J. Dairy Sci. 87:406-412
© American Dairy Science Association, 2004.
Effects of Silage Species and Supplemental Vitamin E on the Oxidative Stability of Milk
R. M. Al-Mabruk1,
N. F. G. Beck1 and
R. J. Dewhurst2
1 Institute of Rural Studies, University of Wales, Aberystwyth SY23 3AL, U.K.
2 Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth SY23 3EB, UK
Corresponding author: R. J. Dewhurst, e-mail: richard.dewhurst{at}bbsrc.ac.uk.
 |
ABSTRACT
|
|---|
Two experiments were conducted to study the effects of feeding legume silages and providing supplemental vitamin E in concentrates on the oxidative stability of milk. In experiment 1, six multiparous Holstein-Friesian cows were offered 1 of 6 silage treatments in a cyclical changeover-design experiment, with four 4-wk periods. The silages were grass, red clover, white clover, alfalfa, grass and red clover mixture (50:50 on a DM basis), and grass and white clover mixture (50:50 on a DM basis). In experiment 2, 8 cows were used in a changeover-design experiment with three 4-wk periods. The 4 treatments were a factorial combination of forages (grass silage or red clover silage) and supplemental vitamin E in the form of all-rac-
-tocopheryl acetate (29 or 290 IU/kg of DM in the concentrate). All forages were offered ad libitum and a flat rate of concentrates (8 kg/d) was fed in both experiments. Red clover silage led to significantly higher forage intakes, milk yields, and milk protein percentage in experiment 2, which was in agreement with results from experiment 1. There was no effect of vitamin E on feed intake, milk production, or milk fat and protein percentage. Red clover silage also led to significant changes in milk fatty acid profiles, particularly increased levels of polyunsaturated fatty acids. Milk samples were stored at 4°C and 20°C and analyzed for -tocopherol and thiobarbituric acid reactive substances at intervals to determine oxidative stability. Diets based on red clover and alfalfa silages were associated with more rapid loss of -tocopherol and increased production of thiobarbituric acid reactive substances during the storage of milk in comparison with diets based on grass silage. The increased oxidative deterioration of milk produced from cows fed red clover silage was avoided by vitamin E supplementation.
Key Words: legume silage • milk oxidation • red clover • vitamin E
Abbreviation key: AS = alfalfa silage, GRCS = mixture of grass silage and red clover silage (50:50 on a DM basis), GWCS = mixture of grass silage and white clover silage (50:50 on a DM basis), GS = grass silage, GS+ = grass silage + supplemental vitamin E, PUFA = polyunsaturated fatty acids, RCS = red clover silage, RCS+ = red clover silage + supplemental vitamin E, TBARS = thiobarbituric acid reactive substances, WCS = white clover silage
|
INTRODUCTION
|
|---|
Legumes are important components of ruminant diets in many parts of the world. They are a vital part of organic (ecological) dairy systems, where they provide the major source of N for the system. Earlier studies have established the high intake characteristics and milk production potential of red clover silage (RCS) in comparison with grass silage (GS; Thomas et al., 1985), alfalfa silage (AS; Broderick et al., 2000, 2001), and corn silage (Hazard et al., 2001).
This article considers the effect of RCS on aspects of milk quality. There have been substantial improvements in the hygienic and keeping qualities of milk over the last century, and the market now expects milk with a long shelf life. The growing importance of extended shelf-life milk products means that this requirement will increase. Recent results from our group (Dewhurst et al., 2003a) showed that feeding RCS led to increased levels of polyunsaturated fatty acids (PUFA), particularly -linolenic acid, in milk. This is a beneficial effect because milk is criticized for its low PUFA and high saturated fatty acid contents in relation to targets for the human diet (Department of Health, 1994). In addition to fish products, ruminant products are one of the few sources of beneficial omega-3 PUFA, which are increasingly regarded as being deficient in the human diet (Simopoulos, 2001). However, this change in fatty acid composition could also have a negative effect on milk, with PUFA making milk more susceptible to oxidation (e.g., Charmley and Nicholson, 1994).
The objective of this work was to confirm the effect of RCS on milk fatty acid profiles and investigate effects on the oxidative stability of milk produced from RCS. The use of supplemental vitamin E (in the concentrates) to overcome problems with RCS feeding was also investigated.
|
MATERIALS AND METHODS
|
|---|
Two experiments were conducted to investigate the effects of legume silages on the oxidative stability of milk. Milk was taken from 2 feeding experiments, 1 of which was described previously (Dewhurst et al., 2003b) and used for evaluations of oxidative stability. Oxidative stability was assessed according to the loss of -tocopherol and appearance of thiobarbituric acid reactive substances (TBARS) during storage at 4°C or 20°C, for 0, 48, and 96 h, respectively.
Animal Feeding Experiments
Experiment 1.
The feeding experiment has been described previously (Dewhurst et al., 2003b). Six multiparous Holstein-Friesian cows (mean initial DIM = 76, SD = 36.0) were allocated to 6 forage treatments according to a 4-period cyclical changeover design with 4-wk periods (Davis and Hall, 1969). The forage treatments were GS, RCS, white clover silage (WCS), AS, and 50:50 (DM basis) mixtures of GS and RCS (GRCS) and GS and WC (GWCS) prepared as big bales. All cows received 8 kg/d of a standard concentrate, which contained (as mixed) 30.0% wheat, 15.0% palm kernel expeller, 14.0% corn gluten feed, 11.0% solvent-extracted rapeseed meal (low glucosinolate, low erucic acid), 9.0% solvent-extracted sunflower meal, 5.0% molasses, 5.0% expeller linseed meal, 5.0% groundnut meal, 2.0% solvent-extracted soybean meal, 1.5% vegetable fat, and 2.5% minerals/vitamins. The mineral/vitamin premix supplied (on a concentrate DM basis): 11,600 IU of vitamin A/kg, 2300 IU of vitamin D3/kg, 35 mg of Cu/kg, 140 mg of Mn/kg, 0.46 mg of Se/kg, and 14 mg/kg of Zn. In addition to the recordings of milk yield and composition already reported (Dewhurst et al., 2003a), samples of evening and morning milk were also taken from each cow during the final week of each period and used immediately for the storage experiments described below. Blood samples were collected from the tail vessels at the end of wk 4 in each period.
Experiment 2.
Eight multiparous Holstein-Friesian cows (initial DIM = 77, SD = 3.2) were randomly assigned to 4 dietary treatments according to a 3-period changeover design (2 incomplete Latin squares) with 4-wk periods. The 4 treatments were a factorial arrangement of forage types (GS or RCS) and concentration of vitamin E in the concentrates (29 or 290 IU of vitamin E/kg of DM in the form of all-rac--tocopheryl acetate). Thus there were 4 treatments, designated: GS, RCS, grass silage + supplemental vitamin E (GS+), red clover silage + supplemental vitamin E (RCS+).
All cows received 8 kg/d of the standard concentrate (as experiment 1 apart from the different concentrations of vitamin E). Concentrates were offered through out-of-parlor feeders with 4 meals spaced at least 4.5 h apart. The silages were prepared from single cuts of ryegrass and red clover during 1999. The cows were housed in a free-stall barn and had ad libitum access to the forages through roughage intake control feeders (Insentec B.V., Marknesse, The Netherlands). Feed intake and milk yields were recorded continuously, with results from the final week of each period used in the statistical analysis. Consecutive evening and morning milk samples were taken from each cow for analysis of fat, protein, and lactose concentrations by infrared milk analyzer (Milkoscan 605; Foss Electric, Hillerød, Denmark). Bulked (by volume) samples were also collected and stored frozen for subsequent fatty acid analysis. Samples of evening and morning milk were also used immediately in the storage experiments described below. Blood samples were collected from the tail vessels at the end of wk 4 in each period.
Milk Stability Experiments
Milk stability experiments were conducted using bulked samples of evening and morning milk taken from each cow. Twenty-milliliter aliquots of each milk sample were stored in glass tubes with teflon-lined screw caps at either 4°C or 20°C and held in the dark for periods of 0, 48, and 96 h (1 tube for each incubation duration). At the end of these incubations, samples were analyzed for content of -tocopherol and TBARS.
Chemical Analysis
Methods of feed processing and chemical analysis in our laboratory have been described previously (Dewhurst et al., 2000). Concentrations of fatty acids in samples of feeds and milk were analyzed according to the methods described by Sukhija and Palmquist (1988). The concentration of -tocopherol in silages was determined according to McMurray et al. (1980). -Tocopherol was extracted and analyzed according to Hidiroglou (1989) for milk and a modification of Hoelher et al. (1998) for plasma (with plasma, deionized water, internal standard in ethanol, ethanol, hexane, and methanol used in the ratios 1:1:1:4:3:3). Thiobarbituric acid-reactive substances in milk were determined according to Vyncke (1975) and are expressed as malondialdehyde equivalents (µg/mL) using 1,1,3,3-tetraethoxypropane as a standard.
Statistical Analysis
Data were analyzed using REML (Genstat 5; Lawes Agricultural Trust, 1997). Preliminary overall REML analysis (storage time x storage temperature x diet) showed that there were highly significant effects (P < 0.001) of storage time and storage temperature, as well as interactions with the effects of diets. Further analysis was conducted using values for each time and temperature combination separately. A fixed effect of diet and a random effect of period + cow were used for experiment 1. A Student-Newman-Keulls test was used to compare treatment means when the overall analysis showed a significant effect of diet. For experiment 2, the model used forage, vitamin E, and their interaction as fixed effects and period + cow as random effects.
|
RESULTS
|
|---|
Feed Composition
Experiment 1.
Details of feed composition were presented earlier (Dewhurst et al., 2003a). The concentration of -tocopherol in GS, RCS, WCS, and AS was 10.7, 16.5, 18.6, and 11.3 mg/kg of DM, respectively.
Experiment 2.
The results of the chemical analysis of the silages used in experiment 2 are shown in Table 1
. The silages were typical of moderate fermentation quality silages, although the N content of the grass silage was at the high end of the normal range. The fatty acid composition of the 2 silages was very similar, with slightly higher concentrations of C18:3 in the grass silage. The concentrate used in experiment 2 contained 4.48% of DM as fatty acids, with 0.83% as C16:0, 1.57% as cis-9 C18:1, 1.18% as C18:2, and 0.15% as C18:3. The concentrations of -tocopherol in GS and RCS are given in Table 1.
Milk Production
Feed intake and milk production results from experiment 2 are shown in Table 2, with detailed fatty acid composition of the milk given in Table 3. Milk fatty acids are presented as proportions of reported fatty acids. Corresponding results for experiment 1 were presented previously (Dewhurst et al., 2003a, 2003b); it is important to note that there was no significant effect of diet on milk fat percentage.
View this table:
[in this window]
[in a new window]
|
Table 2. Effects of legume silages and supplementary vitamin E on feed intake, milk production, and milk composition in Exp. 2.
|
|
View this table:
[in this window]
[in a new window]
|
Table 3. Effects of legume silages and supplementary vitamin E on the fatty acid composition of milk (% total milk fatty acids; Exp. 2).1
|
|
Plasma -Tocopherol
The effects of forage and supplemental vitamin E treatments on concentrations of -tocopherol in plasma are given in Tables 4 and 5 for experiments 1 and 2, respectively.
View this table:
[in this window]
[in a new window]
|
Table 4. Effects of legume silages on the concentration of -tocopherol (µg/mL) in plasma and in milk after storage at 4°C or 20°C for 48 or 96 h (Exp. 1).1
|
|
View this table:
[in this window]
[in a new window]
|
Table 5. Effects of silage crop (grass vs. red clover) and supplemental vitamin E on the concentration of -tocopherol (µg/mL) in plasma and in milk after storage at 4°C or 20°C for 48 or 96 h (Exp. 2).
|
|
Milk Storage Experiments
Concentrations of -tocopherol and TBARS (expressed as malondialdehyde equivalents [µg/mL] using a 1,1,3,3-tetraethoxypropane standard) in fresh and stored milk are shown in Tables 4
and 6, respectively, for experiment 1, and Tables 5 and 7, respectively, for experiment 2.
View this table:
[in this window]
[in a new window]
|
Table 6. Effects of legume silages on the concentration of thiobarbituric acid-reactive substances (expressed as malondialdehyde equivalents in µg/mL) in milk after storage at 4°C or 20°C for 48 or 96 h (Exp. 1).1
|
|
View this table:
[in this window]
[in a new window]
|
Table 7. Effects of silage crop (grass vs. red clover) and supplemental vitamin E on the concentration of thiobarbituric acid-reactive substances (expressed as malondialdehyde equivalents in µg/mL) in milk after storage at 4°C or 20°C for 48 or 96 h (Exp. 2).
|
|
|
DISCUSSION
|
|---|
Feed Intake and Milk Production
Increased voluntary forage (and total) intake and milk production for cows offered RCS rather than GS in experiment 2 are consistent with earlier observations (Thomas et al., 1985; Dewhurst et al., 2003a). Earlier studies showed no effect (e.g., Thomas et al., 1985) or a slight reduction (Hoffman et al., 1998) in milk protein percentage when feeding RCS in place of GS. By contrast, in the current experiment, RCS led to a significant increase in milk protein percentage. It seems likely that the increase in milk protein percentage reflects the improved energy status of cows resulting from the 35% increase in forage voluntary intake. Milk protein percentage was particularly low for cows offered GS, suggesting that low energy status was a dominant issue in determining production responses in this study (Thomson et al., 1985).
Milk Fatty Acids
The 30% increase in the concentration of C18:2 in milk fat and the doubling in concentrations of C18:3 when replacing GS with RCS are consistent with earlier results from this laboratory (Dewhurst et al., 2003a). Similarly, feeding RCS led to lower concentrations of biohydrogenation intermediates (C18:1 trans-11 and conjugated linoleic acid [cis-9, trans-11]) in milk.
The increased concentrations of C18:3 in milk from cows offered RCS occurred despite the slightly lower concentrations of C18:3 in RCS in comparison with GS. In fact, the apparent recovery of C18:3 from feed into milk increased from 5.4% for GS to 9.6% for RCS. The increased apparent recovery of C18:3 into milk and lower levels of biohydrogenation intermediates with RCS confirm the reduction in biohydrogenation noted previously in this laboratory (Dewhurst et al., 2003b; Lee et al., 2003).
-Tocopherol in Forage, Plasma, and Milk
Concentrations of -tocopherol were higher in both of the clover silages (RCS and WCS) in comparison with GS and AS in experiment 1. This pattern was not fully reflected in plasma concentrations of -tocopherol: feeding white clover silage led to the highest concentrations, while concentrations were significantly lower when feeding AS. Concentrations of -tocopherol in plasma were similar for cows fed RCS or GS, despite the higher intakes and higher concentrations of -tocopherol in RCS in experiment 2. The doubling in plasma -tocopherol in response to supplemental vitamin E was highly significant, but proportionately much less than the increase in vitamin E intake (approximately 5-fold). Other workers have shown that vitamin E supplementation increased plasma and milk -tocopherol, but only to a limited extent (e.g., St-Laurent et al., 1990; Jackson et al., 1997).
The concentration of -tocopherol in fresh milk samples generally increased in line with concentrations in plasma (Tables 4 and 5). Cows fed RCS were an exception to this relationship in both experiments, with lower concentrations of -tocopherol in milk relative to plasma concentrations. This suggests that the increased oxidation in these milks started—either in the udder, the collection vessels, or both—before milk samples were analyzed. Similarly, a smaller proportion of -tocopherol intake was yielded in milk when feeding RCS (2.7%) than when feeding GS (5.2%). This effect may also reflect greater oxidation of -tocopherol within the animal and its tissues.
Oxidative Stability of Milk
Diets based on either RCS or AS led to increased levels of TBARS and reduced concentrations of -tocopherol in stored milks. Concentrations of -tocopherol were numerically lower in fresh milk from cows fed RCS or AS, although this difference was not statistically significant owing to large variability. Charmley and Nicholson (1994) also found that significant differences in concentrations of -tocopherol developed during milk storage. Conversely, there were no significant differences in TBARS for milk stored for 96 h at 20°C: this is in agreement with the observations of Lundin and Palmquist (1983) and reflects the fact that all of the milks had deteriorated extensively by this stage.
The consistent and highly significant increase in concentrations of PUFA in milk from cows fed RCS provides an explanation for the reduced oxidative stability of milk. Many earlier studies have shown that increasing concentrations of unsaturated fatty acids in milk, particularly of PUFA C18:2 and C18:3, will increase the susceptibility of milk to oxidation (e.g., Sidhu et al., 1975; Granelli et al., 1998; Timmons et al., 2001).
Supplemental vitamin E led to production of milk from RCS-based diets with the same oxidative stability as was observed for milk from GS-based diets. The effects of supplemental vitamin E on TBARS in stored milk are in agreement with earlier studies (e.g., Lundin and Palmquist, 1983; Atwal et al., 1991).
Legumes are important components of dairy systems where farmers prefer not to use N fertilizer, such as organic (ecological) systems or in regions where N fertilizer is not available at an economic price. Consequently, there may be a need to look for alternative solutions to the problem of milk oxidation for use with organic dairy systems, which do not permit the use of supplemental vitamin E for this purpose. However, results with the mixed forage diet (GRCS) suggest that reduced oxidative stability of milk may not be a major problem with these mixed forage diets that are more typical of organic farms.
|
ACKNOWLEDGEMENTS
|
|---|
The financial support of the Department for Environment, Food, and Rural Affairs, the Milk Development Council, the European Union, and the Government of Libya (studentship for R. A. Al-Mabruk) is gratefully acknowledged.
Received for publication April 22, 2003.
Accepted for publication September 2, 2003.
|
REFERENCES
|
|---|
Atwal, A. S., M. Hidiroglou, and J. K. G. Kramer. 1991. Effects of feeding protec® and -tocopherol on fatty acid composition and oxidative stability of cows’ milk. J. Dairy Sci. 74:140–145.[Abstract]
Broderick, G. A., R. P. Walgenbach, and E. Sterrenburg. 2000. Performance of lactating dairy cows fed alfalfa or red clover silage as the sole forage. J. Dairy Sci. 83:1543–1551.[Abstract]
Charmley, E., and J. W. G. Nicholson. 1994. Influence of dietary fat source on oxidative stability and fatty acid composition of milk from cows receiving a low or high level of dietary vitamin E. Can. J. Anim. Sci. 74:657–665.
Davis, A. W., and W. B. Hall. 1969. Cyclical change-over designs. Biometrika 56:283–293.[Abstract/Free Full Text]
Department of Health. 1994. Nutritional Aspects of Cardio-Vascular Disease. Report on Health and Social Subjects. No. 46. Her Majesty’s Stationery Office, London, UK.
Dewhurst, R. J., W. J. Fisher, J. K. S. Tweed, and R. J. Wilkins. 2003a. Comparison of grass and legume silages for milk production. 1. Production responses with different levels of concentrate. J. Dairy Sci. 86:2598–2611.[Abstract/Free Full Text]
Dewhurst, R. J., R. T. Evans, N. D. Scollan, J. M. Moorby, R. J. Merry, and R. J. Wilkins. 2003b. Comparison of grass and legume silages for milk production. 2. In vivo and in sacco evaluations of rumen function. J. Dairy Sci. 86:2612–2621.[Abstract/Free Full Text]
Dewhurst, R. J., J. M. Moorby, M. S. Dhanoa, R. T. Evans, and W. J. Fisher. 2000. Effects of altering energy and protein supply to dairy cows during the dry period. 1. Intake, body condition, and milk production. J. Dairy Sci. 83:1782–1794.[Abstract]
Granelli, K., P. Barrefors, L. Bjorck, and L. A. Appelqvist. 1998. Further studies on lipid composition of bovine milk in relation to spontaneous oxidised flavour. J. Sci. Food. Agric. 77:161–171.
Hazard T., M. S. Morales, B. N. Butendieck, A. Gomez, and P. M. Mardones. 2001. Evaluation of corn silage and red clover silage mixed in different proportions in the winter feed of dairy cows in Southern Chile. Agric. Tec. 61:306–318.
Hidiroglou, M. 1989. Mammary transfer of vitamin E in dairy cows. J. Dairy Sci. 72:1067–1071.
Hoehler, D., A. A. Frohlich, R. R. Marquardt, and H. Stelsovsky. 1998. Extraction of alpha-tocopherol from serum prior to reversed-phase liquid chromatography. J. Agric. Food. Chem. 46:973–978.
Hoffman, P. C., D. K. Combs, and M. D. Casler. 1998. Performance of lactating dairy cows fed alfalfa silage or perennial ryegrass silage. J. Dairy Sci. 81:162–168.[Abstract]
Jackson, J. A., R. J. Harmon, and Z. Tabeidi. 1997. Effect of dietary supplementation with vitamin E for lactating dairy cows fed tall fescue hay infected with endophyte. J. Dairy Sci. 80:569–572.[Abstract]
Lee, M. R. F., L. J. Harris, R. J. Dewhurst, R. J. Merry, and N. D. Scollan. 2003. The effect of clover silages on long chain fatty acid rumen transformations and digestion in beef steers. Anim. Sci. 76:491–501.
Lundin, P. K., and D. L. Palmquist. 1983. Vitamin E supplementation of high fat diets for dairy cows. J. Dairy Sci. 66:1909–1916.
McMurray, C. H., W. J. Blanchflower, and D. A. Rice. 1980. Influence of extraction techniques on the determination of alpha-tocopherol in animal feedstuffs. J. AOAC 63:1258–1261.
Sidhu, G. S., M. A. Brown, and A. R. Johnson. 1975. Autoxidation in milk rich in linoleic acid. I. An objective method for measuring autoxidation and evaluating antioxidants. J. Dairy Res. 42:185–195.[Medline]
Simopoulos, A. P. 2001. n-3 Fatty acids and human health: Defining strategies for public policy. Lipids 36(Suppl.):S83–S89
St-Laurent, A., M. Hidiroglou, M. Snoddon, and J. W. G. Nicholoson. 1990. Effect of alpha-tocopherol supplementation to dairy cows on milk and plasma -tocopherol concentrations and on spontaneous oxidized flavour in milk. Can. J. Anim. Sci. 70:561–570.
Sukhija, P. S., and D. L. Palmquist. 1988. Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. J. Agric. Food. Chem. 36:1202–1206.
Thomas, C., K. Aston, and S. R. Daley. 1985. Milk production from silage. 3. A comparison of red clover with grass silage. Anim. Prod. 41:23–31.
Timmons, J, S., W. P. Weiss, D. L. Palmquist, and W. P. Harper. 2001. Relationship among dietary roasted soybeans milk components, and spontaneous oxidized flavor of milk. J. Dairy Sci. 84:2440–2449.[Abstract]
Vyncke, W. 1975. Evaluation of the direct thiobarbituric acid extraction method for determining oxidative rancidity in mackerel (Scomber scombrus L.). Fette Seifen Anstrichm. 77:239–240.
This article has been cited by other articles:
|
|
|
|
 
J. A. Voelker Linton and M. S. Allen
Nutrient Demand Interacts with Forage Family to Affect Intake and Digestion Responses in Dairy Cows
J Dairy Sci,
July 1, 2008;
91(7):
2694 - 2701.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Quigley, T. A. Wolfe, and T. H. Elsasser
Effects of Additional Milk Replacer Feeding on Calf Health, Growth, and Selected Blood Metabolites in Calves
J Dairy Sci,
January 1, 2006;
89(1):
207 - 216.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
