Alfa-tocopherol and beta-carotene in roughages and milk in organic dairy herds

Livestock Science 145 (2012) 44–54

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Alfa-tocopherol and beta-carotene in roughages and milk in organic dairy herds Lisbeth Mogensen a,⁎, Troels Kristensen a, Karen Søegaard a, Søren K. Jensen b, Jakob Sehested b a b

Department of Agroecology, Research Centre Foulum, Aarhus University, PO Box 50, DK-8830 Tjele, Denmark Department of Animal Science, Research Centre Foulum, Aarhus University, PO Box 50, DK-8830 Tjele, Denmark

a r t i c l e

i n f o

Article history: Received 2 May 2011 Received in revised form 20 December 2011 Accepted 21 December 2011 Keywords: α-Tocopherol β-Carotene Dairy cows Organic milk production Roughage Vitamin

a b s t r a c t The aim of the present on-farm study was to analyse vitamin content in roughage at harvest and during storage and to analyze milk vitamin content when feeding the roughage to dairy cows. Roughages produced at five organic dairy farms were monitored at harvest and several times during winter as stored silage. As an average of several sampling times, roughage αtocopherol and β-carotene contents (mg per kg DM) during the period when the roughage was fed were, respectively, 30 and 21 in grass–clover silage, 13 and 8 in maize wholecrop silage and 28 and 9 in cereal wholecrop silage. Daily intake of α-tocopherol was 876 mg per cow–431 mg from roughages, 89 mg from concentrates and 356 mg from a vitamin supplement. Milk yield was 25.9 kg energy-corrected milk (ECM) per cow per day with αtocopherol and β-carotene contents (μg/ml) of 0.82 and 0.17. The study additionally showed the following tendency, but due to few observations no final conclusions could be drawn:

• For

grass–clover silage there were generally no losses of vitamins during the ensiling

process and during storage, but there were huge variations between farms.

• For wholecrop silage there was a loss of vitamins during the ensiling process, whereas there were no further losses during the feeding period.

• The output of vitamins in milk was within farm positively correlated to supply of vitamins from roughage. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Organic milk production is based on organic principles and objectives including naturalness and the recycling of nutrients. According to current regulations, the feed used in organic milk production must be 100% organically grown (EC, 1999). However, the integrity of the organic production could be further enhanced if the frequent use of vitamin supplementation (Møller and Laursen, 2007) with artificial fat-soluble vitamins could be substituted by vitamins from natural sources. The use of vitamin supplements reflects the uncertainty regarding the actual content of vitamins in the feed at harvest and after storage during the winter-feeding period. ⁎ Corresponding author. Tel.: + 45 8715 8025; fax: + 45 8715 4798. E-mail address: [email protected] (L. Mogensen). 1871-1413/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2011.12.021

The fat-soluble vitamins, A and E, are essential nutrients and both milk quality and performance of dairy cows depend on a supply of the vitamins or their precursors from feed or a vitamin supplement (Jensen and Nielsen, 1996; Weiss et al., 1990). There are eight different forms of vitamin E (α-tocopherols), which are equally present in synthetic products, whereas only RRR-α-tocopherol is naturally present in the feed plants. The naturally occurring RRR-α-tocopherol has the highest vitamin E activity of the eight forms and is thus the physiologically most important (Bondi, 1987), meaning that animal requirements can be fulfilled from a lower amount of the natural form (e.g. from home-grown roughages) as compared to a synthetic source of α-tocopherols. Vitamin A is synthesised in the cow from carotene precursors of plant origin, but animal requirements can also be fulfilled from vitamin supplements containing synthetic vitamin A

L. Mogensen et al. / Livestock Science 145 (2012) 44–54

and/or carotenes. The vitamin A activity of β-carotene varies between animal species. For cattle the conversion ratio is 5–8 weight units of β-carotene to one unit of vitamin A (Brubacher and Weiser, 1985). The concentrations of α-tocopherol and β-carotene in cattle feed are affected by factors such as plant species, harvest number over season, maturity stage of the plant and pre-wilting. The highest concentrations of β-carotene and α-tocopherol are found in grass, legumes and other green plants, while seeds and wholecrop silages only contain small amounts of vitamins (Putnam and Comben, 1987). Therefore, cattle receiving the majority of the feed from grazing normally have their requirements for fat-soluble vitamins met, whereas cattle fed concentrate and stored roughage may only receive low amounts of fat-soluble vitamins (StLaurent et al., 1990). Also within fresh grass crops the concentrations of α-tocopherol and β-carotene vary. A recent study found the highest concentration of α-tocopherol (mg/ kg DM) in fescue grass (115) followed by bird's-foot trefoil, rye grass and timothy (66–67), white clover and alfalfa (30–33 ) and lowest in red clover (24) (Søegaard et al., 2010). Concentrations of β-carotene (mg/kg DM) were found to be highest in bird's-foot trefoil (87) followed by rye grass and white clover (60–62), timothy, alfalfa and red clover (48–54) and lowest in fescue grass (40). Regarding effect of season, the concentrations of α-tocopherol and βcarotene in a grass–clover mixture were 14% and 25% higher, respectively, in late season (August–October), and 27% and 29% lower, respectively, in June, as compared to the average concentration for the whole season (Jensen et al., 2010b). Also the maturity of the plant at harvest affects the content of vitamins in the silage, and for alfalfa and timothy, the content of α-tocopherol was found to decrease by 20% to 65% depending on whether the crop was harvested at the vegetative stage or at the full flowering stage (Kivimäe and Carpena, 1973). Pre-wilting of the fresh crops in the field caused loss of vitamins. Jensen et al. (2010a) found that αtocopherol in a mixture of perennial rye grass and alfalfa was reduced by 25% after a 24-hour pre-wilting period; and by 16% in white clover, 23% in ryegrass and 41% in red clover (Beeckman et al., 2010). The loss of β-carotene in a mixture of perennial rye grass and alfalfa due to pre-wilting was only 4% (Jensen et al., 2010a). However, knowledge about the actual content of vitamins in roughage at farm level both at harvest and especially after storage during the winter-feeding period is very limited in the literature. The aim of the present study was therefore to analyse factors affecting the vitamin content in roughages at harvest and during storage on-farm and to analyse the vitamin content in the milk from cows fed these roughages. 2. Materials and methods 2.1. The study design The project involved five commercial organic dairy farms with either Holstein Friesian cows or Red Danish Dairy Breed (Table 1). On these farms the two quantitatively most important types of conserved roughage were monitored from harvest in 2007 and until the roughage was fed during

45

the winter 2007/08. On the same day as silage samples were taken, samples from the farm milk tank were collected. These samples of fresh and ensiled roughages and milk were analysed for β-carotene and α-tocopherol content. The sampling day also included registrations of feed composition and intake per cow per day at herd level, including vitamin supplement, if any. Daily intake of β-carotene and α-tocopherol from roughages and supplement was estimated as well as the content of β-carotene and α-tocopherol in the produced milk. A statistical analysis of the correlation between the content of α-tocopherol and β-carotene in milk and roughage was performed. 2.2. Measurements and calculations 2.2.1. Roughage production The two quantitatively most important types of conserved roughage were on all farms first-cut grass clover silage (cut from 21 May to 1 June 2007, aiming for a digestibility of organic matter of 75–80%) followed by either cereal wholecrop silage (farms 1–3) (harvested from 15 to 23 July 2007) or maize wholecrop silage (farms 4–5) (harvested from 10 to 25 October 2007). In the grass-clover fields, the botanical composition covering 1 m 2 was registered by visual evaluation of the sward as the percentages of grass, white clover, red clover, alfalfa, and weeds. Two registrations were made per hectare in the grass–clover field the day before harvest. The height of the grass–clover sward was also measured by using a rising plate metre (30 ∗ 30 cm, 3.8 kg/m 2). In the fields with cereals for wholecrop silage, the botanical composition was registered as the percentage of the soil surface covered by respectively cereals, grass, legumes, and weeds. One registration was made per ha in a 1-m 2 area. In the maize fields, 0.6 registrations were made per ha in an area measuring 4 rows times 5 m. The plant height (from soil surface to the base of the male flower) was measured and the number of plants and cobs per plant were counted. The cobs from 16 plants per registration were used for measuring the weight of the cobs and the degree of filling of the cobs. The latter was registered by counting the relative number of cobs with less than 90% filling (defined as small and white kernels) and an evaluation of the actual filling in these cobs. After cutting, the grass clover was left to wilt before being precision-chopped. All types of silage were stored in either bunker silos or silage piles in the field and covered with plastic. The roughage was carefully packed in the silo by heavy tractors during the entire period of harvest. No additives were used for silage-making. Crop yield was calculated based on the measured volume of the silage pile and an estimate of the density (kg DM/m 3) specific for the type and quality of the roughage. For the grass–clover silage, the time of cutting, and the duration of wilting together with the width of the swath were registered. The type of storage facilities, flour type and the use of cover were described as well. 2.2.2. Vitamin analysis in silage and milk Representative samples for vitamin analysis were taken from the two most important types of roughage on each farm after the following plan: The first sample was taken on

46

L. Mogensen et al. / Livestock Science 145 (2012) 44–54

Table 1 Farm and herd characteristic. Farm no.

1

2

3

4

5

Breeda Cows, no.b Stall Area in rotation, ha Permanent pasture, ha

RDM 62 Loose housing, cubicles 74 11

HF 143 Loose housing, cubicles 160 29

HF 95 Loose housing, cubicles 127 11

HF 156 Loose housing, cubicles 222 0

HF 83 Loose housing, cubicles 87 4

a b

HF = Danish Holstein Friesian, RDM = Red Danish Dairy Breed. Year cow = 365 feeding days.

the day of ensiling from the fresh crop entering the silo. The second sample was taken when the silo was opened to start feeding with the silage. A sample was subsequently taken every six weeks. Each sample was a mix of 10 smaller samples taken from the silage cut surface. All samples were stored on ice during the farm visit and transportation and frozen within few hours, and all the vitamin analyses were made at the same time after collection of all samples. All frozen samples of silage and wilted herbage were freeze-dried and analysed for contents of tocopherols and carotenoids by HPLC after saponification and extraction into heptane as previously described by Jensen et al. (1998). Milk samples for vitamin analysis were taken on the same days as the silage samples were taken for vitamin analysis. Milk samples were taken from the milk tank, thus representing a 24-hour (evening and morning milking) milk production in the herd, and were stored at −20 °C until analysis. Milk samples were thawed and analysed for tocopherol, carotenoids and retinol as previously described by Jensen and Nyholm (1996). 2.2.3. Sampling and analysis of forage quality One representative sample was collected from each type of silage at each farm halfway into the feeding period. Each sample was a mix of 10 smaller samples taken from the silage cut surface. All samples were stored on ice during the farm visit and transportation and frozen within few hours. These samples were analysed for DM (NorFor 60 °C), ash, crude protein, starch (only in wholecrop silage) (Helrich, 1990), NDF (Van Soest, 1994), in-vitro digestibility of organic matter (IVOS) (Tilley and Terry, 1963), as well as pH, lactic acid, acetic acid, and ammonia determined by near-infrared (NIR) spectroscopy at a commercial feed-testing laboratory (Eurofins Steins Laboratories A/S, Holstebro, Denmark). Invivo digestibility of organic matter was calculated based on IVOS (Møller et al., 1989). 2.2.4. Feed intake and milk production Every six weeks, on the same day as the sampling of silage and milk for vitamin analysis, feed intake was recorded as an average for all lactating cows in the herd over a period of 24 h. The roughage mixture was fed ad libitum (or close to ad libitum) once a day on the feeding manger after the morning milking. The amount of roughage/cereals/ concentrate offered on a group basis was weighed (by the scale on the feed-mixer in four herds and on a weighbridge in one herd) and corrected for leftovers, if any, by a visual estimate. Cereals/concentrate was additionally offered in the milking parlour. The amount at herd level was calculated based on registration of

individual allocations. The dry matter intake of each feedstuff was calculated on the basis of feed analyses for the roughage and table values for the concentrated feed. The energy values of each feedstuff were calculated according to Weisbjerg and Hvelplund (1993), with net energy content in one Scandinavian Feed Unit (SFU) of 7890 KJ. The intake of the vitamin mixture per cow per day was based on recordings of the daily amount offered, and as a control and a correction factor the sum of daily intake had to fit the amount of vitamin mixture purchased over a period of three months. Individual milk yield data were collected from the official milk test day recording once a month, representing 24 hours' milk yield with milk from an afternoon milking and from the milking the following morning. Milk concentrations of fat, protein, urea and the somatic cell count (SCC) were analysed by a MilkoScan FT 120 infrared analyser (Foss Electric A/S, Hillerød, Denmark). Energy-corrected milk (ECM) was calculated as defined by Sjaunja et al. (1990). 2.2.5. Calculations The daily intake of α-tocopherol and β-carotene per cow at each farm was calculated from registrations of feed intake and analyses of vitamin content in roughage. For the vitamin mixture, the declared content of vitamins was used. For other types of feed, the vitamin content was based on standard values (Jensen, 2003). The average milk yield per cow per day on the milk test day was calculated as a simple average for the herd. 2.3. Statistical analyses The correlation between the content of α-tocopherol and β-carotene in milk and in roughage was analysed by the CORR procedure (SAS Institute, 1990) for each farm individually. The best estimate for a general correlation across farms was found as the slope (A) from the following model analysed by the GLM procedure (SAS Institute, 1990): Y ¼ farm þ A  vitamins in roughage; Where Y is total vitamin excretion in milk (mg/cow/day) and vitamins in roughage (X) is total supply of vitamins from roughage (mg/cow/day). Farm is the fixed effect of the five different farms. Three observations were included per farm. Furthermore, the correlation between the content of αtocopherol and β-carotene in roughage and different variables describing the quality of the roughage (Tables 2 and 3) was

L. Mogensen et al. / Livestock Science 145 (2012) 44–54

47

Table 2 First cut grass–clover silage from five organic dairy farms: Botanic composition of the sward, harvest and storage conditions, feed quality of the silage. Farm no

1

2

3

4

5

Average

Sward before harvest, Na Height , cm Composition –Grass, % of volume –White clover, % –Red clover, % –Alfalfa, % –Weed, % Harvest conditionb Temperature, °Cc Precipitation, mmc Relative humidityc Global radiation, W/m2c Spring yield, kg DM/ha d Area, ha Time of cutting Width of swath, m Duration of wilting, h DM after wilting, % Storage conditionsb Type Floor type Covering plastic, mm Covering plastic, no. of layer Silage qualityb Dry matter, % NEL, MJ/kg DMe Crude protein, % of DM Ash, % of DM NDF, % of DM Digestibility of organic matter, % pH Lactic acid, % of DM Acetic acid, % of DM Ammonium N, % of N

70 26

184 30

38 17

123 19

56 25

23

57 25 15 0 2

49 23 19 6 3

51 40 6 0 4

57 31 7 0 6

67 19 12 0 2

56 28 12 1 3

13 0 82 17 3181 34.8 11–17 3 49 41.2

12 0 74 26 3266 92.2 8–17 9 32 39.0

13 0 76 21 2103 18.8 10–15 3.5 53 37.6

12 1 76 22 3046 61.5 8–22 7.4 48 40.8

15 0 76 21 5265 28.0 10–18 2 58 41.6

Pile Soil 0.16 2

Silo/pile Concrete/soil 0.19 1

Silo Concrete 0.18 2

Silo Concrete 0.18 2

Silo Concrete 0.15 1

32.3 7.11 14.0 8.6 36.8 78.2 4.1 9.0 1.5 8.0

39.6 6.86 12.1 9.0 36.1 77.2 4.4 6.8 0.5 9.8

35.4 7.66 18.2 9.9 31.3 81.3 3.7 12.4 1.5 7.2

40.9 7.59 12.0 8.2 34.7 81.9 3.9 9.0 0.6 6.3

34.5 6.26 10.7 6.9 45.7 72.9 4.3 3.6 1.4 9.3

a b c d e

3372

48.0 40.0

1.7 36.5 7.04 13.4 8.5 36.9 78.3 4.1 8.2 1.1 8.1

N = number of observations. One observation per farm. Data from 40 ∗ 40 km grid from the period when the grass–clover was lying in swaths. Only the yield from first grass–clover cut is included. Net Energy Lactation (NEL) calculated according to Weisbjerg and Hvelplund (1993).

analysed by the CORR procedure (SAS Institute, 1990) with one observation from each farm.

included or not. The quality of the maize silage was quite similar on the two farms.

3. Results

3.2. Vitamin content in the silage

3.1. Silage composition and quality

Table 4 shows the vitamin content of roughages at harvest after wilting of the grass–clover and the average vitamin content of the different types of silage during the storage and feeding period. The development in the α-tocopherol and β-carotene contents in the grass–clover silage from harvest and during the storage and feeding period is shown in Fig. 1a and b, respectively. The average content of αtocopherol and β-carotene in grass–clover did not reduce from harvest (after wilting) to the start of the feeding period, whereas on average there was a reduction in the content of α-tocopherol and β-carotene during the storage and feeding period. However, large variations were observed between farms and on two farms there was no loss in that period either. The development in the α-tocopherol and β-carotene contents in the wholecrop silages from harvest and during the storage and feeding period is shown in Fig. 1a and b, respectively. For both there was a significant reduction from harvest to the start of the feeding period, but no further

Table 2 shows the botanical composition of the sward, harvest and storage conditions, and the feed quality of the first cut grass–clover silages from the five farms. The grass– clover silage consisted on all farms of a large proportion of grass, some white clover and the smallest proportion was red clover. On all farms the silage was harvested under good weather conditions. There was a large variation between farms in the obtained crop yield of first cut grass–clover, and the digestibility of the produced silage varied from high to medium. Table 3 shows the botanical composition of the fields before harvest, harvest and storage conditions, and the feed quality of cereal wholecrop silage and maize wholecrop silage from three and two farms, respectively. The cereal wholecrop silage included nearly one third weed on all farms. The digestibility of the cereal wholecrop silage was medium on all farms irrespective of whether peas were

48

L. Mogensen et al. / Livestock Science 145 (2012) 44–54

Table 3 Whole crop silage of cereals and maize from five organic dairy farms: botanic composition, description of harvest and storage conditions, and feed quality of the silage. Farm no.

1

2

3

4

5

Average

Average

Cereals

Maize

Type of silage

Barley

Barley/pea

Barley/pea

Maize

Maize

Before harvest, Na Compostion: –Cereals, % –Legumes, % –Undersown, grass% –Undersown, legumes, % –Weed, % Plant height, m Cobs/plant, N Plants, 1000/ha Weight of cobs, g DM Filling of cobs, % Harvest condition b,c Temperature, °C Precipitation, mm Rel. humidity Global radiation, W/m2 Crop yield, kg DM/ha Area, ha DM at harvest, % Storage conditionsc Type Floor type Covering plastic, mm Covering plastic , no. of layer Silage qualityc Dry matter, % NEL, MJ/kg DMd Crude protein, % of DM Ash, % of DM Starch, % of DM NDF, % of DM Om dig. invitro, % pH Lactic acid, % of DM Acetic acid, % of DM Ammonium N, % of N

9

15

17

8

5

74 0 1 3 22

45 21 0 0 35

40 26 0 0 33

a b c d

53 15 1 1 30 2.23 1.33 83 59 98

2.00 0.93 89 51 100

17 0 80 21 6778 9.2 33.4

17 0 74 23 8146 14.5 31.7

15 14 83 18 6282 16.9 26.0

10 0 86 8 10,622 14.2 25.0

5 0 93 6 7240 8.2 30.1

Stack Soil 0.16 2

Stack Soil 0.19 1

Silo Concrete 0.18 2

Silo/ stack Concrete/soil 0.18 2

Silo Concrete 0.15 1

31.8 4.84 13.0 6.3 13.1 41.6 62.0

29.5 4.51 11.5 7.4 19.8 41.5 60.3

27.4 4.99 13.2 5.6 16.2 39.4 62.4

23.6 6.26 8.7 4.3 20.7 47.1 72.9 4.1 5.5 1.9 4.8

29.5 6.63 9.0 4.6 23.4 43.5 75.4 4.0 5.7 1.5 4.9

2.1 1.1 86 55 99

7069

8931

30.4

27.6

1.8

2

29.6 4.78 12.6 6.4 16.4 40.8 61.6

26.6 6.45 8.9 4.5 22.1 45.3 74.2 4.0 5.6 1.7 4.9

N = number of observations. Data from a 40 ∗ 40 km grid from the day the crop was harvested. One observation per farm. Calculated according to Weisbjerg and Hvelplund, 1993.

losses during the storage and feeding period. The αtocopherol content of cereal wholecrop silages was in general reduced by around 50% after harvest, whereas β-carotene was reduced by 28%. An analysis of the correlation between the contents of αtocopherol and β-carotene in grass clover silage and different variables describing the quality of the roughage (one observation per farm) showed a positive correlation between digestibility of organic matter and content of α-tocopherol (ρ = 0.71 P = 0.18) and β-carotene (ρ = 0.93 P = 0.02), and between energy content per kg DM and content of α-tocopherol (ρ = 0.67 P = 0.20) and β-carotene (ρ = 0.92 P = 0.02). Furthermore, a positive correlation was found between the content of white clover and those of α-tocopherol (ρ = 0.61 P = 0.27) and β-carotene (ρ = 0.86 P = 0.06), and a negative correlation between the content of red clover and αtocopherol (ρ = −0.82 P = 0.08) and β-carotene (ρ = −0.74 P = 0.14). Also other variables indicating high-quality silage correlated with the vitamin content in the grass–clover silage. There was a negative correlation between pH and the

content of α-tocopherol (ρ = − 0.70 P = 0.18) and βcarotene (ρ = − 0.83 P = 0.08), and a negative correlation between ammonium and content of α-tocopherol (ρ=−0.86 P=0.06) and β-carotene (ρ=−0.86 P=0.06). 3.3. Feed intake and supply of vitamins from feed Table 5 shows an average daily feed intake of 20 kg DM per day for lactating cows in the herds where roughage made up 74% of the DM intake. Supply of α-tocopherol and β-carotene from roughages accounted for 49% and 51%, respectively, while the supplies from concentrates were 10% and 20%, respectively, of the total supply (Table 5). 3.4. Milk production and vitamin contents in milk Table 6 shows milk production per lactating cow per day during the winter period as well as the concentration of vitamins in milk and the total daily excretion of vitamins in milk. The inter-farm variation in daily milk production was relatively

L. Mogensen et al. / Livestock Science 145 (2012) 44–54

49

Table 4 Concentration of α-tocoferol and β-carotene (mg/kg DM) in different types of silages from five organic dairy farms, at harvest and the average during the feeding period. Farm no. Gras clover silage Av. duration of storage, days α-tocopherol, mg/kg DM –At harvest (after wilting)a –During feeding period, Nb Mean, std dev., (median)c –In % of level at harvest β-carotene, mg/kg DM –At harvest (after wilting)a –During feeding period, N Mean, std dev., (median)c –In % of level at harvest

1

2

3

4

5

Average

288

342

353

216

208

281

23.0 4 22.1 6 (23.1) 96

37.7 2 21.7 4 (21.7) 58

33.4 2 33.9 3 (33.9) 101

25.8 4 45.6 10 (48.0) 176

38.2 4 25.1 6 (24.7) 66

31.6

13.6 4 16.4 5 (14.5) 121

28.2 2 17.5 16 (17.5) 62

26.3 2 27.4 0 (27.4) 104

18.1 4 29.8 6 (31.0) 164

24.0 4 12.6 4 (12.5) 52

22.1

12 99

20.7 9 101

Maize whole crop Av. duration of storage, days α-tocopherol, mg/kg DM –At harvesta –During feeding period, N Mean, std dev., (median)c –In % of level at harvest

178

211

194

41.2 4 16.3 10 (14.2) 40

15.7 4 9.2 2 (8.6) 58

28.5

β-carotene, mg/kg DM –At harvesta –During feeding period, N Mean, std dev., (median)c –In % of level at harvest

10.5 4 14.3 9 (17.5) 135

1.0 4 0.9 0.5 (1.0) 85

5.7

12.7 7 49

7.6 9 112

Whole crop silage Av. duration of storage, days α-tocopherol, mg/kg DM –At harvesta –During feeding period, N Mean, std dev., (median)c –In % of level at harvest

181

185

265

210

28.0 3 15.9 1 (16.0) 57

53.3 3 28.9 8 (27.8) 54

71.7 3 39.4 9 (42.9) 55

51.0

β-carotene, mg/kg DM –At harvesta –During feeding period, N Mean, std dev., (median)c –In % of level at harvest

8.3 3 4.3 0.9 (4.0) 52

10.6 3 8.0 4 (8.0) 75

17.6 3 15.7 6 (19.0) 89

12.2

a b c

28.1 12 55

9.3 6 72

One sample taken when the crops have just entered the silo (for grass–clover after wilting). N = number of observations. From the feeding period.

small (24.4 − 28.2 kg ECM), whereas there was a huge variation between farms in the concentration of α-tocopherol in milk (0.51–1.08 μg/ml). For β-carotene in milk there was an inter-farm variation from 0.15− 0.19 μg/ml. 3.5. Correlation between content of vitamins in roughage and milk In Fig. 2a and b the daily excretion of α-tocopherol and βcarotene in milk is shown as a function of the intake of αtocopherol and β-carotene from the roughage fed during the winter period. The output of α-tocopherol in milk was within farm positively correlated to the supply of αtocopherol from roughage on four out of five farms, but only for farms 3 and 4 was this correlation statistically significant (ρ = 0.99 P = 0.04; ρ = 0.99 P = 0.06, respectively). An estimate for a general correlation across farms showed that an increased input of 100 mg α-tocopherol from roughage resulted in an increased excretion of α-tocopherol in milk of on average 2.3 mg/day.

The output of β-carotene in milk was within farm positively correlated to the supply of β-carotene from roughage on three out of five farms, but was not statistically significant. An estimate for a general correlation across farms showed that an increased input of 100 mg β-carotene from roughage resulted in an increased excretion of β-carotene in milk of on average 0.6 mg/day. 4. Discussion 4.1. Loss of vitamins in the chain from fresh crop to silage feeding The highest concentrations of vitamins are found in the fresh crops. Subsequently, there are several steps in the chain from fresh crops to silage where loss of vitamins can take place: during wilting of grass–clover, during the ensiling process, during storage and feeding, with the result that only a smaller fraction of the vitamins found in the animal feed is transferred to the milk.

50

L. Mogensen et al. / Livestock Science 145 (2012) 44–54

a

Grass-clover silage

60.00

Whole crop silage 80.00

α-tocopherol, mg/kg DM

α-tocopherol, mg/kg DM

70.00

50.00

60.00 40.00 1

50.00

1 2

4

30.00

2 5 3

20.00

40.00

3 4

30.00

5

20.00 10.00

10.00

days after ensiling

0.00

0

50

100

150

b

200

250

300

350

400

450

0.00

500

Days after ensiling

0

50

100

Grass-clover silage

40

150

200

250

300

350

400

Whole crop silage

β-carotene, mg/kg DM

25

β-carotene, mg/kg DM

35 30

20

25

1 4 2 5 3

20 15

1 2 3 4 5

15

10

10 5 5

Days after ensiling

Days after ensiling

0

0

50

100

150

200

250

300

350

400

450

500

0

0

50

100

150

200

250

300

350

400

Fig. 1. a. Content of α-tocopherol (mg/kg DM) in grass–clover silage and wholecrop silage (barley/pea or maize), respectively, at harvest and during the feeding period at five organic dairy farms. b. Content of β-carotene (mg/kg DM) in grass–clover silage and wholecrop silage (barley/pea or maize), respectively, at harvest and during the feeding period at five organic dairy farms.

For grass cut for silage, the first loss of vitamins takes place during the wilting stage in the field. Losses during wilting were not measured in the present study. However, another newly published study showed a 25% loss of α-tocopherol (from 64 to 48 mg/kg), and 4% loss of β-carotene (from 75 to 72 mg/kg DM) during the 24-hour wilting period of a mixture of perennial rye grass and alfalfa (Jensen et al., 2010a). Similarly, Beeckman et al. (2010) found under experimental conditions an average loss of α-tocopherol during wilting of 16% for white clover (from 49 to 41 mg/kg DM), 23% for rye grass (from 156 to 120 mg/kg DM) and 41% for red clover (from 74 to 51 mg/kg DM). On the other hand, Nadeau et al. (2004) found that wilting of cut forage in windrows did not decrease the α-tocopherol and β-carotene contents. From the time when the wilted crop is stored in the silo and during the active fermentation phase of the ensiling process losses of vitamins can take place, and if the silage is not stable after this ensiling process (the stable phase), further losses can take place during the storage period. In the present study, vitamin content was first analysed when crops were placed in the silo and again when starting the feeding, this

period representing the sum of vitamin loss during the ensiling process and the loss in the first part of the storage period. At two farms there was no loss of α-tocopherol or β-carotene in grass clover during the ensiling process and first part of the storage period, whereas on two other farms, 34–48% of the α-tocopherol and β-carotene in the freshly stored grassclover silage was lost during this period. Similar losses of αtocopherol (24%) and β-carotene (20%) during ensiling were found in experiments with red clover/timothy and red clover/fescue grass (Jensen et al., 2010a). Beeckman et al. (2010) found that the α-tocopherol content of ryegrass and red clover decreased during the ensiling process by 16 and 22% respectively, whereas the α-tocopherol content of white clover remained stable. In a Swedish study with grass–clover silage stored in round-bales, α-tocopherol and β-carotene concentrations decreased by an average of 49% (from 35 to 18 mg/kg DM) and 37% (from 19 to 12 mg/kg DM), respectively, during the three-month period from the field-drying to the feeding stage (Nadeau et al., 2004). They concluded that there is a greater risk of α-tocopherol loss in round-baled silage than in silage stored in silos. The present

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Table 5 Daily feed intake (kg DM and % of DM) of different feedstuffs and supply of α-tocopherol and β-carotene (mg/cow/day) from feed and vitamin mixture during the indoor winterfeeding period at five organic dairy farms, average per lactating cow on herd basis. Farm no.

1

2

3

4

5

Average

Feed intake, % of DM, Na Grazing, % Grass–clover silage (1. + other cutb), % Whole crop silage cereals, % Whole crop silage maize, % Grass pellets, % Hay, % Cereals,% Concentrate mixture, % Total, kg DM/cow/day

6 0 21 + 39 18 0 9 0 13 0 19.5

6 0 17 + 52 9 0 7 3 0 12 22.6

4 4 36 + 13 15 0 0 0 22 0 18.4

6 3 32 + 16 3 20 5 0 6 15 20.0

5 0 27 + 15 20 6 0 0 9 23 20.2

2 27 + 27 13 7 4 1 9 12 20.1

316 107 298 721 44 15 41

398 135 455 988 40 14 46

541 73 365 979 55 8 37

598 110 443 1151 52 10 38

305 19 219 543 56 4 40

431 89 356 876 49 10 40

207 258 205 670 31 39 31

289 256 172 717 40 36 24

458 24 150 632 72 4 24

459 156 183 798 58 20 23

148 8 120 276 54 3 43

312 140 166 619 51 20 29

Vitamin supply, mg/cow/day α-tocopherol –Roughage –Cereals and concentratec –Vitamin-mixture –Total daily supply, mg % of α-tocopherol from roughage % of α-tocopherol from cereals/conc. % of α-tocopherol from vit.-mixture β-carotene –Roughage –Cereals and concentratec –Vitamin-mixture –Total daily supply, mg % of β-carotene from roughage % of β-carotene from cereals/conc. % of β-carotene from vit.-mixture a b c

N = number of observations. First cut grass clover silage was analysed for vitamin content—this value for vitamin content was also used for grass clover of later cuts. Mainly caused by a high supply of vitamins from grass pellet, standard values of vitamin content (Jensen, 2003).

investigation showed that for grass–clover silage, the contents of α-tocopherol and β-carotene were on average reduced further during the period when the silage was fed. However, for two farms there was no vitamin loss in that period. On the last farm (No. 4), the values of both α-tocopherol and β-carotene of freshly stored grass–clover were lower than all the measured values during the feeding period, which might indicate that the sample at harvest was not representative for the silage pile. For wholecrop silage (both maize and cereals/peas) there was a clear picture across all farms in that 47% of the αtocopherol and 25% of the β-carotene found in the freshly stored silage was lost during the ensiling process/storage until the start of feeding. There was no further loss of vitamins during the following period when the silage was stored and fed. In general, for grass–clover silage there was huge variation between farms, but it seems to be possible to avoid further vitamin losses (after wilting) during the ensiling process and during storage. For wholecrop silage, there seems to be an unavoidable loss of vitamins during the ensiling process and no further losses during storage. 4.2. Vitamin content in the fed silage The highest concentration of vitamins was found in the grass–clover silage from farms 3 and 4 (Table 4). Characteristic for the grass–clover silage from these farms was a high digestibility of organic matter and high energy content per kg

DM (Table 2) and a relatively low DM yield, especially on farm 3. This indicates that the grass–clover probably was harvested at an early stage of plant development, as the stage of maturity when a plant is harvested is one of the most important factors influencing quality (Rohweder et al., 1978). Earlier studies have found that the content of vitamins in the silage is affected by plant development and the leaf to stem ratio (Kivimäe and Carpena, 1973). For alfalfa and timothy, the content of α-tocopherol decreased by 20–65% when the crop was harvested at the full flowering stage rather than the vegetative stage (Kivimäe and Carpena, 1973). The higher proportion of white clover and lower proportion of red clover in the grass–clover silages from farms 3 and 4 could also have added to the high concentration of vitamins in these silages, as white clover has significantly higher concentrations of both α-tocopherol and β-carotene than red clover (Søegaard et al., 2010). For cereal wholecrop silage, the highest concentration of vitamins was found in the silage from farms 2 and 3. Characteristic for these silages were their high content of legumes and weeds and relatively low content of cereals, which might explain the higher vitamin content as legumes and weeds have significantly higher vitamin contents than cereals (Jensen, 2003). The findings in another study that variation in the digestibility of the wholecrop silage could explain 25–85% of the variation in the α-tocopherol content (Flye and Strudsholm, 1994) would not explain the present case, as digestibility of organic matter did not vary much between farms (from 60.3 to 62.4%).

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Table 6 Milk production per lactating cow per day, concentration of vitamins in milk (mean, std dev, μg/ml) and total daily excretion of vitamins in milk (mg/day) during the indoor winter period (2007/2008) at five organic dairy farms. Farm no.

1

2

3

4

5

Average

Milk production, N Milk, kg ECM, kg Fat % Protein % Urea, mM Somatic cell count, 1000

5 23.7 ± 0.6 24.5 ± 0.6 4.30 ± 0.17 3.39 ± 0.08 3.8 ± 1.4 283 ± 46

5 29.5 ± 1.1 28.2 ± 1.1 3.77 ± 0.07 3.21 ± 0.04 5.2 ± 0.7 191 ± 40

5 24.3 ± 0.4 24.4 ± 0.4 4.10 ± 0.21 3.29 ± 0.15 4.7 ± 0.9 321 ± 54

3 27.3 ± 1.2 27.0 ± 1.2 3.99 ± 0.09 3.26 ± 0.02 – 358 ± 4

5 25.9 ± 0.7 25.3 ± 0.7 4.02 ± 0.11 3.07 ± 0.08 4.0 ± 0.6 309 ± 77

26.1 ± 2.3 25.9 ± 1.7 4.04 ± 0.19 3.24 ± 0.12 4.4 ± 0.7 292 ± 63

Milk concentration, N α-tocopherol, μg/ml mean, std dev (median)

3 0.51 ± 0.17 (0.44)

3 0.66 ± 0.05 (0.65)

3 1.08 ± 0.06 (1.07)

3 0.90 ± 0.14 (0.96)

3 0.97 ± 0.07 (0.93)

0.82 ± 0.23

β-carotene, μg/ml mean, std dev (median) Retinol, μg/ml mean, std dev (median) Total α-tocopherol, mg/day Total β-carotene, mg/day Total retinol, mg/day

0.17 ± 0.02 (0.17)

0.15 ± 0.01 (0.15)

0.19 ± 0.02 (0.19)

0.17 ± 0.03 (0.18)

0.15 ± 0.03 (0.15)

0.17 ± 0.02

0.40 ± 0.09 (0.35)

0.39 ± 0.04 (0.41)

0.47 ± 0.03 (0.46)

0.44 ± 0.02 (0.43)

0.36 ± 0.04 (0.37)

0.41 ± 0.06

12.1 4.0 9.5

19.5 4.4 11.5

26.2 4.6 11.4

24.6 4.6 12.0

25.1 3.9 9.3

21.5 4.3 10.7

N = number of observations.

The average α-tocopherol and β-carotene contents of, respectively, 30 and 21 mg/kg DM in grass–clover silage in this farm study are in agreement with results from recent studies from farms in Sweden (Nadeau et al., 2004) and other Danish experiments (Jensen et al., 2010a). Beeckman et al. (2010) measured about 50 mg α-tocopherol/kg DM in grass–clover silage in unopened silos on private farms with a large variation among farms, and the amount was reduced to about 20 mg/kg DM in opened silos. In lab-scale experiments they found higher levels of α-tocopherol of 102 mg/kg DM in silage of ryegrass, 40 mg/kg DM in red clover and 49 mg/kg DM in white clover (Beeckman et al., 2010). 4.3. Supply of vitamins from the feed ration In the present study, the supply of α-tocopherol from feed corresponds to only 38% of the recommended level of 1360 mg α-tocopherol/day or 2.6 IU/kg BW of total vitamin E (supplemental plus vitamin provided by feedstuffs) (NRC, 2001), which was not reached at any of the farms (despite a vitamin mixture being supplemented). For β-carotene, the average daily intake across farms was 619 mg/cow during the winter period, 453 mg of which came from the feed and 166 mg from the vitamin mixture. The supply from feed could fulfil the recommended level of 160 mg/day or 110 IU/kg BW (NRC, 2001) for all farms without any supplements. 4.4. Vitamin contents in milk The concentration of α-tocopherol in milk found in the present study (0.82 μg/ml) agrees well with the 0.9 μg/ml found in an earlier Danish study by Knudsen et al. (2001) during the indoor winter-feeding period. Also results by Schingoethe et al. (1978) with a total excretion in milk of 26 mg α-tochopherol/cow/day during the indoor feeding period are in agreement with our findings of 22 mg, with the assumption that milk productions are similar. Milk concentrations of β-carotene and retinol (0.17 and 0.41 μg/

ml, respectively) in our study are also in agreement with results from other studies. Jensen (2003) reported values of, respectively, 0.15 μg/ml and 0.44 μg/ml for organically produced milk with Danish Holstein cows during the summer period and Ellis et al. (2007) measured 5.35 μg βcarotene/g milk fat (or 0.22 μg/ml assuming same milk fat percentage as in our study) and 14.11 μg retinol/ g milk fat (or 0.57 μg/ml again assuming same fat percentage) for organic cows in UK during a 12-month period. The content of α-tocopherol in milk is dependent on the content in the cows' feed (Jensen et al., 2005), but the mechanism involved in the translocation of α-tocopherol from feed to milk is poorly understood (Jensen et al., 2005). Within farm, the tendency in the present study was for the concentrations of both α-tocopherol and β-carotene in milk to be positively correlated to the supply in roughage. For example at farm 4, one observation was much lower for both roughage and milk. The lower α-tocopherol supply from roughage was due to a change in the composition of roughage, increasing the proportion of maize wholecrop silage and decreasing the proportion of grass–clover silage which was found to be reflected in a lower α-tocopherol concentration in milk. The lowest concentration of α-tocopherol in milk was seen on farms 1 and 2. Characteristic for these farms was also that they had the lowest concentration of α-tocopherol in both the first cut grass–clover silage and cereal wholecrop silage. Jensen and Nielsen (1996) found that if the natural αtocopherol content in feed was increased from 500 to 1000 mg/cow/day, the content of α-tocopherol in milk rose from 0.6 to 1.1 μg/ml milk. Assuming the same milk production as in the present investigation, this means that an increase in the daily supply of α-tocopherol from feed of 100 mg will result in an increase in α-tocopherol in milk of 2.6 mg, in line with our estimate for a general correlation in the present study. Cows prefer the natural stereoisomer over the synthetic stereoisomers of α-tocopherol (Jensen et al., 2005). Therefore, if an increased daily supply of α-tocopherol originated

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Fig. 2. a. Total amount of α-tocopherol excreted in milk (mg/cow/day) as a function of the supply of α-tocopherol from roughage (mg/cow/day) during the winter period, data from farms 1 to 5. b. Total amount of β-carotene excreted in milk (mg/cow/day) as a function of the supply of β-carotene from roughage (mg/ cow/day) during the winter period, data from farms 1 to 5.

from a synthetic vitamin mixture, the resulting increase in αtocopherol in milk would only be 0.3 mg α-tocopherol for every 100 mg added, according to St-Laurent et al. (1990) and Nicholson and St-Laurent (1991). The study by Knudsen et al. (2001) confirmed that the content of αtocopherol in milk during the winter period was independent of whether the farms used a vitamin supplement or not. 4.5. Farm data The huge amounts of silage produced at farm level makes representative sampling more complicated than with the smaller batches used at experimental level. For example, in the present study the first cut of the grass–clover was harvested from fields measuring between 19 ha and 92 ha per farm and with yields ranging between 2.1 and 5.3 t DM/ha. Even with a large sampling effort, the results indicate that on farm 4 we did not succeed in obtaining a representative sample from the grass–clover at ensiling. Similarly, there were some unexpected results for the dry matter content, where the level was higher in the fresh grass clover after

wilting than in the final silage, especially on farms 1 and 5. This was probably caused by differences in the method used for sampling. During silage stack-filling sampling continued over several hours which may have caused some further drying of the material, whereas final silage samples were taken from the cut surface and immediately stored on ice. But also in general it is rather difficult to get representative samples for dry matter analyses. The differences in DM percentages were not related to problems with silage quality as indicated by the silage quality data. All in all, this type of data and observations give valuable information about the vitamin content in the whole chain from freshly cut crops to the final silage fed to the cows and also how much of the vitamin content ends up in bulk milk on commercial farms. The present study showed that roughage is quantitatively significant for the supply of natural vitamins over a winterfeeding period on commercial farms, and that it is possible for dairy farmers to increase the supply of vitamins by making high-quality grass–clover silage. Grass–clover silage of high digestibility can apparently secure a high content of vitamins in the silage during the entire feeding period. This

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brings us back to the organic dairy farmer and the problem of meeting the vitamin requirements of the cows. Including a large proportion of high-quality silage would appear to be one solution to that problem, but also a way to increase the vitamin content in the milk. 5. Conclusion The average concentrations of α-tocopherol and β-carotene contents (mg per kg DM) were, respectively: 30 and 21 in grass–clover silage, 13 and 8 in maize wholecrop silage, and 28 and 9 in cereal wholecrop silage. The average content of α-tocopherol and β-carotene in milk was 0.82 μg/ml and 0.17 μg/ml, respectively, but with a huge variation between farms. The study also showed the following tendency: For grass–clover silage, there was huge variation between farms, but it seems to be possible to avoid further vitamin losses after wilting, i.e. during the ensiling process and during storage. For the wholecrop silage crop there seems to be an unavoidable loss of vitamins during the ensiling process, whereas there were no further losses during the storage and feeding period. The output of vitamins in milk within farms and overall was positively correlated to the supply of vitamins from roughage. Conflict of interest statement The present article cause no conflict of interest statement. Lisbeth Mogensen Acknowledgement We are grateful to the Danish Ministry of Agriculture, Food and Fisheries for economic support for the present DARCOF III (Danish Agricultural Research Centre for Organic farming) project ‘Increased integrity in organic dairy production through natural sources of vitamins and minerals and non-antibiotic health control’ (2006–2010). We would also like to thank the reviewers for very careful review with many useful and constructive suggestions for improvement of this article. References Beeckman, A., Vicca, J., Van Ranst, G., Janssens, G.P.J., Fievez, V., 2010. Monitoring of vitamin E status of dry, early and mid-late lactating organic dairy cows fed conserved roughages during the indoor period and factors influencing forage vitamin E levels. J. Anim. Physiol. Anim. Nutr. 94, 736–746. Bondi, A.A., 1987. Animal Nutrition. A Wiley-Interscience Publication. John Wiley & Sons. 540 pp. Brubacher, G.B., Weiser, H., 1985. The vitamin A activity of β-carotene. Int. J. Vitam. Nutr. Res. 55, 5–15. EC, 1999. Regulation No 1804/1999 from European Commision, supplementing regulation No 2092/91. Ellis, K.A., Monteiro, A., Innocent, G.T., Grove-White, D., Cripps, P., McLean, W.G., Howard, C.V., Mihm, M., 2007. Investigation of the vitamins A and E and β-carotene content in milk from UK organic and conventional dairy farms. J. Dairy Res. 74, 484–491. Flye, J.C., Strudsholm, F., 1994. LK- meddelelse 105. Landbrugets Rådgivningscenter. 2 pp.

Helrich, K., 1990. Official Methods of the Analysis of the AOAC, 15th edition. Association of the Official Analytical Chemists. 684 pp. Jensen, S.K., 2003. Absorption og omsætning af vitaminer. In: Hvelplund, T., Nørgaard, P. (Eds.), Kvægets ernæring og fysiologi. Bind 1—Næringsstofomsætning og fodervurdering: DJF Report—Husbandry, No 53, pp. 375–388. Jensen, S.K., Nielsen, K.N., 1996. Tocopherols, retinol, β-carotene and fatty acids in fat globule membrane and fat globule core in cow's milk. J. Dairy Res. 63, 565–574. Jensen, S.K., Nyholm, K., 1996. Distribution of tocopherols, retinol, βcarotene and fatty acids among fat globule membrane and fat globule core in cow's milk. J. Dairy Res. 63, 565–574. Jensen, S.K., Jensen, C., Jakobsen, K., Engberg, R.M., Andersen, J.O., Lauridsen, C., Sørensen, P., Skibsted, L.H., Bertelsen, G., 1998. Supplementation of broiler with retinol acetate, beta-carotene or canthaxanthin: effect of vitamin status and oxidative status of broilers in vivo on meat stability. Acta Agric. Scand. Sect. A. Anim. Sci. 48, 28–37. Jensen, S.K., Kristensen, N.B., Lauridsen, C., Sejrsen, K., 2005. Enrichment of cow's milk with natural or synthetic vitamin E. 10th Symposium: Vitamins and additives in nutrition of man and animal, pp. 78–83. 28–29 September, Jena/Thuringia, Germany. Jensen, S.K., Hymøller, L., Søegaard, K., Lindqvist, H., Nadeau, E., 2010a. Vitaminer og fedtsyrer i hø og ensilage – hvad sker der ved forvejring og lagring? ICROFS nyt. 2. 2010, pp. 6–7http://www.icrofs.dk/ pdf/icrofsnyt/2010. Jensen, S.K., Søegaard, K., Sehested, J., Lindqvist, H., Nadeau, E., 2010b. Indflydelse af høstmetode og konservering på vitamin—og fedtsyreindhold. Økologisk græsmarksproduktion og udnyttelser til mælkeproduktion. : Internal Report Husbandry, No 27. Faculty of Agricultural Sciences, Aarhus University, pp. 15–20. Kivimäe, A., Carpena, C., 1973. The level of vitamin E content in some conventional feeding stuffs and the effects of generic variety, harvesting, processing and storage. Acta Agric. Scand. 19, 162–168. Knudsen, B.S., Hermansen, J.E., Jensen, S.K., Kristensen, T., 2001. E-vitamin til malkekøer. DJF-Report No. 27. 76 pp. Møller, J., Laursen, P.H., 2007. Stor forskel på udgift til mineraler og vitaminer. Kvæginfo No. 1779. www.landbrugsinfo.dk/kvaeg. Møller, E., Andersen, P.E., Witt, N., 1989. En sammenligning af in vitro opløselighed og in vivo fordøjelighed af organisk stof i grovfoder. 13. Beretning fra Fællesudvalget for Statens Planteavls- og Husdyrbrugsforsøg. . 23 pp. Nadeau, E., Johansson, B., Jensen, S.K., Olsson, G., 2004. Vitamin content of forage as influenced by harvest and ensiling techniques. Grassland Sci. Eur. 9, 891–893. Nicholson, J.W.G., St-Laurent, A.M., 1991. Effect of forage type and supplemental dietary E-vitamin on milk oxidative stability. Can. J. Anim. Sci. 71, 1181–1186. NRC, 2001. Nutrient Requirements of Dairy Cattle, Seventh Revised Edition. The National Academy of Sciences, Washington DC. Putnam, M.E., Comben, N., 1987. Vitamin E—review article. Vet. Rec. 121, 541–545. Rohweder, D.A., Barnes, R.F., Jorgensen, N., 1978. Proposed hay grading standards based on laboratory analyses for evaluation quality. J. Dairy Sci. 47, 747–759. SAS Institute, 1990. 4th edition. SAS/STAT User's Guide, Vol. 2. Vers. 6. Gary, NC, pp. 893–1686. Schingoethe, D.J., Parsons, J.G., Ludens, F.C., Tucker, W.L., Shave, H.J., 1978. Evitamin status of dairy cows fed stored feeds continuously or pastured during summer. J. Dairy Sci. 61 (11), 1582–1589. Sjaunja, L.O., Baevre, L., Junkkarinen, L., Pedersen, J., Setälä, J.A., 1990. Nordic proposal for an energy-corrected milk (ECM) formula. 27th session, Paris 2–6 July. ICRPMA. Søegaard, K., Jensen, S.K., Sehested, J., 2010. Vitaminer, mineraler og foderværdi af græsmarksarter. Økologisk græsmarksproduktion og udnyttelser til mælkeproduktion. : Internal Report Husbandry, No 27. Faculty of Agricultural Sciences, Aarhus University, pp. 15–20. St-Laurent, A.M., Hidiroglou, M., Snoddon, M., Nicholson, J.W.G., 1990. Effect of α-tocopherol supplementation to dairy cows on milk and plasma αtocopherol concentrations and on spontaneous oxidized flavour in milk. Can. J. Anim. Sci. 70, 561–570. Tilley, J.M.A., Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Bri. Grassl. Soc. 18, 104–111. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, Second edition. Cornell University Press, Ithaca and London. 476 pp. Weisbjerg, M.R., Hvelplund, T., 1993. Bestemmelse af nettoenergiindhold (FEk) i forhold til kvæg. Forskningsrapport, nr. 3. Statens Husdyrbrugsforsøg. 39 pp. Weiss, W.P., Hogan, J.S., Smith, K.L., Hoblet, K.H., 1990. Relationships among Selenium, E-vitamin, and mammary gland health in commercial dairy herds. J. Dairy Sci. 73 (2), 371–390.