J. Dairy Sci. 88:2037-2042
© American Dairy Science Association, 2005.
Effects of Dietary Sources of Vegetable Oils on Performance of High-Yielding Lactating Cows and Conjugated Linoleic Acids in Milk
H. C. Zheng1,
,
J. X. Liu1,
J. H. Yao2,
Q. Yuan2,
H. W. Ye3,
J. A. Ye1 and
Y. M. Wu1
1 Institute of Dairy Science, Zhejiang University, Hangzhou 310029, China
2 Hangzhou Station for Detection of Agricultural Products, Hangzhou 310020, China
3 Hangzhou Zhengxing Animal Industry Company, Hangzhou 311301, China
Corresponding author: Jian-Xin Liu; e-mail: liujx{at}zju.edu.cn.
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ABSTRACT
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This study was conducted to examine the effects of dietary supplementation with vegetable oils on performance of high-yielding lactating cows and conjugated linoleic acid (CLA) content in milk fat. Twelve lactating Holstein cows in early lactation (30 to 45 d postpartum) were used in a triple 4 x 4 Latin square design. In each period, the cows in each group were fed the same basal diet and received one of the following treatments: 1) control (without oil), 2) 500 g of cottonseed oil, 3) 500 g of soybean oil, and 4) 500 g of corn oil. Each experimental period lasted for 3 wk, with the first 2 wk used for adaptation to the diet. Supplementation with vegetable oils tended to increase milk yield, with the highest milk yield in the cottonseed oil group (35.0 kg/d), compared with the control (34.4 kg/d). Milk fat percentage was decreased, but there were few effects on percentage and yield of milk protein as well as milk fat yield. The cows fed added soybean oil produced milk with the highest content of trans-11 C18:1 (23.8 mg/g of fat), which was twice that of the control (12.6 mg/g of fat). Content of cis-9, trans-11 CLA in milk fat increased from 3.5 mg/g in the control to 6.0, 7.1, and 10.3 mg/g for the cows fed oils from cottonseed, corn, and soybean, respectively. A significant linear relationship existed between trans-11 C18:1 and cis-9, trans-11 CLA. Supplementation with oils doubled the content of total fatty acids in blood plasma, with little difference between different vegetable oil sources. Octadecenoic acid content was significantly higher in blood plasma of animals fed added oils from cottonseed and soybean than those fed with corn oil and control. The plasma trans-11 C18:1 content was significantly higher in the oil-added animals than in control. Supplementation of vegetable oils tended to improve milk production of lactating cows, and the CLA content in milk fat was significantly increased. Soybean oil seemed to be the optimal source to increase CLA production.
Key Words: vegetable oil • milk performance • conjugated linoleic acid • lactating cow
Abbreviation key: CLA = conjugated linoleic acid.
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INTRODUCTION
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Conjugated linoleic acid (CLA) represents a mixture of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. The CLA are effective as anticarcinogenic, antidiabetic, and antilipogenic agents in the diet of laboratory animals (see review by Pariza et al., 2001). Recently, it has been demonstrated that trans-10, cis-12 C18:2 is responsible for reduced lipogenesis in the rodent (Pariza et al., 2001). In contrast, milk fat-derived cis-9, trans-11 C18:2 prevented growth of human mammary cancer cells more effectively than did synthetic trans-10, cis-12 C18:2 (O’Shea et al., 2000).
Ruminant meat and milk are the predominant natural sources of the CLA. Cis-9, trans-11 CLA, however, accounts for nearly 90% of total CLA in milk fat from cows fed typical diets (see review by Bauman et al., 1999). Trans-10, cis-12 C18:2 represented less than 2% of total CLA (Piperova et al., 2000). The cis-9, trans-11 CLA can be formed as a result of incomplete biohydrogenation of dietary fatty acids and by desaturase action on trans-11 C18:1 in the rumen. It can also arise from isomerization via cis-12, trans-11 isomerase produced by rumen bacteria (Kepler and Tove, 1967). In the bovine mammary gland (Bauman et al., 1999) or human tissues (Pariza et al., 2001), trans-11 C18:1 (another intermediate of biohydrogenation) can be a source for endogenous synthesis of cis-9, trans-11 CLA via 
9-desaturase.
Typically, milk fat contains between 3 and 6 mg of CLA/g of fat, but the levels of CLA in milk can vary widely among herds (Kelly and Bauman, 1996). The substantial variation in content of CLA in milk fat between herds suggests that diet has a major influence. Kelly et al. (1998) demonstrated that dietary supplementation of vegetable oils high in linoleic acid gave the greatest response, and there is a clear dose-dependent increase in milk fat content of CLA. The objective of this study was to compare the effect of dietary supplementation of different plant oils on CLA content in milk fat. The vegetable oils examined were similar in linoleic acid content but varied in proportions of C18 mono-, di-, and trienes.
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MATERIALS AND METHODS
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Animals and Diets
Twelve multiparous Holstein cows in early lactation (between 30 and 45 d postpartum) were used in a triple 4 x 4 Latin square design to evaluate responses to supplementary vegetable oils. Treatments included a control diet and diets supplemented with oil from cottonseed, soybean, or corn. The vegetable oils were acquired from a local supermarket. Each experimental period lasted 1 wk, and was preceded by a 2-wk period of adaptation to the diet. Net energy content of diets was calculated from the ingredient composition. Diets were formulated to meet energy and protein requirements (NRC, 2001) of lactating cows producing 35 kg of milk and consuming 24 kg of DM/d (Table 1
). Oils were added at 500 g/d per head, i.e., at a level of about 2% of dietary DM, resulting in a dietary ether extract content of 6.7%. The oil was added to the concentrate daily by pouring it on and mixing at feeding to ensure its equal distribution in the ration. The cows were fed and milked 3 times a day.
Sampling, Measurements, and Analyses
Forages and concentrates were sampled on d 1, 3, and 5 of each experimental period. Samples were immediately dried in a forced-air oven at 60°C, and stored in sealed plastic containers at room temperature until analyzed. In preparation for analyses, dried forages and concentrates were ground first through a 2-mm screen (Wiley; Arthur H. Thomas, Philadelphia, PA), and then through a 1-mm screen in a Cyclotec mill (Tecator 1093, Hoganas, Sweden). Forages and concentrates were analyzed for ADF (Robertson and Van Soest, 1981), NDF (Van Soest et al., 1991), and total N (AOAC, 1990; method no. 988.05).
Four 50-mL aliquots of milk were collected at each milking (0700, 1400, and 2100 h) on d 1, 3, 5, and 7 of each experimental period. One aliquot containing Bromopol (milk preservative; D&F Control Systems, San Ramon, CA) was stored at 4°C until analyzed for fat, protein, and lactose by infrared analysis (Laporte and Paquin, 1999) with a 4-channel spectrophotometer (MilkoScan, Foss Electric, Hillerød, Denmark). The second aliquot without Bromopol was stored at –20°C until the end of the experiment, then thawed and centrifuged at 10,000 x g for 1 h to harvest milk fat for fatty acid analysis.
For fatty acid analysis in total plasma, blood samples (10 mL) were obtained from the coccygeal artery immediately after collection of milk samples on d 7. Blood was transferred to tubes containing 286 IU of heparin in 100 µL of sterile saline and centrifuged at 3000 x g for 15 min to harvest plasma. Plasma was stored at –20°C until lipid extraction and fatty acid analysis.
Total lipids from plasma (2 mL) were extracted with chloroform:methanol (2:1, vol/vol). Fatty acids in vegetable oils, milk fat, and blood plasma were methylated by in situ transesterification with 0.5 N methanolic NaOH followed by 14% boron trifluoride in methanol (Loor and Herbein, 2001). Samples were injected by autosampler into a Hewlett-Packard 6890A gas chromatograph equipped with a flame-ionization detector (Hewlett-Packard, Sunnyvale, CA). Methyl esters from all samples were separated on a 0.32 mm x 30 m x 0.25 µm i.d. fused silica capillary column (HP-Innowax, Hewlett-Packard). Pure methyl ester standards (Sigma, St. Louis, MO) were used to identify peaks, and determine correction factors for individual fatty acids. For vegetable oils and milk fatty acid analysis (1 µL of methyl esters in hexane injected at a 1:5 split ratio), the injector temperature was maintained at 220°C and the detector temperature maintained at 225°C. For fatty acids in vegetable oils, the oven temperature was 200°C (held for 20 min); for fatty acids in milk fat, the oven temperature was 50°C (held for 1 min), and then increased at 20°C/min to 220°C (held for 10 min). Ultra-pure nitrogen was used as the carrier gas. All fatty acids eluted at a flow of 2.5 (for vegetable oils) and 1.9 mL/min (for milk fat).
Analysis of fatty acids in total plasma and lipid fractions required injection of 1 µL of methyl-esters in hexane with split ratio of 1:20. The injector temperature was maintained at 225°C and the detector temperature at 250°C. The initial column temperature was 205°C (held for 5 min), and then increased at 2°C/min to 220°C (held for 10 min). Ultrapure nitrogen was used as the carrier gas. All fatty acids eluted at a flow of 0.8 mL/min.
Statistical Analyses
Data were processed using the GLM procedure of SPSS (2001) using a linear model with the effects of diet, square, period within square, and cow within square. The interaction between square and treatment was tested, found to be nonsignificant and then removed from the model. Main effects (diet) were tested using Duncan’s multiple range procedure using the GLM procedure of SPSS (2001).
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RESULTS
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The 3 vegetable oils used in the study were chosen because they had similar linoleic acid content although they differed in C16:0, C18:1, and C18:3 (Table 2). The DM intake was relatively constant during each period and did not differ among the 4 treatments (P > 0.1). Across all treatments, DM intake averaged 23.93 kg/d.
Milk yields were not different for type of oil fed (Table 3), although they were relatively higher in cows fed vegetable oil diets (P > 0.05). Milk fat percentage was lowest when cows received the soybean oil diet, significantly different from the other diets (3.05 vs. 3.44, P < 0.05), whereas milk fat yield showed no significant differences between the 4 diets. Contents and yield of protein and lactose remained unchanged for all treatments.
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Table 3. Dry matter intake and milk production, composition, and component yields by Holstein cows influenced by supplementation of different vegetable oils.
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Myristic and palmitic acids contents were decreased (P < 0.05) by supplementation with vegetable oils (Table 4). These fatty acid contents in milk fat did not differ due to oil sources. Stearic acid was higher when supplemented with oil from cottonseed or corn (P < 0.05). Oleic acid was significantly increased (P < 0.05) and was relatively higher in response to soybean oil. The cows supplemented with soybean oil produced milk with highest content of trans-11 C18:1, twice that in the control, followed by the animals on corn and cottonseed oil diets. A similar response was observed for the cis-9, trans-11 CLA in milk fat, which increased to 10.3, 7.1, and 6.0 mg/g of fat for the animals supplemented with soybean oil, corn oil, and cottonseed oil, respectively, from 3.5 mg/g for the control. A significant linear relationship existed between trans-11 C18:1 and cis-9, trans-11 CLA (R2 = 0.664, P < 0.05) (Figure 1).
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Table 4. Contents of medium- and long-chain fatty acids in milk fat from Holstein dairy cows influenced by supplementation of different vegetable oils.
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Figure 1. Linear relationship between concentrations of trans-11 C18:1 and cis-9, trans-11 conjugated linoleic acids (CLA) in milk fat from Holstein dairy cows.
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Supplementation with vegetable oils almost tripled the content of fatty acids in blood plasma (P < 0.01), with little difference between different oil sources (Table 5). When cows received the cottonseed oil, palmitic acid content in blood plasma was significantly higher (237.3 mg/g of fat, P < 0.05), whereas content of C18:2 was lower (410.6 mg/g of fat, P < 0.05) than for the other treatments. Content of trans-11 C18:1 was higher (P < 0.05) for oil-supplemented diets than for the control, with higher content for the soybean oil diet than for the cottonseed oil diet (P < 0.05). The content of oleic acid in blood plasma for cottonseed oil or soybean oil was significantly higher than that for corn oil and control, with no difference between the latter 2 groups.
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Table 5. Contents of medium- and long-chain fatty acids in blood plasma of Holstein dairy cows influenced by supplementation with vegetable oils.
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DISCUSSION
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Our experiment evaluated the quantitative significance of ruminal availability of oleic or linoleic acid in secretion of conjugated linoleic acid in milk. Fatty acid profiles in blood plasma and milk fat were used to assess changes in the production of hydrogenation intermediates in the rumen.
Contents of C14:0 and C16:0 in milk fat were significantly lower in response to supplemented vegetable oils. Milk fat contains fatty acids derived from de novo synthesis by the mammary gland (C4:0 to C14:0 plus a portion of C16:0) and from mammary uptake of preformed fatty acid (a portion of C16:0 and all longer chain fatty acids). Exogenous fatty acids compete for esterification with newly synthesized short-chain fatty acids in mammary cells, and could lead to feedback inhibition of lipogenic enzymes (Palmquist et al., 1993). Supplemental cis-9 C18:1 was preferentially incorporated into the sn-2 position of the milk fat triglyceride at the expense of C16:0 (DePeters et al., 2001), effectively lowering its content and increasing cis-9 C18:1. Comparable responses in milk triglyceride composition have been observed in cows fed on high linoleic oil (Christie, 1981; Palmquist et al., 1993). Thus, greater uptake and secretion of dietary and rumen-derived fatty acids may account for the majority of the reduction in de novo synthesis in cows fed unsaturated oils (Palmquist et al., 1993).
All of the vegetable oil diets resulted in milk fat contents of cis-9, trans-11 C18:2 that were 2- to 3-fold higher than that in the control. Content in the soybean oil treatment was significantly higher than for the other treatments (Table 3
). Previous work has suggested that the biohydrogenation sequence of linoleic acid can lead to an increase in CLA levels in milk fat (McGuire et al., 1996). The 3 vegetable oils used in this study have similar contents of linoleic acid, but resulted in different CLA contents in milk fat. This result indicated that other fatty acids might contribute to CLA production. A strong linear relationship between trans-11 C18:1 and CLA in milk fat was found in our study (R2 = 0.664, P < 0.05), which agreed with other studies (Lawless et al., 1998; Griinari and Bauman, 1999; Secchiari et al., 2003), suggesting that CLA is predominantly synthesized in the mammary cells from trans-11 C18:1.
Linolenic acid (cis-9, cis-12, cis-15 C18:3) has been shown to be converted to cis-9, trans-11, cis-15 conjugated triene, then to trans-11, cis-15 C18:2, and finally to an octadecenoic acid that is either trans-11, trans-15, or cis-15 (Harfoot and Hazelwood, 1988). Harfoot and Hazelwood (1988) confirmed that biohydrogenation of oleic acid by mixed ruminal microbes involves the formation of several positional isomers of trans monoenes (including trans-11 C18:1) rather than only direct biohydrogenation to form stearic acid. Consequently, trans-11 C18:1 is a common intermediate in the biohydrogenation of linoleic acid, 
-linolenic acid, and oleic acid. Cis-9, trans-11 CLA content in milk fat was highest (P < 0.05) in response to soybean oil (Table 4), although soybean oil had moderate C18:2 and C18:1, compared with the other 2 vegetable oils used in this study (Table 2). The fact that soybean oil was rich in C18:3 may explain this observation. The C18:2 content in corn oil was 44 mg/g of fat lower than that in cottonseed oil (Table 2), but the cis-9, trans-11 CLA content in milk fat was slightly higher in response to corn oil than to cottonseed oil (Table 4), which may be due to the higher C18:1 content in corn oil than in cottonseed oil.
Dietary supplementation with vegetable oils may alter rumen fermentation, with decreased acetic acid production (Onetti et al., 2001), which could cause the milk fat depression syndrome. Several mechanisms have been proposed to explain how lipids interfere with ruminal fermentation (Jenkins, 1993). The lipid "coating" theory and direct antimicrobial effects have received the most attention. Antimicrobial effects of lipids in the rumen have many similarities to cytotoxic effects of fatty acids on membrane function of eukaryotic cells, such as uncoupling of oxidative phosphorylation. Long-chain unsaturated fatty acids may readily attach to lipid bilayers in biological membranes because of their hydrophobic and amphiphilic nature. The longer the chains and the more double bonds that fatty acids have, the easier it is for them to attach and destroy the membrane of bacteria. This is most likely why the milk fat percentage is the lowest in response to soybean oil.
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CONCLUSIONS
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Secretion of unsaturated fatty acids and rumen-derived trans C18:1 in milk may be increased by inclusion of high-oil feed ingredients in dairy diets. Trans-11 C18:1 is a common intermediate in the biohydrogenation of linoleic acid, -linolenic acid, and oleic acid. Cis-9, trans-11 CLA in milk fat seemed predominantly synthesized in the mammary gland from trans-11 C18:1. The transfer ratio of C18:1, C18:2, C18:3 to trans-11 C18:1 should be studied further.
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ACKNOWLEDGEMENTS
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The authors gratefully acknowledge Hu Zhan-li, Lin Gui-you, and Zhong Xiao-sheng for their assistance in animal feeding and care.
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FOOTNOTES
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*Research supported in part by Ministry of Science and Technology of China and by Zhejiang Provincial Department of Science and Technology, China.
Current address: Institute of Animal Science & Veterinary Medicine, ZAAS, Hangzhou 310021, China. 
Received for publication December 20, 2004.
Accepted for publication February 23, 2005.
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ABSTRACT
INTRODUCTION
