coulon and Remond (1991) in their meta analysis. Increased milk fat content with elevated concentrate feeding is somewhat anomalous as the opposite effect is generally observed (Chilliard et al., 2000). However, this effect is sometimes observed in early lactation, as in the current experiment, and is likely due to low energy status in cows on a pure forage diet (Laird et al., 1981; Aston et al., 1995). The unsupplemented cows lost weight and were in negative energy balance (Table 7). Cows in negative energy balance synthesize less shortand medium chain FA as the mammary de novo synthesis of these short-chained FA is inhibited by uptake of long-chain FA (C16:0, C18:0, C18:1c-9) mobilized from adipose tissue (Palmquist et al., 1993). The assumption appears to be confirmed by the lower concentrations of short- and medium-chained FAs (b C16) and higher content of C16:0 in the milk fat produced on the

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forage only diets. Mobilization of adipose FA increases the milk content of C18:0 and C18:1c-9 as well (Chilliard et al., 2000). However, in our study the supplemented cows had significantly higher milk content of C18:0 than those on forage only (Table 8), which was likely due to higher dietary supply of C18:0 precursors. Supplementation increased the total fat intake considerably (Table 6), and the dietary intake of the sum of C18:1c9 and C18:2n-6 was on average 197 and 59 g/day for supplemented and unsupplemented cows, respectively. Dietary C18:1c9 and C18:2n-6 is extensively biohydrogenated to C18:0 as the end product (Harfoot and Hazlewood, 1997). Thus, the likely reason for higher milk content of C18:0 with supplementation was higher dietary supply of C18:0 precursors (C18:1c9 and C18:2n-6). Although extensive rumen hydrogenation, some dietary unsaturated FA escapes the rumen, explaining higher content of C18:2n-6 and lower content of C18:3n-3 in milk from cows offered concentrates than on forage only diet. This effect of grain supplementation on milk C18:2n-6 and C18:3n-3 is in accordance with others (Dewhurst et al., 2003). RSC diets increased milk concentration of C18:3n-3 despite similar diet concentration and lower intake of C18:3n-3 in comparison with WCS, and the effect of RCS was independent of concentrate supply. There was no significant difference in live weight change between forage types either. Thus, the effect of RCS cannot be explained by higher intake of C18:3n-3 or by greater supply of C18:3n-3 from mobilized adipose tissue. However, apparent recovery of C18:3n-3 from feed to milk was on average 4.3% for WCS and 5.7% for RCS. Dewhurst et al. (2003) also observed higher C18:3n-3 recovery on red clover than on white clover silages and this is likely due to less biohydrogenation on RSC than on WCS diets (Lee et al., 2003). The action of the enzyme polyphenol oxidase, in red clover, that inhibits lipolysis by producing electrophilic quinones has been suggested to be the mechanism of this reduced biohydrogenation (Lee et al., 2004). 4.5. N utilisation As the dietary CP content was similar, no difference between treatments in the apparent N utilisation from feed to milk (g N in milk per g N intake) was to be expected (Frank and Swensson, 2002). Increased feed intake in the supplemented cows increased milk production, explaining that despite higher N intake they were as N efficient as the cows on only forage diets. The CP level was optimal to what has been found to maximise nitrogen utilisation without decreased produc-

Fig. 1. N conversion efficiency of feed N into milk N (g/g) for the experimental treatments with white (WCS) and red clover (RCS)-grass silages without (0) and with (1) concentrate supplementation in two years (yr1 and yr2). The relationship is described by the equations: N efficiency = − 0.2968 N intake + 437.8, r2 = 0.91 for treatments with concentrate supplementation and N efficiency = − 0.904 N intake + 618, r2 = 0.88 for the treatments without supplementation.

tion. However, lower milk urea content in supplemented cows (Table 7) suggests improved capture of ammonia N by rumen microbes (Broderick and Clayton, 1997). There was a negative relationship between N intake and utilisation within each concentrate supplementation level groups (Fig. 1). N utilisation decreased by 0.90 and 0.30 g per g extra N intake for the treatments without and with concentrate supplementation, respectively. The latter rate is close to rates reported previously (Castillo et al., 2000; Bertilsson and Murphy, 2003; Steinshamn et al., 2006). The different response to increasing N intake indicates that N utilisation in cows on high forage ration, even at lower N intakes, is more sensitive to changes in dietary N concentrations than in diets where easily fermentable carbohydrates are included. 4.6. Fertility Sehested et al. (2003) observed increased calving interval with decreasing feeding level in organic production. The number of cows in the present experiment was low and health and reproductive data should be treated with caution. However, there were no tendencies of negative effect of low feeding level on reproductive performance. Reksen et al. (2001) found that Norwegian Red cattle dairy cows, from the same herd as in the present study, adjusted to low energy supply (40% less than recommended) without delay in onset of postpartum ovarian activity. Therefore, a likely reason for the relatively high fertility in the present study is the ability of the used cattle breed to adjust to less energydense rations, or that the only forage diets supplied sufficient nutrients with regard to reproduction. The RCS diets contained high amount of isoflavonoids (Steinshamn et al., 2008), suggested to affect fertility

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negatively (Kallela et al., 1984), but had no apparent negative effect. 5. Conclusions Harvested at the same date in a short term rotational ley system in cool temperate growing conditions, white clover gave similar total DM yield but lower clover DM yields than red clover when cultivated with the same grass species. Silage prepared from the two crops revealed that white and red clovers have widely the same effect on intake and most production parameters in early lactating dairy cows. Red clover based diets yielded milk with higher contents of PUFA, particularly C18:3n-3, and lower n-6/n-3 FA ratio independent of concentrate level. Supplementation reduced silage DMI by 0.32 and 0.26 kg/kg and increased ECM production by 0.96 and 0.87 kg/kg concentrate DM for WCS and RCS, respectively. Only forage diets gave lower milk fat and protein content, but higher milk PUFA content and a more beneficial (lower) n-6/n-3 FA ratio. The N utilisation by cows on only forage diet was more sensitive to changes in dietary N content than those fed concentrate, and supplementation with energy concentrate should be considered when offering silage based diets with high crude protein content. Type of silage or concentrate supplementation had no apparent negative effect on health or reproduction. Therefore, depending on the price ratio of organic produced concentrate and milk, only forage diets may be an alternative to meet the requirement of a 100% organic feed ration for winter bred dairy cows. Acknowledgments The authors are grateful to the staff at the Animal Production Centre, Norwegian University of Life Sciences for technical assistance and care of the experimental animals and Claes-Gøran Fristedt at the Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences for feed and milk sampling. Thanks are also due to Karl N. Kerner for the improvement of our English. This work was financially supported by the Norwegian Agricultural Authority. References Abberton, M.T., Marshall, A.H., 2005. Progress in breeding perennial clovers for temperate agriculture. J. Agric. Sci. 143, 117–135. Al Mabruk, R.M., Beck, N.F.G., Dewhurst, R.J., 2004. Effects of silage species and supplemental vitamin E on the oxidative stability of milk. J. Dairy Sci. 87, 406–412.

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