Conjugated Linoleic Acid (CLA) Content of Milk from Cows Offered ...

Conjugated Linoleic Acid (CLA) Content of Milk from Cows Offered Diets Rich in Linoleic and Linolenic Acid1 T. R. Dhiman,* L. D. Satter,* M. W. Pariza,† M. P. Galli,*,2 K. Albright,† and M. X. Tolosa‡ *US Dairy Forage Research Center USDA, Agricultural Research Service, †Food Research Institute, University of Wisconsin, Madison 53706 ‡Animal, Dairy and Veterinary Sciences Department, Utah State University, Logan 84322-4815

ABSTRACT Two experiments were conducted to determine the conjugated linoleic acid (CLA) content of milk from cows offered diets rich in linoleic and linolenic acid. In experiment 1, 36 cows were assigned to a control and five treatment groups. Cows in the control group received a diet containing 51% forage and 49% grain on a dry matter basis. In the treatment group, grain was partly replaced by either 18% raw cracked soybeans, 18% roasted cracked soybeans, 3.6% soybean oil, 2.2% linseed oil, or 4.4% linseed oil. Experimental diets were fed for 5 wk. Average CLA contents in milk fat from wk 2 through 5 were 0.39% in control and 0.37, 0.77, 2.10, 1.58, and 1.63% of total fatty acids in the raw soybean, roasted soybeans, soybean oil, 2.2% linseed oil, and 4.4% linseed oil treatments, respectively. In experiment 2, 36 cows were assigned to a control and 5 treatment groups. Cows in the control group received a diet containing 55% forage and 45% grain. In the treatment groups, grain was partly replaced by soybean oil at 0.5, 1.0, 2.0, 4.0, or by linseed oil at 1.0% of the dietary dry matter. Experimental diets were fed for 5 wk. Average CLA contents in milk fat from wk 2 through 5 were 0.50% in control and 0.75, 0.76, 1.45, 2.08, and 0.73% of total fatty acids in 0.5, 1.0, 2.0, 4.0 soybean oil and 1.0% linseed oil treatments, respectively. Diets rich in linoleic or linolenic acid can increase CLA content of milk when dietary oil is accessible to the rumen microorganisms.

Received May 25, 1999. Accepted January 4, 2000. Corresponding author: T. R. Dhiman; e-mail: [email protected]. 1 Trade names and the names of commercial companies are used in this report to provide specific information. Mention of a trade name or manufacturer does not constitute a guarantee or warranty of the product by the USDA or an endorsement over products not mentioned. 2 Present address: Billinghurst 2559, 3rd Floor, Capital Federal (1425), Argentina. 2000 J Dairy Sci 83:1016–1027

(Key words: conjugated linoleic acid, milk fat, cow, soybean) Abbreviation key: CLA = conjugated linoleic acid, LO1 = linseed oil at 1%, LO2 = linseed oil at 2.2%, LO4 = linseed oil at 4.4%, RAWSB = raw cracked soybeans, RSB = roasted cracked soybeans, SO = soybean oil, SO1 = soybean oil at 0.5%, SO2 = soybean oil at 1%, SO3 = soybean oil at 2%, SO4 = soybean oil at 4%. INTRODUCTION Conjugated linoleic acid (CLA) is a naturally occurring fatty acid in foods derived from ruminants. Conjugated linoleic acid is a term used for a mixture of positional and geometric isomers of linoleic acid (cis9, cis-12 octadecadienoic acid) that contain conjugated unsaturated double bonds. More than 82% of the CLA in dairy products is the cis-9, trans-11 isomer (2). Conjugated linoleic acid has been shown to have anticarcinogenic properties (11, 12, 16, 25) and possibly other effects that would be positive for human health. If these effects are found to occur in humans, increases in the concentration of CLA would increase the nutritive and therapeutic value of milk and milk products. Average CLA content in milk varies between 0.3 and 0.6% of total fatty acids (19). Typical consumption of CLA by humans is lower (on an equivalent BW basis) than the dose that has been shown to be effective in reducing tumors in animal models (17). Intake of CLA can be increased by increasing the consumption of foods from ruminant origin or by increasing the CLA content of foods derived from ruminant origin. The latter approach is more practical. Kepler and Tove (20) identified the cis-9, trans-11 isomer of C18:2 fatty acid as an intermediate in the biohydrogenation of linoleic acid by the rumen bacterium Butyrivibrio fibrisolvens. In the rumen, dietary lipids are hydrolyzed, and resulting unsaturated fatty acids are converted to saturated fatty acids by the rumen microorganisms (13). When the dietary supply of unsaturated fatty acids is high, or the biohydrogenation pro-

1016

CONJUGATED LINOLEIC ACID IN MILK

cess may be incomplete, the CLA can escape the rumen and become available for absorption in the lower digestive tract, thus providing a source of CLA to the mammary gland. Dhiman et al. (5) have shown that cattle grazing pasture had higher CLA content in milk than did cattle fed pasture plus grain. Feeding full fat extruded soybeans, full fat extruded cottonseed, or sunflower oil increased the CLA content in milk (6, 19). Pasture grasses are rich in linolenic acid (C18:3). The oil in soybeans, cottonseed, and sunflower are rich sources of linoleic acid (C18:2). An important question is whether the CLA found in milk from cows grazing grass or fed soybeans is related to dietary intake of linoleic and linolenic acid. The objective of this study was to determine the CLA content of milk from cows offered diets rich in linoleic or linolenic acid. MATERIALS AND METHODS Two experiments were conducted to determine the CLA content of milk from cows offered feed sources rich in linoleic acid and linolenic acid. The experimental details for each experiment are given below. Experiment 1 The main objective of this experiment was to study the influence of feeding soybean seeds, soybean oil, or linseed oil as a dietary source of linoleic and linolenic acid on CLA content of cow’s milk. Cows and treatments. Thirty-six primiparous Holstein dairy cows in midlactation were grouped into six blocks according to milk yield. Cows within each block were randomly assigned to six treatments. At the start of the experiment cows averaged 128 DIM (range 63 to 205 d) and were yielding 33.2 kg/d of milk (range 29.9 to 37.6 kg/d). Cows were weighed on two successive days at the beginning of the experiment after the morning milking. Average BW of the animals at the start of the experiment was 540 kg (SD = 35 kg). Animals were fed a basal control diet or diets containing either raw cracked soybeans (RAWSB), roasted cracked soybeans (RSB), soybean oil (SO), linseed oil at 2.2% (LO2), or linseed oil at 4.4% of dietary DM (LO4). To prepare cracked soybeans, soybeans were passed through a roller mill to provide a particle size of 25% or less than that of the whole soybean seed. Ingredient composition of the diets is given in Table 1. Diets contained 51% forage and 49% of a grain mixture, with alfalfa silage and corn silage fed as the forage sources in a 3:1 ratio (DM basis). Diets were balanced for minerals and vitamins and were formulated to meet the nutrient demand of cows according to the NRC (23) recommendations.

1017

Cows were housed in a tie-stall barn and were fed individually a TMR once daily. Orts were restricted to 5 to 10% of intake on an as-fed basis. The experimental period was 7 wk; the first 2 wk of the experiment served as a pretrial period and the next 5 wk as a trial period. During the pretrial period, cows in all groups received the basal control diet. Measurements were made during wk 2 (pretrial period), 4, 5, 6, and 7 of the experiment and are designated as sampling wk 1 through 5, respectively. Sampling, analyses, and calculations. Daily feed offered and orts for individual cows were recorded. Orts were mixed for each treatment and a representative sample was frozen daily. Samples of alfalfa silage and corn silage were frozen daily, and samples of individual TMR ingredients were taken once weekly. Weekly composite samples of forage were used for NDF, ADF, and CP analysis. The DM content of the feed ingredients was determined by oven-drying at 60°C for 48 h. Dietary formulations were adjusted weekly (if necessary) to account for small changes in ingredient DM content. Dried feed samples from each week during the trial period were ground through a Wiley mill (1-mm screen; Arthur H. Thomas, Philadelphia, PA) and were analyzed for NDF (30), ADF (7), and CP with a Leco Nanalyzer (Model FP-2000, Leco Corporation, St. Joseph, MI). During analysis, the samples were further dried at 105°C for 8 h to determine absolute DM. Chemical analyses were expressed on this final DM. The chemical composition of the TMR was calculated from the chemical composition of individual ingredients. Daily DMI for individual cows was calculated by subtracting the weekly mean of orts from the weekly mean of feed offered. The NEL and RUP contents of the diet were calculated by using NEL and RUP values for individual ingredients (23). Dietary ingredients were analyzed for fatty acid content and composition by the procedure of Sukhija and Palmquist (29). Chemical composition of the diets is given in Table 1. Mean DM content of the diets ranged between 56.1 and 57.1%. The NEL content of the diets containing oil seeds or free oil was high due to the higher energy value of the oil (Table 1). Average protein content of the RAWSB and RSB treatment diets was higher than in the other diets due to slightly higher than expected protein content of the soybeans. The calculated RUP content as a percentage of total protein in the RSB diet was higher than the RAWSB because the heat treatment of soybeans generally increases the RUP content (27). The NDF and ADF contents of the diets were within the recommended ranges (23) for lactating cows. Total fatty acid content was higher in the treatment diets containing oil seeds or free oil compared with the control (Table 2). As expected, the diet containing LO4 Journal of Dairy Science Vol. 83, No. 5, 2000

1018

DHIMAN ET AL. Table 1. Ingredient and chemical composition of the diets (experiment 1). Diet1 Control

RAWSB

RSB

SO

LO2

LO4

(%, DM basis) Ingredient Alfalfa silage Corn silage High moisture ear corn Soybean meal Raw cracked soybean Roasted cracked soybeans2 Soybean oil Linseed oil Dicalcium phosphate Limestone Mg oxide3 Trace-mineralized salt4 Chemical DM NEL, Mcal/kg DM5 CP RUP5 NDF ADF

34.0 17.0 31.9 15.5 ... ... ... ... 0.35 0.70 0.05 0.50

34.0 17.0 27.2 2.2 18.0 ... ... ... 0.35 0.70 0.05 0.50

34.0 17.0 27.2 2.2 ... 18.0 ... ... 0.35 0.70 0.05 0.50

34.0 17.0 27.5 16.3 ... ... 3.6 ... 0.35 0.70 0.05 0.50

34.0 17.0 29.2 16.0 ... ... ... 2.2 0.35 0.70 0.05 0.50

34.0 17.0 26.5 16.5 ... ... ... 4.4 0.35 0.70 0.05 0.50

56.1 1.64 17.9 5.6 29.2 21.0

56.5 1.69 18.6 5.1 28.8 20.8

57.1 1.69 19.0 7.1 28.8 20.8

56.7 1.78 17.9 5.6 28.5 20.6

56.5 1.73 17.9 5.6 28.7 20.8

56.9 1.81 17.9 5.6 28.3 20.6

1 Cows were fed a control diet, or diets containing either raw cracked soybeans (RAWSB), roasted cracked soybeans (RSB), soybean oil (SO), linseed oil at 2.2% (LO2), or linseed oil at 4.4% (LO4) of diet DM. The control diet was also used for feeding during the pretrial period. Vitamin supplement was added to provide additional at a rate of 146,785 IU of vitamin A/d per cow, 48,928 IU of vitamin D/d per cow, and 489 IU of vitamin E/d per cow. 2 Soybeans were roasted at 146°C and steeped for 30 min using a Jet-Pro Roaster (Jet Pro Co., Springfield, OH). 3 Contained minimum of 58.2% Mg. 4 Composition: 95 to 97% NaCl, 0.55% Zn, 0.55% Mn, 0.35% Fe, 0.14% Cu, 0.008% I, 0.006% Se, and 0.002% Co. 5 Calculated with NEL and RUP values of NRC (23). The RUP values used for high moisture ear corn and roasted soybeans were 45 and 50% of total CP, respectively.

had the highest fatty acid content (6.9% of dietary DM). Adding soybean seeds or oil increased the proportion of long-chain fatty acids (C18:0, C18:1, and C18:2). Linseed oil is a rich source of C18:3 fatty acid, and, therefore, the diets containing linseed oil (LO2 and LO4) had higher proportions of C18:3 compared with other diets. Daily milk weights were recorded. Duplicate milk samples (with bronopol-B2 preservative and without

preservative) were collected from two consecutive a.m. and p.m. milkings during wk 2, 4, 5, 6, and 7 of the experiment. Milk samples with preservative were analyzed for fat, protein, lactose, and SNF by the AgSource Cooperative Services (Milk Analysis Laboratory, Menomonie, WI) by infrared procedures with a Fossomatic605 utilizing a B filter (Foss Electric, Hillerød, Denmark). Final milk composition was expressed based on

Table 2. Fatty acid composition and total fatty acid content of diets (experiment 1). Fatty acid Treatment1

C14:0

C16:0

C16:1

C18:1

C18:2

C18:3

Total fatty acids2

Control RAWSB RSB SO LO2 LO4

0.5 0.2 0.2 0.2 0.3 0.2

21.1 14.9 15.1 16.1 14.7 12.2

% of reported fatty acids 0.7 1.8 18.4 0.3 3.2 21.4 0.3 3.2 21.7 0.3 3.2 21.1 0.4 2.7 17.7 0.3 3.0 17.5

47.0 50.6 50.7 50.1 32.0 26.2

10.5 9.4 8.8 9.0 32.2 40.6

% of DM 2.7 5.7 5.9 6.1 4.8 6.9

C18:0

1 Cows were fed a control diet, or diets containing either raw cracked soybeans (RAWSB), roasted cracked soybeans (RSB), soybean oil (SO), linseed oil at 2.2% (LO2), or linseed oil at 4.4% (LO4) of diet DM. 2 Sum of C14:0 to C18:3.

Journal of Dairy Science Vol. 83, No. 5, 2000


weighted a.m. and p.m. milk yields. Daily fat and protein yields were calculated by multiplying the milk yield with fat and protein concentration of the respective week on an individual cow basis. Weekly composite milk samples containing no preservative were analyzed for fatty acid composition, including CLA content. To determine fatty acid composition, milk fat was extracted by boiling in a detergent solution as described by Hurley et al. (15), weighed, capped under argon gas, and stored if needed at –20°C until further analysis. Fat samples were analyzed for fatty acids in a gas chromatograph as described by Dhiman et al. (6). Relative yield of CLA was calculated by multiplying the fat yield with CLA concentration of the respective week on an individual cow basis. Statistical analysis. The data for production variables and fatty acid composition from sampling wk 2 through 5 were analyzed as a randomized block design with repeated measures using the ANOVA procedures of Minitab (21). The model used was: Yijk = µ + Ti + Bj + TBij + Wk + BWjk + TWik + Eijk Where: Yijk = dependent variable for cow receiving treatment i in block j during week k. µ = population mean, Ti = treatment effect, Bj = block effect, TBij = treatment × block effect. This was used as an error term for the treatment effect. Wk = week effect, BWjk = block × week effect. This was used as an error term for the week effect. TWik = treatment by week effect. Eijk = residual error. This was used as an error term for the treatment × week interaction effect. The data were analyzed for treatment differences within each week when treatment × week interaction was significant. Least squares means were compared by using a protected least significant difference test. Significance was declared at P < 0.05 unless otherwise noted. Experiment 2 Experiment 2 was a follow up to experiment 1, and the objective was to determine the influence of feeding graded concentrations of soybean oil to dairy cows on the CLA content of milk.

1019

Cows and treatments. Thirty-six midlactation Holstein dairy cows were fed a control diet for 2 wk. At the end of the pretrial period, cows were grouped into six blocks according to milk yield at wk 2. Cows within each block were randomly assigned to six treatments. The experimental period was 5 wk. At the start of the experimental period mean milk production was 29.0 kg/d (range 25.4 to 33.6 kg/d). Cows were weighed 2 d in a row at the beginning of the experiment after the morning milking, and an average BW of the animals at the start of the experiment was 567 kg (SD = 46 kg). Cows were fed either a control diet, or control diet supplemented with soybean oil at 0.5% (SO1), 1.0% (SO2), 2.0% (SO3), 4.0% (SO4), or linseed oil at 1.0% (LO1) of dietary DM. Ingredient composition of the experimental diets is given in Table 3. The oil was added to the diet by replacing high moisture ear corn. Diets contained 55% forage and 45% grain mix, with alfalfa silage and corn silage, providing forage in a 2:1 ratio (DM basis). Soybean meal was used as a protein supplement. Mineral and vitamin additions were the same in all diets. The diets were balanced to meet the nutrient demand of cows according to NRC (23) recommendations. Cows were housed in a tie-stall barn and were fed individually a TMR once daily. The chemical composition of diets is given in Table 3. The DM content of the diets ranged from 49.9 to 50.5% during the experimental period. Diets containing oil had a higher content of NEL than did the control diet. The diets were isonitrogenous and had similar RUP contents, and the NDF and ADF contents of the diets were within normal recommended ranges for lactating cows (23). The fatty acid composition and total fatty acid content of diets are given in Table 4. As expected, the diets containing oil had higher concentrations of total fatty acids. Soybean oil is a rich source of long-chain fatty acids, and therefore increasing amounts of soybean oil in the SO1, SO2, SO3, and SO4 treatments increased the proportion of C16:0, C18:0, C18:1, and C18:3 compared with the control diet. Diets containing soybean oil had a larger proportion of C18:2 fatty acid, and diets containing linseed oil (LO1) had a larger proportion of C18:3. The SO4 treatments had the highest concentration of total fat in the diet (6.3% on a DM basis). Dietary fat in excess of 7% of the diet DM has been shown to inhibit microbial activity in the rumen (24), so total dietary fat in the SO4 treatment was close to the suggested safe limit for inclusion of fat in dairy diets. Sampling, analysis, and calculations. The procedures used for feeding, sampling and analyses were the same as described in experiment 1. Measurements were made during wk 2 (pretrial period), 4, 5, 6, and 7 of the experiment and were designated as sampling Journal of Dairy Science Vol. 83, No. 5, 2000

1020

DHIMAN ET AL. Table 3. Ingredient and chemical composition of the diet (experiment 2). Diet1 Composition

Control

SO1

SO2

SO3

SO4

LO1

(%, DM basis) Ingredient Alfalfa silage Corn silage High moisture ear corn Soybean meal Soybean oil Linseed oil Dicalcium phosphate Limestone Mg oxide2 Trace-mineralized salt3 Chemical DM NEL, Mcal/kg DM4 CP RUP4 NDF ADF

37.0 18.0 28.6 14.8 ... ... 0.40 0.70 0.05 0.50

37.0 18.0 28.0 14.9 0.5 ... 0.40 0.70 0.05 0.50

37.0 18.0 27.4 15.0 1.0 ... 0.40 0.70 0.05 0.50

37.0 18.0 26.1 15.3 2.0 ... 0.40 0.70 0.05 0.50

37.0 18.0 23.7 15.7 4.0 ... 0.40 0.70 0.05 0.50

37.0 18.0 27.4 15.0 ... 1.0 0.4 0.70 0.05 0.50

50.1 1.62 18.1 5.66 27.2 20.1

49.9 1.64 18.1 5.66 27.1 20.0

50.2 1.66 18.1 5.65 27.1 20.0

50.3 1.70 18.2 5.66 27.0 20.0

50.5 1.78 18.2 5.64 26.7 19.9

50.2 1.66 18.1 5.65 27.1 20.0

1 Cows were fed a control diet, or diets containing either soybean oil at 0.5% (SO1), 1.0% (SO2), 2.0% (SO3), 4.0% (SO4), or linseed oil at 1.0% (LO1) of diet DM. Vitamin supplement was added to provide additional at a rate of 146,785 IU of vitamin A/d per cow, 48,928 IU of vitamin D/d per cow, and 489 IU of vitamin E/d per cow. 2 Contained minimum 58.2% Mg. 3 Composition: 95 to 97% NaCl, 0.55% Zn, 0.55% Mn, 0.35% Fe, 0.14% Cu, 0.008% I, 0.006% Se, and 0.002% Co. 4 Calculated with NEL and RUP values of NRC (23).

wk 1 through 5, respectively. Data for production and fatty acid variables from sampling wk 2 through 5 were analyzed by using the statistical model identical to the one described in experiment 1. RESULTS AND DISCUSSION Experiment 1 There was no week × treatment interaction effect for intake and production variables except for milk protein content. Therefore, mean DMI, milk yield, and milk composition data from sampling wk 2 through 5

are given in Table 5. The DMI was not different among treatments; however, intakes were numerically lower in the SO and LO4 treatments compared with other treatments. These results suggest that soybean oil in free form at 3.6% or linseed oil at 4.4% of dietary DM had no negative influence on feed intake. However, Mohamed et al. (22) observed negative effects of feeding 4% oil in the diet on feed intake, and attributed it to a reduction in digestibility of DM by free oil. Cows in all treatments had similar milk yields, although 3.5% FCM yield was lower in the SO and LO4 treatments compared with the other treatments (Table

Table 4. Fatty acid composition and total fatty acid content of diets (experiment 2). Fatty acid Treatment

C14:0

C16:0

C16:1

C18:1

C18:2

C18;3

Total fatty acids2

Control SO1 SO2 SO3 SO4 LO1

0.7 0.6 0.5 0.4 0.3 0.5

17.8 16.7 15.9 14.7 13.6 14.3

% of reported fatty acids 0.5 2.8 18.4 0.4 3.0 19.0 0.4 3.2 19.4 0.3 3.4 20.1 0.2 3.6 20.7 0.4 3.2 19.0

47.8 49.0 49.8 50.9 52.0 38.5

12.0 11.3 10.8 10.2 9.6 24.1

% of DM 2.4 2.9 3.4 4.4 6.3 3.4

1

C18:0

1 Cows were fed a control diet or diets containing either soybean oil at 0.5% (SO1), 1.0% (SO2), 2.0% (SO3), 4.0% (SO4), or linseed oil at 1.0% (LO1) of diet DM. 2 Sum of C14:0 to C18:3.


1021

CONJUGATED LINOLEIC ACID IN MILK Table 5. Dry matter intake, milk yield, and milk composition of cows fed diets rich in linoleic and linolenic acid (experiment 1). Treatment1

P Value

Item

Control

RAWSB

RSB

SO

LO2

LO4

SEM

Trt

Trt × wk2

DMI, kg/d Milk yield, kg/d 3.5% FCM,3 kg/d Milk fat, % Milk fat yield, kg/d Milk protein yield, kg/d Milk lactose, % Milk SNF, % CLA yield,4 g/d

21.6 29.6 29.2a 3.41a 1.00 0.99 4.95 9.05 4.0d

22.0 29.8 29.9a 3.53a 1.05 0.99 5.01 9.07 3.8d

21.3 31.6 30.6a 3.34a 1.05 0.98 4.95 8.81 8.1c

20.2 29.0 25.9b 2.82b 0.83 0.95 4.87 8.91 16.9a

21.1 31.1 30.3a 3.35a 1.04 0.99 5.10 9.04 16.5a

20.0 30.3 25.2b 2.47b 0.74 0.99 5.10 9.04 12.5b

0.7 0.9 1.2 0.18 0.2 0.03 0.07 0.14 1.3

0.3 0.3 0.01 0.01 0.01 0.9 0.2 0.8 0.01

0.1 0.1 0.1 0.4 0.2 0.2 0.07 0.6 0.2

a,b,c,d Means with unlike superscripts within a row differ according to P value indicated. Mean value represents average from sampling wk 2 through 5. 1 Cows were fed a control diet, or diets containing either raw cracked soybeans (RAWSB), roasted cracked soybeans (RSB), soybean oil (SO), linseed oil at 2.2% (LO2), or linseed oil at 4.4% (LO4) of diet DM. 2 Treatment × week interaction effect. 3 3.5% FCM = 0.432 (kilograms of milk) + 16.2 (kilograms of fat). 4 Calculated by multiplying fat yield by the conjugated linoleic acid (CLA) concentration in milk fat for corresponding week. This was done on individual cow basis.

5). This reduction in FCM was due to depression in milk fat content. Milk fat depression commonly occurs when diets high in free oil are fed (1). The lower milk fat content could have been due to the increased dietary supply of C18:2 and C18:3 fatty acids, because increased dietary supply of C18:2 and C18:3 fatty acids has been shown to increase C18:1 fatty acid in milk through ruminal biohydrogenation (4). In another study, supplementation with soybean oil caused a marked increase in C18:1 fatty acid in the rumen fluid (22). A trans isomer of C18:1 fatty acid has been shown to decrease milk fat content (26). The cis and trans fatty acids in milk fat were not measured in the present study. The mechanism by which the trans isomer of C18:1 fatty acid reduces milk fat content is not known. Feeding additional fat through heat-treated soybeans in the RSB treatment did not affect milk yield. Cows used in this study were primiparous, and the magnitude of response in milk yield to fat supplementation is generally lower in primiparous cows than in mature cows (10). Cows fed fat as oilseeds in the RAWSB or RSB or as free oil in the LO2 treatments had similar milk fat content. The addition of polyunsaturated oils in free form tends to depress milk fat percentage (28), whereas supplementation of oil through seeds maintains or increases milk fat content (3, 22). The oil in seeds may have been released slowly during ruminal digestion, possibly reducing the accumulation and amount of C18:1 trans fatty acids leaving the rumen, thus reducing the potential for milk fat depression with the RAWSB and RSB treatments (1, 9, 22). Banks et al. (1) reported that offering soybean oil 24 times per day had less effect on milk fat percent-

age than if oil was offered twice daily. It appears that the manner in which oil is fed to the dairy cow determines its effect on milk fat content. Interestingly, linseed oil at 2.2% of dietary DM (LO2) did not depress milk fat content. However, linseed oil at 4.4% of dietary DM (LO4) resulted in severe milk fat depression. These results suggest that the hydrogenating ability of rumen microorganisms can be exceeded when large amounts of oil are presented to the rumen. Reduction in milk fat content in the SO and LO4 treatments resulted in decreased milk fat yield. The week × treatment interaction effect was significant for milk protein content, suggesting that the protein in milk varied during the sampling week among different treatments. Therefore, weekly mean milk protein contents from sampling wk 1 through 5 are shown in Figure 1. Analysis of milk protein data separately for wk 2, 3, 4, and 5 showed no significant differences among treatments within each week. Depression in milk protein content is often observed when dairy cows are fed fat (8). Milk protein content did not change because of fat feeding in this study. Dietary fat has been shown to adversely affect microbial fermentation and microbial protein yield in the rumen, thereby decreasing the supply of protein available for utilization by the cow (18). Diets fed in this experiment may have supplied adequate protein for the production level of the cows so that no negative effect on milk protein content appeared. Milk protein yield, lactose, and SNF content of milk were not different among treatments. Week × treatment interaction effect was significant for milk fatty acids, except for C18:0. Therefore, the Journal of Dairy Science Vol. 83, No. 5, 2000

1022

DHIMAN ET AL.

weekly means for milk fatty acids during sampling wk 1 through 5 are presented in Figure 2. Addition of oil through seeds or as a free oil reduced the proportions of medium-chain fatty acids (C12:0, C14:0, and C16:0) in milk fat compared with milk fat from the cows in control treatment. This decrease was proportional to the amount of oil supplied through the diet. Reduction in the proportion of C12:0, C14:0, and C16:0 fatty acids was greater in the RSB compared with RAWSB. The magnitude of decrease in C16:0 fatty acid was greater than in C12:0 and C14:0. The proportions of C16:0 decreased 29% in treatments compared with the control. In another study, feeding roasted soybeans decreased the proportion of C16:0 fatty acid by 27% (4). The proportions of C16:1 decreased in the RAWSB and RSB treatments compared with the control. An increased supply of dietary long-chain fatty acids has been shown to increase their secretion in milk fat and inhibit de novo synthesis of medium-chain fatty acids in the mammary gland (9), and as a result, milk is low in mediumchain fatty acids. The proportions of C18:0, C18:1, and C18:2 fatty acids increased when fat was supplemented through oil seeds or as a free oil (Figure 2). The average increases were 41, 43, and 65% for C18:0, C18:1, and C18:2 fatty acids, respectively. Increases in the proportion of C18:1 in milk fat can be obtained by feeding lipids rich in unsaturated 18-carbon fatty acids (4, 9). The increases in C18:2 were relatively high in RAWSB and RSB treatments compared with the other treatments. However, the increase in C18:2 fatty acid was the same for the RAWSB and RSB treatments. As mentioned earlier,

Figure 1. Milk protein content of cows offered diets rich in linoleic and linolenic acid. Sampling week 1 through 5 represents pretrial period and week 4 through 7 of the experiment, respectively. Cows were fed a control diet (—), or diets containing either raw cracked soybeans (䊏), roasted cracked soybeans (▲), soybean oil (✶), linseed oil at 2.2% (◆), or linseed oil at 4.4% (●) of diet DM. The least significant difference for treatment means was 0.34. Journal of Dairy Science Vol. 83, No. 5, 2000

oil is released slowly from oil seeds in the rumen and has relatively less probability of biohydrogenating than does free oil. Feeding oil through seeds makes more unsaturated oil available in the small intestine for absorption. Therefore, increased C18:2 in milk fat of cows fed the RAWSB and RSB treatments probably reflects its high transfer efficiency from the diet to the milk fat (4). The proportions of C18:3 in milk fat were increased with the RAWSB, RSB, and LO4 treatments compared with the control (Figure 2). The high content of C18:3 might be expected in milk fat of these cows based on the high content of C18:3 in the diet. Cows on the SO treatment did not show a change in the proportion of C18:3 in milk despite a similar dietary supply of C18:3 in the SO, RAWSB, and RSB treatments (207, 206, and 193 g /d of C18:3 in RAWSB, RSB, and SO treatments, respectively). This may be due to greater ruminal biohydrogenation of C18:3 when it was supplied in the form of free oil compared with oilseeds. The CLA content of milk was increased with the RSB, SO, LO2, and LO4 treatments compared with control (Figure 2). The average increase was 97, 438, 305, and 318% for the RSB, SO, LO2, and LO4 treatments, respectively. Feeding soybeans in raw cracked form (RAWSB) had no effect on CLA content of milk, possibly because of a relatively low release of oil from raw soybeans in the rumen compared with heattreated soybeans. Heat treatment makes soybeans brittle, and as a result, may increase the release of oil. Soybean oil at 3.6% of dietary DM in SO treatment significantly increased the CLA yield in milk compared with linseed oil at 4.4% of dietary DM in LO4 treatment. These results suggest that soybean oil is more efficient in increasing CLA in milk than linseed oil. In a study reported by Kelly et al. (19), the CLA concentration in milk fat increased 500% by feeding 5.3% oil, but at the same time milk fat concentration dropped from 3.38 to 2.25%. For potential economic and anticarcinogenic effects of CLA, the goal is to increase CLA concentration and the absolute amounts secreted in milk while maintaining normal milk composition. Feeding heat-treated cracked soybeans doubled (97% increase) the CLA content of milk without altering milk composition. Assuming the cost of heat treating soybeans at $20/tonne, the cost of feeding heat-treated soybeans to dairy cows would be $0.07/d per cow. The return will depend upon the value of CLA enriched milk. However, enhancing CLA content in milk through heat-treated soybeans appears to be the more cost-effective option. Results from this experiment suggest that feeding soybean oil or linseed oil to lactating dairy cows would increase the amount of CLA


1023

Figure 2. Milk fatty acid composition (% of total fatty acids) of cows offered diets rich in linolenic and linoleic acid (experiment 1). Sampling week 1 through 5 represents pretrial period and week 4 through 7 of the experiment, respectively. Cows were fed a control diet (—), or diets containing either raw cracked soybeans (䊏), roasted cracked soybeans (▲), soybean oil (✶), linseed oil at 2.2% (◆), or linseed oil at 4.4% (●) of diet DM. The least significant difference for treatment means were 0.62, 1.18, 2.94, 0.48, 2.45, 3.50, 0.79, 0.14, and 0.44 for C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, and CLA.


1024

DHIMAN ET AL. Table 6. Dry matter intake, milk yield, milk composition, and milk fatty acid composition of cows fed diets rich in linoleic and linolenic acid (experiment 2). Treatment1

P value

Item

Control

SO1

SO2

SO3

SO4

LO1

SEM

Trt

Trt × wk2

DMI, kg/d Milk yield, kg/d 3.5% FCM,3 kg/d Milk fat, % Milk fat yield, kg/d Milk protein, % Milk protein yield, kg/d Milk lactose, % Milk SNF, % CLA yield,4 g/d Milk fatty acid, % of total fatty acids reported C16:0 C18:0 C18:2 C18:3

20.6 27.4 27.0 3.44a 0.94 3.53 0.96 4.99 9.19 4.8d

21.7 27.9 28.3 3.60a 1.00 3.50 0.97 4.98 9.15 7.1c

20.6 28.3 28.5 3.56a 1.00 3.44 0.97 4.98 9.11 8.5c

19.7 28.3 25.0 2.80b 0.79 3.47 0.98 4.96 9.10 13.8b

21.1 28.5 26.1 2.93b 0.85 3.59 1.01 5.00 9.25 18.1a

21.7 28.4 29.2 3.72a 1.05 3.45 0.97 5.08 9.19 7.5c

0.7 1.0 1.5 0.2 0.1 0.1 0.03 0.08 0.14 1.1

0.3 1.0 0.3 0.01 0.1 0.9 0.8 0.9 0.9 0.01

0.4 0.2 0.4 0.12 0.4 0.1 0.06 0.2 0.06 0.2

40.8a 9.8b 2.8ab 0.63b

38.3ab 10.9ab 2.8ab 0.65b

36.5b 13.1a 2.8ab 0.62b

34.0bc 11.5ab 3.3a 0.61b

31.8c 13.0a 3.0ab 0.53b

37.5ab 13.0a 2.6b 0.82a

1.1 0.7 0.2 0.03

0.01 0.01 0.06 0.01

0.5 0.5 0.5 0.8

a,b,c,d Means with unlike superscripts within a row differ according to P value indicated. Mean value represents average from sampling wk 2 through 5. 1 Cows were fed a control diet, or diets containing either soybean oil at 0.5% (SO1), 1.0% (SO2), 2.0% (SO3), 4.0% (SO4), or linseed oil at 1.0% (LO1) of diet DM. 2 Treatment × week effect. 3 3.5% FCM = 0.432 (kilograms of milk) + 16.2 (kilograms of fat). 4 Calculated by multiplying fat yield by the conjugated linoleic acid (CLA) concentration in milk fat for the corresponding week. This was done on individual cow basis.

in milk fat, provided that the oil is accessible to the rumen microorganisms for biohydrogenation. Experiment 2 The treatment × week interaction effect was not significant for production variables, therefore the mean values from sampling wk 2 through 5 are given in Table 6. The DMI, milk yield, and FCM yield were the same in all treatments. Feeding fat has been reported to increase FCM yield by 1.0 to 1.5 kg/d (24). This failure to see an increase in milk yield from supplemental fat could be caused by the negative effects of free oil on the rumen microbial population (18) or because cows were in mid to late lactation. Cows in the SO3 and SO4 treatments had lower milk fat content compared with other treatments (Table 6). As discussed in experiment 1, the low milk fat content is characteristic of milk fat depression that commonly occurs when diets high in free oil are fed (1, 22). The amount of oil supplied with treatments SO1, SO2, and LO1 did not reduce milk fat content. Reduction in milk fat content of cows in the SO3 and SO4 treatments resulted in decreased milk fat yield (P < 0.1) compared with other treatments. Milk protein content and yield were similar among treatments (Table 6). It is possible that dietary oil addition may adversely affect AA available for milk Journal of Dairy Science Vol. 83, No. 5, 2000

protein synthesis in some situations (18, 22). However, diets in this experiment probably supplied sufficient protein to prevent any apparent negative effect on milk protein content. Milk lactose and SNF contents were not different among treatments. The average proportions of C16:0, C18:0, C18:2, and C18:3 in milk fat from sampling week 2 through 5 are given in Table 6. Five out of nine fatty acids had significant treatment × week interaction effects. Therefore, the data for all fatty acids is presented graphically in Figure 3. Figure 3 presents data from sampling week 1 through 5. The proportions of C12:0, C14:0, and C16:0 fatty acids decreased in milk fat of cows fed oil compared with the control treatment, and this decrease was proportional to the amount of oil in the diet. Cows on the SO4 treatment had 50, 28, and 22% decreases in the proportions of C12:0, C14:0, and C16:0 fatty acids, respectively. The proportion of C16:1 did not show any trend with the treatments. The average C16:1 content in milk fat was 2.3% of the total fatty acids. Others have reported similar values for C16:1 in milk fat (19). As discussed earlier, the dietary supply of long-chain fatty acids has been shown to decrease the proportions of medium-chain fatty acids and increase the long-chain fatty acids in milk fat (9). The proportions of C18:0 and C18:1 were higher in milk fat from cows fed oils (Table 6 and Figure 3). The C18:3 contents were highest in the


1025

Figure 3. Milk fatty acid composition (% of total fatty acids) of cows offered diets rich in linoleic and linolenic acids (experiment 2). Sampling week 1 through 5 represents pretrial period and week 4 through 7 of the experiment, respectively. Cows were fed a control diet (—), or diets containing either soybean oil at 0.5% (䊏), 1.0% (▲), 2.0% (✶), 4.0% (◆), or linseed oil at 1.0% (●) of diet DM. The least significant difference for treatment means were 0.73, 1.19, 3.61, 0.56, 2.68, 3.26, 0.56, 0.12, and 0.45 for C12:0, C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, and CLA.

1026

DHIMAN ET AL.

LO1 treatment, reflecting the large amount of C18:3 in linseed oil. The proportions of C18:3 were the same in milk fat of control cows and cows receiving soybean oil. The CLA content of milk fat was increased by 237 and 314% in cows fed soybean oil at 2 (SO3) or 4% (SO4) of diet DM compared with control, respectively (Figure 3). The CLA contents of milk in SO1, SO2, and LO1 treatments were not different than control. Similar milk yield, but a large increase in the CLA content, resulted in increased daily yield of CLA per cow in the SO3 and SO4 treatments (Table 6). Increasing soybean oil in the diet did not result in linear increase in CLA content of milk. The appearance of dietary long-chain fatty acids in the milk fat depends on the extent of their biohydrogenation in the rumen and consequently their availability in the small intestine for absorption. Unsaturated fatty acids in the rumen are generally converted to saturated fatty acids within a short time through ruminal biohydrogenation (14). However, the extent of lipolysis and biohydrogenation decreases with increasing amounts of substrate (18). Therefore, greater response at a higher concentration of soybean oil in this study may have been caused by incomplete biohydrogenation and increased escape of CLA from the rumen to the lower digestive tract. CONCLUSIONS The results from experiments 1 and 2 show that feeding lipid sources rich in C18:2 or C18:3 either as seeds or free oil will increase the CLA content of milk when oil is accessible to the rumen microorganisms for biohydrogenation. Enhancing CLA content of milk through feeding heat-treated soybeans seems to be an economical option. Another option to increase CLA content in milk is to feed soybean or linseed oil. Feeding free oil decreased milk fat content, but the large increase in CLA content of milk from cows fed oils resulted in increased daily yield of CLA per cow. Using current prices in North America, soybean oil is cheaper than linseed oil. Feeding soybean oil at 2% of the dietary DM resulted in a 237% increase in CLA content of milk compared with the control. ACKNOWLEDGMENT The authors acknowledge the assistance given by the farm crew at the US Dairy Forage Research Center farm at Prairie du Sac, WI. The authors thank Donald V. Sisson (Department of Mathematics and Statistics, Utah State University) for statistical analysis of the data. Journal of Dairy Science Vol. 83, No. 5, 2000

REFERENCES 1 Banks W., J. L. Clapperton, M. E. Kelly, A. G. Wilson, and R.J.M. Crawford. 1980. The yield, fatty acid composition and physical properties of milk fat obtained by feeding soya oil to dairy cows. J. Sci. Food Agric. 31:368–374. 2 Chin, S. F., W. Liu, J. M. Storkson, Y. L. Ha, and M. W. Pariza. 1992. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J. Food Compos. Anal. 5:185–197. 3 DePeters, E. J., S. J. Taylor, A. A. Franke, and A. Aguirre. 1985. Effects of feeding whole cottonseed on composition of milk. J. Dairy Sci. 68:897–902. 4 Dhiman, T. R., K. V. Zanten, and L. D. Satter. 1995. Effect of dietary fat source on fatty acid composition of cow’s milk. J. Sci. Food Agric. 69:101–107. 5 Dhiman, T. R., G. R. Anand, L. D. Satter, and M. Pariza. 1999. Conjugated linoleic acid content of milk from cows fed different diets. J. Dairy Sci. 82:2146–2156. 6 Dhiman, T. R., E. D. Helmink, D. J. McMahon, R. L. Fife, and M. W. Pariza. 1999. Conjugated linoleic acid content of milk and cheese from cows fed extruded oilseeds. J. Dairy Sci. 82:412–419. 7 Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS-USDA, Washington, DC. 8 Grummer, R. R. 1988. Influence of prilled fat and calcium salt of palm oil fatty acids on ruminal fermentation and nutrient digestibility. J. Dairy Sci. 71:117–123. 9 Grummer, R. R. 1991. Effect of feed on the composition of milk fat. J. Dairy Sci. 74:3244–3257. 10 Grummer, R. R. 1992. Feeding strategies for supplemental fat. Pages 248–259 in Large Dairy Herd Management. H. H. Van Horn and C. J. Wilcox, ed. Am. Dairy Sci. Assoc., Champaign, IL. 11 Ha, Y. L., N. K. Grimm, and M. W. Pariza. 1987. Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis 8:1881–1887. 12 Ha, Y. L., J. Storkson, and M. W. Pariza. 1990. Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res. 50:1097–1101. 13 Harfoot, C. G., and G. P. Hazlewood. 1988. Lipid metabolism in the rumen. Pages 285–322 in The Rumen Microbial Ecosystem. P. N. Hobson, ed. Elsevier Science Publishers Ltd., Essex, England. 14 Hawke, J. C., and W. R. Silcock. 1970. The in vitro rates of lipolysis and biohydrogenation in rumen contents. Biochim. Biophys. Acta. 218:201–212. 15 Hurley, W. L., G. J. Warner, and R. R. Grummer. 1987. Changes in triglyceride fatty acid composition of mammary secretions during involution. J. Dairy Sci. 70:2406–2410. 16 Ip, C., S. F. Chin, J. A. Scimeca, and M. W. Pariza. 1991. Mammary cancer prevention by conjugated dienoic derivative of linoleic acid. Cancer Res. 51:6118–6124. 17 Ip, C., M. Singh, H. J. Thompson, and J. A. Scimeca. 1994. Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Res. 54:1212–1215. 18 Jenkins, T. C. 1993. Lipid metabolism in the rumen. J. Dairy Sci. 76:3851–3863. 19 Kelly, M. L., J. R. Berry, D. A. Dwyer, J. M. Griinari, P. Y. Chouinard, M.E.V. Amburgh, and D. E. Bauman. 1998. Dietary fatty acid sources affect conjugated linoleic acid concentrations in milk from lactating dairy cows. J. Nutr. 128:881–885. 20 Kepler, C. R., and S. B. Tove. 1967. Biohydrogenation of unsaturated fatty acids. J. Biol. Chem. 242:5686–5692. 21 Minitab. 1992. Minitab Reference Manual. Release 9. Minitab Inc., State College, PA. 22 Mohamed, O. E., L. D. Satter, R. R. Grummer, and F. R. Ehle. 1988. Influence of dietary cottonseed and soybean on milk production and composition. J. Dairy Sci. 71:2677–2688.

CONJUGATED LINOLEIC ACID IN MILK 23 National Research Council. 1989. Nutrient Requirements of Dairy Cattle. 6th rev. ed. Natl. Acad. Sci., Washington, DC. 24 Palmquist, D. L. 1984. The use of fats in diets for lactating dairy cows. Pages 357–381 in Fats in Animal Nutrition. J. Wiseman, ed. Butterworths, Boston. 25 Parodi, P. W. 1997. Cow’s milk fat components as potential anticarcinogenic agents. J. Nutr. 127:1055–1060. 26 Romo, G. A., D. P. Casper, R. A. Erdman, and B. B. Teter. 1996. Abomasal infusion of cis or trans fatty acid isomers and energy metabolism of lactating dairy cows. J. Dairy Sci. 79:2005–2015. 27 Satter, L. D., T. R. Dhiman, and J. T. Hsu. 1994. Use of heat processed soybeans in dairy rations. Pages 19–28 in Proc. Cor-

1027

nell Nutr. Conf. Feed Manuf., Rochester, NY. Cornell Univ., Ithaca, NY. 28 Selner, D. R., and L. H. Schultz. 1980. Effects of feeding oleic acid or hydrogenated vegetable oils to lactating cows. J. Dairy Sci. 63:1235–1241. 29 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. 30 Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597.
