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Fatty Acid and Triglyceride Composition of Milk Fat from Lactating Holstein Cows in Response to Supplemental Canola Oil
J. Dairy Sci. 84:929–936 American Dairy Science Association, 2001.
Fatty Acid and Triglyceride Composition of Milk Fat from Lactating Holstein Cows in Response to Supplemental Canola Oil E. J. DePeters,* J. B. German,† S. J. Taylor,* S. T. Essex,* and H. Perez-Monti* Departments of Animal Science* and Food Science and Technology† University of California, Davis 95616-8521
ABSTRACT
INTRODUCTION
The objective was to determine the influence of dietary lipid on total and sn-2 fatty acid composition and triglyceride structure of milk fat in lactating Holstein cows. Five primiparous Holstein cows surgically fitted with ruminal and duodenal cannulas were used in a 4 × 5 incomplete Latin square. All cows received a basal diet. Treatments consisted of a basal diet with no supplemental canola oil (control), basal diet with canola oil added to the concentrate portion of the diet to provide 1.6% fat, basal diet with 330 g of canola oil infused directly into the rumen, and basal diet with 330 g of canola oil infused directly into the abomasum. Canola oil treatments decreased palmitic acid and increased oleic acid content of milk fat compared with the control. Stearate was higher when canola oil was rumen available compared with control and abomasal infusion. Abomasal infusion increased linoleic and linoleic acids in milk fat compared with the other treatments. The sn2 fatty acid composition reflected total fatty acid composition. All canola oil treatments reduced palmitic acid and increased oleic acid content at the sn-2 position. Changes in sn-2 composition reflect specificity of the acyl transferases and substrate concentration. Triglyceride composition reported as carbon number was altered by canola oil. Triglycerides in carbon number C50, C52, and C54 were increased while C32, C34, and C36 were decreased. (Key words: fatty acid, sn-2, triglyceride, milk fat)
Opportunities exist to modify the composition of milk fat from cattle (Baer, 1991; Banks, 1987; Banks et al., 1989) and to improve milk fat’s nutritional (Baer, 1991; Ney, 1991) and manufacturing (Baer, 1991; Banks et al., 1983; Banks et al., 1989) properties. Feed and animal factors influence the composition of milk fat (Christie, 1979; Grummer, 1991; Palmquist et al., 1993). Dietary lipids modify the composition of bovine milk fat. Supplementing the diet of cows with lipid decreased the C16:0 and medium chain fatty acids (C10:0, C12:0, and C14:0) and increased the C18:0 and C18:1 content of milk fat (Baer, 1991; DePeters et al., 1989; Palmquist et al., 1993). Even though the reduced environment associated with the microbial population in the rumen promotes the hydrogenation of unsaturated fatty acids consumed in the diet (Baldwin, 1984), 20% of more of the fatty acids in the milk fat of dairy cows are monounsaturated. The oleic acid in milk fat is, in part, a consequence of the stearoyl-coenzyme A (CoA) desaturase in the intestinal epithelium and mammary tissue (Banks, 1987). In contrast to fatty acids, the influence of diet on triglyceride structure has not been well studied. Differences in triglyceride structure were associated with pasture and barn feeding periods, but no information on diets was provided (Precht and Frede, 1996). Supplementing lactating cows with lipids altered triglyceride structure (Banks et al., 1989), which may have contributed to differences in melting point observed at 5°C. Improving the nutritional value and manufacturing properties of milk fat may be achieved by altering the diet of the cow. The relationship of the fatty acid composition of milk fat and human health, for example coronary heart disease, has received considerable attention (Ney, 1991). Feeding lipid protected from ruminal hydrogenation increased the unsaturated fatty acid composition of meat and milk lipids (Garrett et al., 1976), and consumption of the polyunsaturated meats and dairy products by young adults lowered plasma cholesterol in the high subgroup consisting of individuals with the highest initial plasma cholesterol levels (Hodges et
Abbreviation key: CoA = coenzyme A, OA = canola oil infused into the abomasum, OD = canola oil added to the diet, OR = canola oil infused into the rumen, NO = no supplemental oil.
Received May 25, 2000. Accepted November 10, 2000. Corresponding author: E. J. DePeters; e-mail: ejdepeters@ucdavis. edu.
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al., 1975). More recently, a protected lipid supplement high in oleic acid increased the unsaturated fatty acid content of milk fat (Noakes et al., 1996). Consumption of the dairy products higher in unsaturated fatty acids by humans lowered total and low-density lipoprotein cholesterol. The stereospecific distribution of fatty acids in lipids may contribute to human health (Kritchesvky, 1988; Small, 1991). Increasing the amount of palmitic acid in the sn-2 position of cottonseed oil by randomization significantly increased cottonseed oil’s atherogenic properties in rabbits (Kritchevsky et al., 1988). Redgrave et al. (1988) demonstrated that the fractional removal rate of triglycerides and cholesteryl esters from plasma of rats was affected by triglyceride structure. Similarly, fractional removal rates of triglycerides were lower for 1,3-dioleoly-2-stearoyl glycerol and 1,3-dioleolyl-2-palmitoyl glycerol compared with triolein, demonstrating that the stereochemistry of acyl glycerols affected lipid metabolism (Redgrave et al., 1988; Small, 1991). Feeding lactating cows linoleic acid protected from ruminal hydrogenation significantly increased the linoleic acid content at the sn-2 position of milk fat (Christie and Clapperton, 1982), indicating that it was possible to modify the stereospecific distribution of fatty acids in milk fat. Manufacturing properties of milk fat were also influenced by supplementing the diet of cows with lipid (Banks et al., 1980). Tong et al. (1995) supplemented lactating cows with either tallow or a palm oil-based product (calcium salts of long chain fatty acids). Tallow increased oleic acid and decreased palmitic acid content of milk fat, and tallow supplementation created a softer butter. The softer butter was associated with a decreased concentration of saturated triglycerides in the carbon chain lengths between 42 and 50 (German et al., 1995). Triglyceride composition of milk fat has received less attention than total fatty acid composition. However, Banks et al. (1989) noted that fatty acid composition of milk fat only could not account for differences observed in manufacturing properties of milk fat from two breeds. Few researchers have evaluated changes in both fatty acid composition and triglyceride structure of milk fat from lactating cows in response to dietary manipulation. The objective of this study was to determine the influence of dietary lipid on total and sn-2 fatty acid composition and triglyceride structure of milk fat in lactating Holstein cows. METHODS Five primiparous Holstein cows surgically fitted with ruminal and duodenal cannulas were used in a 4 × 5 Journal of Dairy Science Vol. 84, No. 4, 2001
Table 1. Ingredient and chemical composition of the basal diet. Item
Basal diet
Ingredient (%) Alfalfa hay, chopped Barley, steam-rolled Beet pulp, shredded Corn, dry-rolled Molasses, cane Sodium bicarbonate Monosodium phosphate Trace mineral salt Soybean meal Chromic oxide
46.6 25.2 13.9 6.0 5.9 0.7 0.7 0.5 0.4 0.1
Chemical (% DM) CP Ash ADF Total fatty acids
17.4 9.5 16.7 2.0
incomplete Latin square. Ruminal cannulas were 10.2 cm i.d. (Bar Diamond, Parma, ID). At the start of the study cows averaged 172 DIM (SD 18 d). All cows received the basal diet, which was fed as a TMR (Table 1). Treatments consisted of 1) basal diet with no supplemental oil (NO), 2) basal diet with canola oil added to the concentrate portion of the diet to provide 1.6% fat (OD), 3) basal diet with approximately 330 g of canola oil infused directly into the rumen (OR), and 4) basal diet with approximately 330 g of canola oil infused directly into the abomasum (OA). Canola oil was high in oleic acid content and contained 5.57 (C16:0), 2.41 (C18:0), 43.03 (C18:1 cis 9+10), 26.47 (C18:2), and 7.99 (C18:3) g/100 g of fat. For OR and OA treatments, cows were fitted with a harness containing two small pouches, one on each side of the cow. One pouch carried a polypropylene bottle with a weighed amount of canola oil. The other pouch contained a battery-powered peristaltic universal micro-metering pump (Everest Electronics, Seaford, South Australia) to allow continuous infusion of canola oil for OR and OA. Tygon tubing (1/ 8 cm i.d., 1/4 cm o.d., 1/16 cm wall; Tygon R-1000 labultraflex, Fisher Scientific, Santa Clara, CA) from the infusion pump was passed through the ruminal cannula into the rumen for OR. Treatments OD and OR represented supplemental oil which was potentially exposed to ruminal biohydrogenation. For OA, tubing from the infusion pump was first passed through the ruminal cannula and then through the reticulo-omasal orifice with final placement in the abomasum (Clark et al., 1977). Treatment OA prevented ruminal biohydrogenation of the unsaturated fatty acids in canola oil. Each treatment period was 14 d long. Cows were housed in an open corral with access to an exercise area and bedded free stalls. Cows were trained to use Calan electronic feeding gates (American Calan, Inc., Northwood, NH), which allowed individual feeding of each
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animal. Intake of the basal diet fed in all treatments was measured. Cows were milked twice daily, and milk yield was measured. Milk samples were collected from consecutive evening and morning milkings the last week of each period and preserved with 2-bromo-2-nitro-propane-1-3-diol (Dairy & Food Labs, Inc., Modesto, CA). Diet samples were processed and analyzed similar to methods reported by Crocker et al. (1998) for DM, CP, ash, acid detergent fiber, and total fatty acids. Fatty acid composition of milk fat was determined by gas chromatography of methyl esters. Fat was extracted according to Erickson and Dunkley (1964) and prepared according to Crocker et al. (1998). Using glass graduated pipets, 10 mg of the extracted fat was aliquoted into a Teflon-lined, screw-cap glass culture tube. The solvent was evaporated under a stream of nitrogen at 60°C followed by a 1.0 ml wash of iso-octane (2,2,4 trimethylpentane), which was also evaporated under nitrogen at 60°C. A 2.0-ml aliquot of iso-octane was added to the culture tube with vortexing. Methyl esters were formed by the addition of 100 µl of 3 M potassium hydroxide, vortexing, and allowed to stand at room temperature for 10 min. Esters were washed and separated by the addition of 2.0 ml of saturated NaCl and 8.5 ml of deionized water. Tubes were shaken with a Burell wrist action shaker for 10 min, centrifuged at 1750 rpm for 10 min to separate the methyl esters in the upper solvent layer, and then aliquoted into 1.8-ml gas chromatography autosampler vials. The ester mixture was analyzed with a Supelco 2560, 100-m capillary column with a 0.25 mm i.d. and a 0.20-micron film thickness. Hydrogen was used as the carrier gas with a linear flow rate set at 27 cm/s with a column head pressure of 33 psi. A Hewlett Packard 5890 gas chromatograph equipped with a 7673A auto-injector injected 1.0 µL of sample that was split 125 times. Injector and detector temperatures were set at 220°C, and the column temperature was programmed at 75°C for 10 min and then increased to 175°C for 38 min at 20°C/min. A flame-ionization detector with a signal range of one was used. Esters were integrated and quantified with version 6.07 of Chrome Perfect (Justice Innovations, Inc., Palo Alto, CA) software interfaced with a Hewlett Packard 3393A integrator. The sn-2 fatty acids in milk fat were separated by thin-layer chromatography followed by methylation. Milk fat was extracted according to Erickson and Dunkley (1964). Using glass graduated pipets, 100-mg aliquot of lipid extract was placed into a Teflon-lined, screw-cap glass culture tube. Solvent was evaporated under a stream of nitrogen at 60°C. The 2-monoacylglycerols were formed and separated by the method described by Hendriske and Harwood (1986), with the following modifications. An enzyme reaction time of 1
h was used, and Whatman, LK6F Linear-K, silica gel, 5 × 20 cm, 250-micron chromatography plates were used. The monoacylglycerol band was scrapped and transferred to a 12 mm × 100 mm screw-top, Teflon-lined culture tube. Two hundred microliters of methanol:hexane (4:1), containing 0.05 mg of nonadecanoic acid (C19:0), was added to each tube and vortexed. Methylation was achieved by adding 5.0 µl of acetyl chloride slowly down the side of the culture tube to prevent superheating and subsequent popping. Tubes were sealed under nitrogen and incubated for 1 h at 100°C. After cooling at room temperature, 500 µL of 6% K2CO3 was added, followed by vortexing and the addition of 200 µL of hexane. Tubes were centrifuged for 5 min at 1750 rpm, and the upper hexane layer was removed and placed in a 1.8-ml gas chromatograph auto sampler vial. The solvent was evaporated under nitrogen and brought up to a final volume of 75 µL with hexane. Esters were determined and quantified as described earlier. Triglyceride profiles of milk fat were obtained by high temperature polar capillary gas chromatography (Varian 3400, Walnut Creek, CA). A 30-m DB-HT17 (J& W, Sacramento, CA) capillary column with a relatively polar stationary phase was used to separate intact triglycerides on the basis of total chain length and unsaturation. A solvent programmable injector (Varian 8100, Walnut Creek, CA) was used to introduce the triglyceride samples dissolved in octane onto the column using a 10-cm, 0.5-mm i.d. fused silica tube as a retention gap. The sample injection volume was 0.4 µl with a solvent plug size of 0.5 µl. The initial injector temperature was set to 130°C and was programmed to rise immediately on injection to 355°C at 115°/min. The column temperature was programmed from 130 to 355°C at 20°/min and held constant at 355°C for an additional 20 min. The detector temperature was set at 355°C. Standard monoacid and mixed chain triglycerides (Nu chek, Elysian, MN), as well as hydrogenated milk fat samples were used to determine the retention times of specific triglycerides. The location of specific fatty acids on the triglyceride was not resolved using these analyses. However, certain specific molecular species were inferred from the known positional specificity of bovine milk fat triglycerides. Data were analyzed by the general line models procedure of SAS (1998) for a 4 × 5 incomplete Latin square design using the following model described by Hicks (1993): Yijk = µ + Ti + Pj + Ck + Eijk where, Journal of Dairy Science Vol. 84, No. 4, 2001
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DEPETERS ET AL.
Yijk µ Ti Pj Ck Eijk
= = = = = =
observation; mean; treatment, i = 1,2,3,4; period, j = 1,2,3,4; cow, k = 1,2,3,4,5; and residual error.
OA was higher than NO but lower than either OD or OR. Since OA prevented ruminal biohydrogenation of fatty acids, the slightly higher C18:0 in milk fat may be a consequence of the C18:0 contained in the infused canola oil. In the small intestine the additional C18:0 from OA would be available for absorption and milk fat synthesis. All canola oil treatments increased oleic acid (C18:1 cis 9+10) content compared to NO. The higher oleic acid content of OD and OR may be a consequence of mammary stearoyl-CoA desaturase which desaturates C18:0 to C18:1. The higher C18:1 trans content of milk fat for OD and OR, in particular the trans 11 isomer, indicated that the canola oil was subjected to ruminal biohydrogenation. This trans isomer of oleic acid has been associated with ruminal biohydrogenation (Harfoot, 1978). The C18:1 trans content of OA was similar to NO; an indication that ruminal biohydrogenation of fatty acids was prevented by the infusion method. Milk fat composition of C18:2 and C18:3 was higher for only OA. Abomasal infusion prevented ruminal biohydrogenation of unsaturated fatty acids and provided unsaturated fatty acids postruminally for milk fat synthesis. In contrast, OD and OR did not prevent hydrogenation of polyunsaturated fatty acids. The fatty acid composition at the sn-2 position of milk fat was influenced by treatment (Table 4). All canola oil treatments reduced the proportion of C16:0 and increased the proportion of C18:0 and C18:1 cis 9+10 esterified at the sn-2 position compared to NO. The sn-2 content of C18:2 and C18:3 was increased dramatically for OA compared with the other three treatments. Changes in the fatty acid esterified at the sn-2 position are similar to changes in total fatty acid composition of milk fat. Fatty acid distribution in triglycerides is non random (Blank and Privett, 1964). The ratio of a fatty acid in the sn-2 position to the proportion of the same fatty acid in total fatty acids (all positions) was calculated. Only those fatty acids present in the sn-2 position were considered for the total fatty acids and the distribution recalculated. The ratio for C14:0 and C16:0 for all treatments was greater than 1, suggesting
Means were separated by Duncan’s multiple range test with significance declared at P ≤ 0.05. The ratio of a fatty acid in the sn-2 position to the proportion of the same fatty acid in the total fatty acids for all positions was calculated and compared using Student’s t-test with the assumption of unequal variances. A one-sample Student’s t-test was calculated to compare if the ratio of the sn-2 FA to the total FA was equal to one (SAS, 1998). RESULTS AND DISCUSSION Intake of DM was higher for OD and OR compared with NO and OA (Table 2). Yield of milk was affected by treatment and was highest for OD and OR and lowest for NO and OA. The additional intake of energy provided by canola oil may have supported higher milk yield. Infusion of canola oil increases the amount of long chain fatty acids available to the mammary gland for milk fat synthesis and reduces de novo synthesis of fatty acids from acetate in the mammary gland. The latter may spare energy for milk synthesis. Milk fat percentage was highest for OA and lowest for OR. Yields of milk fat were similar across treatments. Total fatty acid composition of milk fat was influenced by treatment (Table 3). Abomasal infusion of canola oil decreased C14:0 content of milk fat compared with the other treatments. Content of C16:0 was lowest for OA, highest for NO, and intermediate for OD and OR. A similar response occurred for C16:1 cis. Stearate was highest for OD and OR and lowest for NO. The higher C18:0 for OD and OR may be a consequence or ruminal biohydrogenation of unsaturated fatty acids contained in the canola oil. The stearate content of milk fat for
Table 2. Production performance of cows. Treatment1 Item DMI, kg/d Milk, kg/d 4% FCM, kg/d Fat, % Fat, kg/d
NO
OD a
18.0 25.0a 23.6 3.69ab 0.92
OR b
19.6 28.6b 26.4 3.50bc 0.99
OA b
19.5 29.0b 26.2 3.40c 0.97
SE a
17.7 26.6a 25.8 3.83a 1.01
0.36 0.56 0.73 0.07 0.04
Means in the same row with different superscripts differ P < 0.05. NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b 1
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FATTY ACID AND TRIGLYCERIDE OF MILK FAT Table 3. Total fatty acid composition of milk fat (g/100 g of fat). Treatment1 Fatty acid
NO
OD
OR
OA
SE
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C16:1 C16:1 C18:0 C18:1 C18:1 C18:1 C18:2 C18:3
3.62 2.68 1.66 4.39 5.58 14.77a 37.47a 0.39 2.40a 6.17a 0.13a 0.76a 15.10a 3.61a 1.29a
3.84 2.65 1.61 4.01 4.76 14.32a 30.07b 0.39 1.82b 9.57b 0.26b 1.02ab 20.97b 3.29a 1.43a
3.72 2.68 1.66 4.15 5.01 14.33a 30.13b 0.39 2.00b 9.04b 0.32c 1.37b 20.40b 3.41a 1.42a
3.39 2.51 1.57 3.93 4.59 12.34b 27.22c 0.32 1.47c 6.89c 0.14a 0.61a 21.70b 9.54b 3.79b
0.14 0.05 0.04 0.16 0.23 0.28 0.78 0.02 0.08 0.39 0.03 0.13 0.69 0.49 0.19
trans cis trans 9 trans 11 cis 9+10
Means in the same row with different superscripts differ (P < 0.05). NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b,c 1
a preference for these fatty acids at the sn-2 position compared with C10:0 and C12:0 with ratios less than 1. Marshall and Knudsen (1977) reported that chain length specificity of the 1-acylglycerolphosphate acyltransferase was C16 > C14 > C12 > C10 > C8, which agreed with the fatty acid esterified at the sn-2 position observed in the present study. An acyl specificity of C16:0 ∼ C14:0 ∼ C12:0 ∼ C18:2 > C18:3 ∼ C18:0 ∼ C20:4 > C18:1 it was observed for the human lysophosphatidic acid acyltransferase (hLPAATα), and acyl specificity of hLPAATα was affected by acyl concentration (Aguado and Campbell, 1998). The ratio of the 18 C unsaturated fatty acids were found at a lower proportion at the sn2 position. Reske et al. (1997) suggested that in plant oils, triglyceride synthesis was driven by mass action; the amount of substrate available was likely more im-
portant than the specificity of the acylytransferases. Recently, Mistry (1999) compared the fatty acid composition of bovine and ovine milk fat and proposed that substrate availability in the ruminant mammary gland may be the determining factor for fatty acid esterification at the sn-2 position. The proportion of C18:2 and C18:3 at the sn-2 position increased for abomasal compared with ruminal infusion of canola oil while the ratio did not change supports the suggestion that substrate availability along with acyltransferase specificity is important in determining the fatty acid at the sn-2 position, which was proposed for plant lipids (Lassner et al., 1995; Reske et al., 1997). Treatments affected the triglyceride structure of milk fat (Table 5). Canola oil additions decreased triglycerides in the C32, C34, C36, C42, and C44 groups and
Table 4. Fatty acid composition at the sn-2 position of milk fat (g/100 g of sn-2 fat). Treatment1 Fatty acid
NO
OD
OR
OA
SE
C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:1 C18:1 C18:2 C18:3
0.32 3.81a 24.08 54.64a 1.80a 4.28a 0.18a 0.32a 7.91a 2.27a 0.39a
0.05 2.34b 23.58 48.32b 1.14b 9.51b 0.39bc 0.58ab 11.66b 2.22a 0.30a
0.12 2.83ab 23.26 47.01b 1.43b 9.12b 0.54c 0.81b 12.18bc 2.33a 0.44a
0.10 3.32ab 19.90 45.25b 1.06b 7.93b 0.29ab 0.35a 14.05c 6.44b 1.32b
0.11 0.42 1.34 1.18 0.11 0.78 0.05 0.09 0.65 0.48 0.08
cis trans 9 trans 11 cis 9+10
Means in the same row with different superscripts differ P < 0.05. NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b,c 1
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DEPETERS ET AL. Table 5. Triglyceride structure of milk fat (g/100 g of fat). Treatment1 Carbon no.
NO
OD
OR
OA
SE
C26 C28 C30 C32 C34 C36 C38 C40 C42 C44 C46 C48 C50 C52 C54 Total
1.55 1.95 2.71 5.12a 10.24a 15.54a 14.43 10.52 8.76a 8.29a 7.75 5.64 3.68a 1.12a 0.12a 97.44
1.55 1.88 2.69 4.76b 9.46b 14.51ab 14.92 11.29 7.97b 7.47b 7.43 6.03 4.62b 1.81b 0.34b 96.71
1.55 1.91 2.73 4.80b 9.39b 14.21b 14.91 11.34 8.10b 7.03b 7.65 6.06 4.69b 1.85b 0.33b 96.56
1.57 2.01 3.32 4.63b 8.71c 13.83b 15.08 11.51 7.85b 7.32b 7.55 5.72 5.05b 2.17b 0.41b 96.73
0.05 0.09 0.37 0.10 0.18 0.34 0.20 0.25 0.18 0.24 0.24 0.18 0.21 0.13 0.05 0.41
Means in the same row with different superscripts differ P < 0.05. NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b,c 1
increased triglycerides in C40, C50, and C52 groups. Canola oil treatments provided 18 C fatty acids, which were available for milk fat synthesis. Long chain fatty acids were reported to reduce de novo fatty acid synthesis in the mammary gland (Grummer, 1991; Palmquist et al., 1993). There were trends for lower C10:0 and C12:0 and significantly lower C16:0 with all canola oil treatments compared with NO. The higher proportion of long chain fatty acids and the lower proportion of medium chain fatty acids associated with canola oil treatments would explain a portion of the changes in carbon number for triglyceride structure. Banks et al. (1989) and Precht (1991) noted that triglyceride composition of milk fat was influenced by diet. The distributions between C52 and C54 in milk fat were for example, affected by added dietary fat and, to a greater extent, with severe underfeeding (Precht, 1991). Supplementing the diet of lactating cows with oils decreased the proportions of triglycerides from 30 to 36 and 42 to 46 and increased triglycerides from 50 to 54 (Banks et al., 1989). Triglycerides did not differ with type of oil fed even though the fatty acid composition of the milk was influenced by the type of dietary oil (Banks et al., 1989). No changes in the proportion of triglycerides associated with C26, C28, C30, C38, C46, and C48 occurred (Table 5). Although total carbon number was not different, the individual peaks of distribution of acyl-C groups were different. For example, the total area for C38 was not affected by treatment, but canola oil treatments decreased acyl-C groups associated with peaks 1, 2, 3, and 4. In contrast, peaks 4 and 6 of C38 were increased with canola oil treatments. Site of canola oil Journal of Dairy Science Vol. 84, No. 4, 2001
addition also influenced acyl-C groups. Treatments OD and OR increased peak 5 of C38 compared with NO: OA did not influence this peak. Infusing canola oil into the abomasum increased acyl-C groups associated with peaks 7, 8, and 10. The exact changes occurring in triglyceride structure can not be described by the techniques used in the present study. However, the data do demonstrate that although the total proportion of triglycerides within a carbon number do not change, the distribution of the acyl-C groups may change. Precht and Frede (1996) evaluated the correlation coefficients between triglycerides and solid fat contents at different temperatures between 0 and 30°C. For C40, C50, C52, and C54 triglycerides, the correlation coefficient was negative. These researchers attributed this change in sign from positive to negative to be a result of different contents of C16 and C18:1 fatty acids comprising the different acyl-C-groups with the same carbon number. Their observations support our findings that triglyceride content did not change for some carbon numbers, but acyl-C groups were different. Similarly, Banks et al. (1989) observed that type of oil fed influenced fatty acid composition but not triglyceride composition of milk fat. However, differences in butter time were observed, indicating that the manufacturing properties of the milk fats were altered. Changes in specific acyl-C groups within triglycerides were not measured and may account for differences in butter time, although little research has been done in this area. In the present study, triglyceride structure was affected by diet of the cow. Recently, Ruiz-Sala et al. (1996) reported that milk fat from cows was higher in long chain and unsaturated
FATTY ACID AND TRIGLYCERIDE OF MILK FAT
triglycerides compared with the milk fat from ewes. However, information on the diets fed to the sheep, goats, and cattle was not provided. Since diet affects triglyceride structure, species comparison of fatty acid and triglyceride composition of milk fat may be confounded by diet. CONCLUSIONS Supplementing the diet of lactating dairy cows with canola oil modified the total and the sn-2 fatty acid composition and triglyceride structure of milk fat. Canola oil when either added to the diet or infused into the rumen increased the C18:0 and C18:1 and decreased the C16:0 contents of milk fat. Infusing canola oil into the abomasum to prevent ruminal biohydrogenation of unsaturated fatty acids increased the C18:1, C18:2, and C18:3 and decreased the C14:0 and C16:0 contents of milk fat compared with NO. Changes in the fatty acid esterified at the sn-2 position of milk fat in response to canola oil supplementation of the diet reflect changes in total fatty acid composition. Triglyceride composition of milk fat was affected by diet. Triglycerides in the C32, C34, C36, C42, and C44 groups decreased and C40, C50, and C52 groups increased with canola oil supplementation. Although some triglycerides were not affected by treatment, within a specific triglyceride, acyl-C groups were different. The study of triglyceride composition of milk fat from cattle, and possibly other ruminants, must consider dietary differences. The impact of changes in fatty acid and triglyceride compositions on the nutritional and manufacturing studies of milk fat are not known and require further study. ACKNOWLEDGMENTS The authors thank Susannah Taylor, Lisa Crocker, and Megan Osborne for their technical assistance, B. Kreuscher, F. Stewart, and W. Paroczai for care of the cows, and L. George, School of Veterinary Medicine, for the surgical preparation of the animals. Scott Essex expresses appreciation to the American Registry of Professional Animal Scientists, California Chapter, for their financial support through the Dr. Antionio A. Jimenez Memorial Award. Financial support was provided by the California Dairy Research Foundation (Davis), the California Agricultural Experiment Station (Davis), and NuAg Systems (Middletown, CA). The research is a contribution of Regional Research Project W-181. REFERENCES Aguado, B., and R. D. Campbell. 1998. Characterization of a human lysophosphatidic acid acyltransferease that is encoded by a gene
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located in the class III region of the human major histocompatibility complex. J. Biol. Chem. 273:4096–4105. Baer, R. J. 1991. Alteration of the fatty acid content of milk fat. J. Food Prot. 54:383–386. Baldwin, R. L. 1984. Digestion and metabolism of ruminants. BioScience 34:244–249. Banks, W. 1987. Opportunities for varying the composition of cows’ milk. J. Soc. Dairy Technol. 40:96–99. 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. Banks, W., J. L. Clapperton, D. D. Muir, and A. K. Girdler. 1989. Whipping properties of cream in relation to milk composition. J. Dairy Res. 56:97–105. Banks, W., J. L. Clapperton, and W. Steele. 1983. Dietary manipulation of the content and fatty acid composition of milk fat. Proc. Nutr. Soc. 42:399–406. Blank, M. L., and O. S. Privett. 1964. Structure of milk fat triglycerides. J. Dairy Sci. 47:481–488. Christie, W. W. 1979. The effects of diet and other factors on the lipid composition of ruminant tissues and milk. Prog. Lipid Res. 17:245–277. Christie, W. W., and J. L. Clapperton. 1982. Structures of the triglycerides of cows’ milk, fortified milks (including infant formulae), and human milk. J. Soc. Dairy Technol. 35:22–24. Clark, J. H., H. R. Spires, R. G. Derrig, and M. B. Bennink. 1977. Milk production, nitrogen utilization and glucose synthesis in lactating cows infused postruminally with caseinate and glucose. J. Nutr. 107:631–644. Crocker, L. M., E. J. DePeters, J. G. Fadel, H. Perez-Monti, S. J. Taylor, J. A. Wyckoff, and R. A. Zinn. 1998. Influence of processed corn grain in diets of dairy cows on digestion of nutrients and milk composition. J. Dairy Sci. 81:2394–2407. DePeters, E. J., S. J. Taylor, and R. L. Baldwin. 1989. Effect of dietary fat in isocaloric rations on the nitrogen content of milk from Holstein cows. J. Dairy Sci. 72:2949–2957. Erickson, D. R., and W. L. Dunkley. 1964. Spectrophotometric determination of tocopherol in milk and milk lipides. Anal. Chem. 36:1055–1058. Garrett, W. N., Y. T. Yang, W. L. Dunkley, and L. M. Smith. 1976. Increasing the polyunsaturated fat content of beef and lamb. J. Anim. Sci. 42:845–853. German, J. B., C. Simoneau, E. DePeters, P. S. Tong, J. Mattas, and J. Sullivan. 1995. Effects of feeding supplemental fat to dairy cattle on milk fat composition and properties. Part 2. Triglyceride distribution and milk fat functionality. J. Dairy Sci. (Suppl. 1)78:134. (Abstr.) Grummer, R. R. 1991. Effect of feed on the composition of milk fat. J. Dairy Sci. 74:3244–3257. Harfoot, C. G. 1978. Lipid metabolism in the rumen. Prog. Lipid Res. 17:21–54. Hendrikse, P. W., and J. L. Harwood. 1986. Analytical methods. Pages 249–286 in The Lipid Handbook. F. D. Gunstone, J. L. Harwood, and F. B. Padley, eds. Chapman and Hall, New York, NY. Hicks, C. R. 1993. Fundamental Concepts in the Design of Experiments. 4th ed. Saunders College Publishing, San Francisco, CA. Hodges, R. E., A. F. Salel, W. L. Dunkley, R. Zelis, P. F. McDonagh, C. Clifford, R. K. Hobbs, L. M. Smith, A. Fan, D. T. Mason, and C. Lykke. 1975. Plasma lipid changes in young adult couples consuming polyunsaturated meats and dairy products. Am. J. Clin. Nutr. 28:1126–1140. Kritchevsky, D. 1988. Effects of triglyceride structure on lipid metabolism. Nutr. Rev. 46:177–181. Kritchevsky, D., S. A. Tepper, S. Wright, A. Kuksis, and T. A. Hughes. 1998. Cholesterol vehicle in experimental atherosclerosis. 20. Cottonseed oil and randomized cottonseed oil. Nutr. Res. 18:259–264. Lassner, M. W., C. K. Levering, H. M. Davies, and D. S. Knutzon. 1995. Lysophosphatidic acid acyltransferase from Meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol. 109:1389–1394. Journal of Dairy Science Vol. 84, No. 4, 2001
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Marshall, M. O., and J. Knudsen. 1977. The specificity of 1-acylsn-glycerol 3-phosphate acyltransferase in microsomal fractions from lactating cow mammary gland towards short, medium and long chain acyl-CoA esters. Biochim. Biophys. Acta 489:236–241. McCrae, I., F. Harding, and D. D. Muir. 1987. Milk compositional quality—opportunities for manipulation. J. Soc. Dairy Technol. 40:57–58. Mistry, D. H. 1999. Cloning of the bovine and the sheep lysophosphatidic acid acyltransferase (LPAAT). M.S. Thesis, Univ. CA, Davis 95161. Ney, D. M. 1991. Potential for enhancing the nutritional properties of milk fat. J. Dairy Sci. 74:4002–4012. Noakes, M., P. J. Nestel, and P. M. Clifton. 1996. Modifying the fatty acid profile of dairy products through feedlot technology lowers plasma cholesterol of humans consuming the products. Am. J. Clin. Nutr. 63:42–46. Palmquist, D. L., A. D. Beaulieu, and D. M. Barbano. 1993. Feed and animal factors influencing milk fat composition. J. Dairy Sci. 76:1753–1771. Precht, D. 1991. Detection of adulterated milk fat by fatty acid and triglyceride analysis. Fat Sci. Technol. 93:538–544.
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Precht, D., and E. Frede. 1996. Determination of the solid fat content in milk fat by gas chromatographic triglyceride analysis. Fat Sci. Technol. 9:324–330. Redgrave, T. G., D. R. Kodali, and D. M. Small. 1988. The effect of triacyl-sn-glycerol structure on the metabolism of chylomicrons and triacylglycerol-rich emulsions in the rat. J. Biol. Chem. 263:5118–5123. Reske, J., J. Siebrecht, and J. Hazebroek. 1997. Triacylglycerol composition and structure in genetically modified sunflower and soybean oils. J.A.O.C.S. 74:989–998. Ruiz-Sala, P., M.T.G. Hierro, I. Martinez-Castro, and G. Santa-Maria. 1996. Triglyceride composition of ewe, cow, and goat milk fat. J. Amer. Oil Chem. Soc. 73:283–293. SAS User’s Guide: Statistics, Release 7.0 Edition. 1998. SAS Inst. Inc., Cary, NC. Small, D. M. 1991. The effects of glyceride structure on absorption and metabolism. Annu. Rev. Nutr. 11:413–434. Tong, P. S., J. Mattas, J. S. Herring, E. DePeters, and J. B. German. 1995. Effects of feeding supplemental fat to dairy cattle on milk fat composition and properties. Part 1. Fatty acid composition and butter manufacture. J. Dairy Sci. 78(Suppl. 1):134. (Abstr.)
Fatty Acid and Triglyceride Composition of Milk Fat from Lactating Holstein Cows in Response to Supplemental Canola Oil E. J. DePeters,* J. B. German,† S. J. Taylor,* S. T. Essex,* and H. Perez-Monti* Departments of Animal Science* and Food Science and Technology† University of California, Davis 95616-8521
ABSTRACT
INTRODUCTION
The objective was to determine the influence of dietary lipid on total and sn-2 fatty acid composition and triglyceride structure of milk fat in lactating Holstein cows. Five primiparous Holstein cows surgically fitted with ruminal and duodenal cannulas were used in a 4 × 5 incomplete Latin square. All cows received a basal diet. Treatments consisted of a basal diet with no supplemental canola oil (control), basal diet with canola oil added to the concentrate portion of the diet to provide 1.6% fat, basal diet with 330 g of canola oil infused directly into the rumen, and basal diet with 330 g of canola oil infused directly into the abomasum. Canola oil treatments decreased palmitic acid and increased oleic acid content of milk fat compared with the control. Stearate was higher when canola oil was rumen available compared with control and abomasal infusion. Abomasal infusion increased linoleic and linoleic acids in milk fat compared with the other treatments. The sn2 fatty acid composition reflected total fatty acid composition. All canola oil treatments reduced palmitic acid and increased oleic acid content at the sn-2 position. Changes in sn-2 composition reflect specificity of the acyl transferases and substrate concentration. Triglyceride composition reported as carbon number was altered by canola oil. Triglycerides in carbon number C50, C52, and C54 were increased while C32, C34, and C36 were decreased. (Key words: fatty acid, sn-2, triglyceride, milk fat)
Opportunities exist to modify the composition of milk fat from cattle (Baer, 1991; Banks, 1987; Banks et al., 1989) and to improve milk fat’s nutritional (Baer, 1991; Ney, 1991) and manufacturing (Baer, 1991; Banks et al., 1983; Banks et al., 1989) properties. Feed and animal factors influence the composition of milk fat (Christie, 1979; Grummer, 1991; Palmquist et al., 1993). Dietary lipids modify the composition of bovine milk fat. Supplementing the diet of cows with lipid decreased the C16:0 and medium chain fatty acids (C10:0, C12:0, and C14:0) and increased the C18:0 and C18:1 content of milk fat (Baer, 1991; DePeters et al., 1989; Palmquist et al., 1993). Even though the reduced environment associated with the microbial population in the rumen promotes the hydrogenation of unsaturated fatty acids consumed in the diet (Baldwin, 1984), 20% of more of the fatty acids in the milk fat of dairy cows are monounsaturated. The oleic acid in milk fat is, in part, a consequence of the stearoyl-coenzyme A (CoA) desaturase in the intestinal epithelium and mammary tissue (Banks, 1987). In contrast to fatty acids, the influence of diet on triglyceride structure has not been well studied. Differences in triglyceride structure were associated with pasture and barn feeding periods, but no information on diets was provided (Precht and Frede, 1996). Supplementing lactating cows with lipids altered triglyceride structure (Banks et al., 1989), which may have contributed to differences in melting point observed at 5°C. Improving the nutritional value and manufacturing properties of milk fat may be achieved by altering the diet of the cow. The relationship of the fatty acid composition of milk fat and human health, for example coronary heart disease, has received considerable attention (Ney, 1991). Feeding lipid protected from ruminal hydrogenation increased the unsaturated fatty acid composition of meat and milk lipids (Garrett et al., 1976), and consumption of the polyunsaturated meats and dairy products by young adults lowered plasma cholesterol in the high subgroup consisting of individuals with the highest initial plasma cholesterol levels (Hodges et
Abbreviation key: CoA = coenzyme A, OA = canola oil infused into the abomasum, OD = canola oil added to the diet, OR = canola oil infused into the rumen, NO = no supplemental oil.
Received May 25, 2000. Accepted November 10, 2000. Corresponding author: E. J. DePeters; e-mail: ejdepeters@ucdavis. edu.
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al., 1975). More recently, a protected lipid supplement high in oleic acid increased the unsaturated fatty acid content of milk fat (Noakes et al., 1996). Consumption of the dairy products higher in unsaturated fatty acids by humans lowered total and low-density lipoprotein cholesterol. The stereospecific distribution of fatty acids in lipids may contribute to human health (Kritchesvky, 1988; Small, 1991). Increasing the amount of palmitic acid in the sn-2 position of cottonseed oil by randomization significantly increased cottonseed oil’s atherogenic properties in rabbits (Kritchevsky et al., 1988). Redgrave et al. (1988) demonstrated that the fractional removal rate of triglycerides and cholesteryl esters from plasma of rats was affected by triglyceride structure. Similarly, fractional removal rates of triglycerides were lower for 1,3-dioleoly-2-stearoyl glycerol and 1,3-dioleolyl-2-palmitoyl glycerol compared with triolein, demonstrating that the stereochemistry of acyl glycerols affected lipid metabolism (Redgrave et al., 1988; Small, 1991). Feeding lactating cows linoleic acid protected from ruminal hydrogenation significantly increased the linoleic acid content at the sn-2 position of milk fat (Christie and Clapperton, 1982), indicating that it was possible to modify the stereospecific distribution of fatty acids in milk fat. Manufacturing properties of milk fat were also influenced by supplementing the diet of cows with lipid (Banks et al., 1980). Tong et al. (1995) supplemented lactating cows with either tallow or a palm oil-based product (calcium salts of long chain fatty acids). Tallow increased oleic acid and decreased palmitic acid content of milk fat, and tallow supplementation created a softer butter. The softer butter was associated with a decreased concentration of saturated triglycerides in the carbon chain lengths between 42 and 50 (German et al., 1995). Triglyceride composition of milk fat has received less attention than total fatty acid composition. However, Banks et al. (1989) noted that fatty acid composition of milk fat only could not account for differences observed in manufacturing properties of milk fat from two breeds. Few researchers have evaluated changes in both fatty acid composition and triglyceride structure of milk fat from lactating cows in response to dietary manipulation. The objective of this study was to determine the influence of dietary lipid on total and sn-2 fatty acid composition and triglyceride structure of milk fat in lactating Holstein cows. METHODS Five primiparous Holstein cows surgically fitted with ruminal and duodenal cannulas were used in a 4 × 5 Journal of Dairy Science Vol. 84, No. 4, 2001
Table 1. Ingredient and chemical composition of the basal diet. Item
Basal diet
Ingredient (%) Alfalfa hay, chopped Barley, steam-rolled Beet pulp, shredded Corn, dry-rolled Molasses, cane Sodium bicarbonate Monosodium phosphate Trace mineral salt Soybean meal Chromic oxide
46.6 25.2 13.9 6.0 5.9 0.7 0.7 0.5 0.4 0.1
Chemical (% DM) CP Ash ADF Total fatty acids
17.4 9.5 16.7 2.0
incomplete Latin square. Ruminal cannulas were 10.2 cm i.d. (Bar Diamond, Parma, ID). At the start of the study cows averaged 172 DIM (SD 18 d). All cows received the basal diet, which was fed as a TMR (Table 1). Treatments consisted of 1) basal diet with no supplemental oil (NO), 2) basal diet with canola oil added to the concentrate portion of the diet to provide 1.6% fat (OD), 3) basal diet with approximately 330 g of canola oil infused directly into the rumen (OR), and 4) basal diet with approximately 330 g of canola oil infused directly into the abomasum (OA). Canola oil was high in oleic acid content and contained 5.57 (C16:0), 2.41 (C18:0), 43.03 (C18:1 cis 9+10), 26.47 (C18:2), and 7.99 (C18:3) g/100 g of fat. For OR and OA treatments, cows were fitted with a harness containing two small pouches, one on each side of the cow. One pouch carried a polypropylene bottle with a weighed amount of canola oil. The other pouch contained a battery-powered peristaltic universal micro-metering pump (Everest Electronics, Seaford, South Australia) to allow continuous infusion of canola oil for OR and OA. Tygon tubing (1/ 8 cm i.d., 1/4 cm o.d., 1/16 cm wall; Tygon R-1000 labultraflex, Fisher Scientific, Santa Clara, CA) from the infusion pump was passed through the ruminal cannula into the rumen for OR. Treatments OD and OR represented supplemental oil which was potentially exposed to ruminal biohydrogenation. For OA, tubing from the infusion pump was first passed through the ruminal cannula and then through the reticulo-omasal orifice with final placement in the abomasum (Clark et al., 1977). Treatment OA prevented ruminal biohydrogenation of the unsaturated fatty acids in canola oil. Each treatment period was 14 d long. Cows were housed in an open corral with access to an exercise area and bedded free stalls. Cows were trained to use Calan electronic feeding gates (American Calan, Inc., Northwood, NH), which allowed individual feeding of each
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animal. Intake of the basal diet fed in all treatments was measured. Cows were milked twice daily, and milk yield was measured. Milk samples were collected from consecutive evening and morning milkings the last week of each period and preserved with 2-bromo-2-nitro-propane-1-3-diol (Dairy & Food Labs, Inc., Modesto, CA). Diet samples were processed and analyzed similar to methods reported by Crocker et al. (1998) for DM, CP, ash, acid detergent fiber, and total fatty acids. Fatty acid composition of milk fat was determined by gas chromatography of methyl esters. Fat was extracted according to Erickson and Dunkley (1964) and prepared according to Crocker et al. (1998). Using glass graduated pipets, 10 mg of the extracted fat was aliquoted into a Teflon-lined, screw-cap glass culture tube. The solvent was evaporated under a stream of nitrogen at 60°C followed by a 1.0 ml wash of iso-octane (2,2,4 trimethylpentane), which was also evaporated under nitrogen at 60°C. A 2.0-ml aliquot of iso-octane was added to the culture tube with vortexing. Methyl esters were formed by the addition of 100 µl of 3 M potassium hydroxide, vortexing, and allowed to stand at room temperature for 10 min. Esters were washed and separated by the addition of 2.0 ml of saturated NaCl and 8.5 ml of deionized water. Tubes were shaken with a Burell wrist action shaker for 10 min, centrifuged at 1750 rpm for 10 min to separate the methyl esters in the upper solvent layer, and then aliquoted into 1.8-ml gas chromatography autosampler vials. The ester mixture was analyzed with a Supelco 2560, 100-m capillary column with a 0.25 mm i.d. and a 0.20-micron film thickness. Hydrogen was used as the carrier gas with a linear flow rate set at 27 cm/s with a column head pressure of 33 psi. A Hewlett Packard 5890 gas chromatograph equipped with a 7673A auto-injector injected 1.0 µL of sample that was split 125 times. Injector and detector temperatures were set at 220°C, and the column temperature was programmed at 75°C for 10 min and then increased to 175°C for 38 min at 20°C/min. A flame-ionization detector with a signal range of one was used. Esters were integrated and quantified with version 6.07 of Chrome Perfect (Justice Innovations, Inc., Palo Alto, CA) software interfaced with a Hewlett Packard 3393A integrator. The sn-2 fatty acids in milk fat were separated by thin-layer chromatography followed by methylation. Milk fat was extracted according to Erickson and Dunkley (1964). Using glass graduated pipets, 100-mg aliquot of lipid extract was placed into a Teflon-lined, screw-cap glass culture tube. Solvent was evaporated under a stream of nitrogen at 60°C. The 2-monoacylglycerols were formed and separated by the method described by Hendriske and Harwood (1986), with the following modifications. An enzyme reaction time of 1
h was used, and Whatman, LK6F Linear-K, silica gel, 5 × 20 cm, 250-micron chromatography plates were used. The monoacylglycerol band was scrapped and transferred to a 12 mm × 100 mm screw-top, Teflon-lined culture tube. Two hundred microliters of methanol:hexane (4:1), containing 0.05 mg of nonadecanoic acid (C19:0), was added to each tube and vortexed. Methylation was achieved by adding 5.0 µl of acetyl chloride slowly down the side of the culture tube to prevent superheating and subsequent popping. Tubes were sealed under nitrogen and incubated for 1 h at 100°C. After cooling at room temperature, 500 µL of 6% K2CO3 was added, followed by vortexing and the addition of 200 µL of hexane. Tubes were centrifuged for 5 min at 1750 rpm, and the upper hexane layer was removed and placed in a 1.8-ml gas chromatograph auto sampler vial. The solvent was evaporated under nitrogen and brought up to a final volume of 75 µL with hexane. Esters were determined and quantified as described earlier. Triglyceride profiles of milk fat were obtained by high temperature polar capillary gas chromatography (Varian 3400, Walnut Creek, CA). A 30-m DB-HT17 (J& W, Sacramento, CA) capillary column with a relatively polar stationary phase was used to separate intact triglycerides on the basis of total chain length and unsaturation. A solvent programmable injector (Varian 8100, Walnut Creek, CA) was used to introduce the triglyceride samples dissolved in octane onto the column using a 10-cm, 0.5-mm i.d. fused silica tube as a retention gap. The sample injection volume was 0.4 µl with a solvent plug size of 0.5 µl. The initial injector temperature was set to 130°C and was programmed to rise immediately on injection to 355°C at 115°/min. The column temperature was programmed from 130 to 355°C at 20°/min and held constant at 355°C for an additional 20 min. The detector temperature was set at 355°C. Standard monoacid and mixed chain triglycerides (Nu chek, Elysian, MN), as well as hydrogenated milk fat samples were used to determine the retention times of specific triglycerides. The location of specific fatty acids on the triglyceride was not resolved using these analyses. However, certain specific molecular species were inferred from the known positional specificity of bovine milk fat triglycerides. Data were analyzed by the general line models procedure of SAS (1998) for a 4 × 5 incomplete Latin square design using the following model described by Hicks (1993): Yijk = µ + Ti + Pj + Ck + Eijk where, Journal of Dairy Science Vol. 84, No. 4, 2001
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Yijk µ Ti Pj Ck Eijk
= = = = = =
observation; mean; treatment, i = 1,2,3,4; period, j = 1,2,3,4; cow, k = 1,2,3,4,5; and residual error.
OA was higher than NO but lower than either OD or OR. Since OA prevented ruminal biohydrogenation of fatty acids, the slightly higher C18:0 in milk fat may be a consequence of the C18:0 contained in the infused canola oil. In the small intestine the additional C18:0 from OA would be available for absorption and milk fat synthesis. All canola oil treatments increased oleic acid (C18:1 cis 9+10) content compared to NO. The higher oleic acid content of OD and OR may be a consequence of mammary stearoyl-CoA desaturase which desaturates C18:0 to C18:1. The higher C18:1 trans content of milk fat for OD and OR, in particular the trans 11 isomer, indicated that the canola oil was subjected to ruminal biohydrogenation. This trans isomer of oleic acid has been associated with ruminal biohydrogenation (Harfoot, 1978). The C18:1 trans content of OA was similar to NO; an indication that ruminal biohydrogenation of fatty acids was prevented by the infusion method. Milk fat composition of C18:2 and C18:3 was higher for only OA. Abomasal infusion prevented ruminal biohydrogenation of unsaturated fatty acids and provided unsaturated fatty acids postruminally for milk fat synthesis. In contrast, OD and OR did not prevent hydrogenation of polyunsaturated fatty acids. The fatty acid composition at the sn-2 position of milk fat was influenced by treatment (Table 4). All canola oil treatments reduced the proportion of C16:0 and increased the proportion of C18:0 and C18:1 cis 9+10 esterified at the sn-2 position compared to NO. The sn-2 content of C18:2 and C18:3 was increased dramatically for OA compared with the other three treatments. Changes in the fatty acid esterified at the sn-2 position are similar to changes in total fatty acid composition of milk fat. Fatty acid distribution in triglycerides is non random (Blank and Privett, 1964). The ratio of a fatty acid in the sn-2 position to the proportion of the same fatty acid in total fatty acids (all positions) was calculated. Only those fatty acids present in the sn-2 position were considered for the total fatty acids and the distribution recalculated. The ratio for C14:0 and C16:0 for all treatments was greater than 1, suggesting
Means were separated by Duncan’s multiple range test with significance declared at P ≤ 0.05. The ratio of a fatty acid in the sn-2 position to the proportion of the same fatty acid in the total fatty acids for all positions was calculated and compared using Student’s t-test with the assumption of unequal variances. A one-sample Student’s t-test was calculated to compare if the ratio of the sn-2 FA to the total FA was equal to one (SAS, 1998). RESULTS AND DISCUSSION Intake of DM was higher for OD and OR compared with NO and OA (Table 2). Yield of milk was affected by treatment and was highest for OD and OR and lowest for NO and OA. The additional intake of energy provided by canola oil may have supported higher milk yield. Infusion of canola oil increases the amount of long chain fatty acids available to the mammary gland for milk fat synthesis and reduces de novo synthesis of fatty acids from acetate in the mammary gland. The latter may spare energy for milk synthesis. Milk fat percentage was highest for OA and lowest for OR. Yields of milk fat were similar across treatments. Total fatty acid composition of milk fat was influenced by treatment (Table 3). Abomasal infusion of canola oil decreased C14:0 content of milk fat compared with the other treatments. Content of C16:0 was lowest for OA, highest for NO, and intermediate for OD and OR. A similar response occurred for C16:1 cis. Stearate was highest for OD and OR and lowest for NO. The higher C18:0 for OD and OR may be a consequence or ruminal biohydrogenation of unsaturated fatty acids contained in the canola oil. The stearate content of milk fat for
Table 2. Production performance of cows. Treatment1 Item DMI, kg/d Milk, kg/d 4% FCM, kg/d Fat, % Fat, kg/d
NO
OD a
18.0 25.0a 23.6 3.69ab 0.92
OR b
19.6 28.6b 26.4 3.50bc 0.99
OA b
19.5 29.0b 26.2 3.40c 0.97
SE a
17.7 26.6a 25.8 3.83a 1.01
0.36 0.56 0.73 0.07 0.04
Means in the same row with different superscripts differ P < 0.05. NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b 1
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FATTY ACID AND TRIGLYCERIDE OF MILK FAT Table 3. Total fatty acid composition of milk fat (g/100 g of fat). Treatment1 Fatty acid
NO
OD
OR
OA
SE
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C16:1 C16:1 C18:0 C18:1 C18:1 C18:1 C18:2 C18:3
3.62 2.68 1.66 4.39 5.58 14.77a 37.47a 0.39 2.40a 6.17a 0.13a 0.76a 15.10a 3.61a 1.29a
3.84 2.65 1.61 4.01 4.76 14.32a 30.07b 0.39 1.82b 9.57b 0.26b 1.02ab 20.97b 3.29a 1.43a
3.72 2.68 1.66 4.15 5.01 14.33a 30.13b 0.39 2.00b 9.04b 0.32c 1.37b 20.40b 3.41a 1.42a
3.39 2.51 1.57 3.93 4.59 12.34b 27.22c 0.32 1.47c 6.89c 0.14a 0.61a 21.70b 9.54b 3.79b
0.14 0.05 0.04 0.16 0.23 0.28 0.78 0.02 0.08 0.39 0.03 0.13 0.69 0.49 0.19
trans cis trans 9 trans 11 cis 9+10
Means in the same row with different superscripts differ (P < 0.05). NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b,c 1
a preference for these fatty acids at the sn-2 position compared with C10:0 and C12:0 with ratios less than 1. Marshall and Knudsen (1977) reported that chain length specificity of the 1-acylglycerolphosphate acyltransferase was C16 > C14 > C12 > C10 > C8, which agreed with the fatty acid esterified at the sn-2 position observed in the present study. An acyl specificity of C16:0 ∼ C14:0 ∼ C12:0 ∼ C18:2 > C18:3 ∼ C18:0 ∼ C20:4 > C18:1 it was observed for the human lysophosphatidic acid acyltransferase (hLPAATα), and acyl specificity of hLPAATα was affected by acyl concentration (Aguado and Campbell, 1998). The ratio of the 18 C unsaturated fatty acids were found at a lower proportion at the sn2 position. Reske et al. (1997) suggested that in plant oils, triglyceride synthesis was driven by mass action; the amount of substrate available was likely more im-
portant than the specificity of the acylytransferases. Recently, Mistry (1999) compared the fatty acid composition of bovine and ovine milk fat and proposed that substrate availability in the ruminant mammary gland may be the determining factor for fatty acid esterification at the sn-2 position. The proportion of C18:2 and C18:3 at the sn-2 position increased for abomasal compared with ruminal infusion of canola oil while the ratio did not change supports the suggestion that substrate availability along with acyltransferase specificity is important in determining the fatty acid at the sn-2 position, which was proposed for plant lipids (Lassner et al., 1995; Reske et al., 1997). Treatments affected the triglyceride structure of milk fat (Table 5). Canola oil additions decreased triglycerides in the C32, C34, C36, C42, and C44 groups and
Table 4. Fatty acid composition at the sn-2 position of milk fat (g/100 g of sn-2 fat). Treatment1 Fatty acid
NO
OD
OR
OA
SE
C10:0 C12:0 C14:0 C16:0 C16:1 C18:0 C18:1 C18:1 C18:1 C18:2 C18:3
0.32 3.81a 24.08 54.64a 1.80a 4.28a 0.18a 0.32a 7.91a 2.27a 0.39a
0.05 2.34b 23.58 48.32b 1.14b 9.51b 0.39bc 0.58ab 11.66b 2.22a 0.30a
0.12 2.83ab 23.26 47.01b 1.43b 9.12b 0.54c 0.81b 12.18bc 2.33a 0.44a
0.10 3.32ab 19.90 45.25b 1.06b 7.93b 0.29ab 0.35a 14.05c 6.44b 1.32b
0.11 0.42 1.34 1.18 0.11 0.78 0.05 0.09 0.65 0.48 0.08
cis trans 9 trans 11 cis 9+10
Means in the same row with different superscripts differ P < 0.05. NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b,c 1
Journal of Dairy Science Vol. 84, No. 4, 2001
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DEPETERS ET AL. Table 5. Triglyceride structure of milk fat (g/100 g of fat). Treatment1 Carbon no.
NO
OD
OR
OA
SE
C26 C28 C30 C32 C34 C36 C38 C40 C42 C44 C46 C48 C50 C52 C54 Total
1.55 1.95 2.71 5.12a 10.24a 15.54a 14.43 10.52 8.76a 8.29a 7.75 5.64 3.68a 1.12a 0.12a 97.44
1.55 1.88 2.69 4.76b 9.46b 14.51ab 14.92 11.29 7.97b 7.47b 7.43 6.03 4.62b 1.81b 0.34b 96.71
1.55 1.91 2.73 4.80b 9.39b 14.21b 14.91 11.34 8.10b 7.03b 7.65 6.06 4.69b 1.85b 0.33b 96.56
1.57 2.01 3.32 4.63b 8.71c 13.83b 15.08 11.51 7.85b 7.32b 7.55 5.72 5.05b 2.17b 0.41b 96.73
0.05 0.09 0.37 0.10 0.18 0.34 0.20 0.25 0.18 0.24 0.24 0.18 0.21 0.13 0.05 0.41
Means in the same row with different superscripts differ P < 0.05. NO = No canola oil, OD = canola oil added to the diet, OR = canola oil infused ruminally, and OA = canola oil infused abomasally. a,b,c 1
increased triglycerides in C40, C50, and C52 groups. Canola oil treatments provided 18 C fatty acids, which were available for milk fat synthesis. Long chain fatty acids were reported to reduce de novo fatty acid synthesis in the mammary gland (Grummer, 1991; Palmquist et al., 1993). There were trends for lower C10:0 and C12:0 and significantly lower C16:0 with all canola oil treatments compared with NO. The higher proportion of long chain fatty acids and the lower proportion of medium chain fatty acids associated with canola oil treatments would explain a portion of the changes in carbon number for triglyceride structure. Banks et al. (1989) and Precht (1991) noted that triglyceride composition of milk fat was influenced by diet. The distributions between C52 and C54 in milk fat were for example, affected by added dietary fat and, to a greater extent, with severe underfeeding (Precht, 1991). Supplementing the diet of lactating cows with oils decreased the proportions of triglycerides from 30 to 36 and 42 to 46 and increased triglycerides from 50 to 54 (Banks et al., 1989). Triglycerides did not differ with type of oil fed even though the fatty acid composition of the milk was influenced by the type of dietary oil (Banks et al., 1989). No changes in the proportion of triglycerides associated with C26, C28, C30, C38, C46, and C48 occurred (Table 5). Although total carbon number was not different, the individual peaks of distribution of acyl-C groups were different. For example, the total area for C38 was not affected by treatment, but canola oil treatments decreased acyl-C groups associated with peaks 1, 2, 3, and 4. In contrast, peaks 4 and 6 of C38 were increased with canola oil treatments. Site of canola oil Journal of Dairy Science Vol. 84, No. 4, 2001
addition also influenced acyl-C groups. Treatments OD and OR increased peak 5 of C38 compared with NO: OA did not influence this peak. Infusing canola oil into the abomasum increased acyl-C groups associated with peaks 7, 8, and 10. The exact changes occurring in triglyceride structure can not be described by the techniques used in the present study. However, the data do demonstrate that although the total proportion of triglycerides within a carbon number do not change, the distribution of the acyl-C groups may change. Precht and Frede (1996) evaluated the correlation coefficients between triglycerides and solid fat contents at different temperatures between 0 and 30°C. For C40, C50, C52, and C54 triglycerides, the correlation coefficient was negative. These researchers attributed this change in sign from positive to negative to be a result of different contents of C16 and C18:1 fatty acids comprising the different acyl-C-groups with the same carbon number. Their observations support our findings that triglyceride content did not change for some carbon numbers, but acyl-C groups were different. Similarly, Banks et al. (1989) observed that type of oil fed influenced fatty acid composition but not triglyceride composition of milk fat. However, differences in butter time were observed, indicating that the manufacturing properties of the milk fats were altered. Changes in specific acyl-C groups within triglycerides were not measured and may account for differences in butter time, although little research has been done in this area. In the present study, triglyceride structure was affected by diet of the cow. Recently, Ruiz-Sala et al. (1996) reported that milk fat from cows was higher in long chain and unsaturated
FATTY ACID AND TRIGLYCERIDE OF MILK FAT
triglycerides compared with the milk fat from ewes. However, information on the diets fed to the sheep, goats, and cattle was not provided. Since diet affects triglyceride structure, species comparison of fatty acid and triglyceride composition of milk fat may be confounded by diet. CONCLUSIONS Supplementing the diet of lactating dairy cows with canola oil modified the total and the sn-2 fatty acid composition and triglyceride structure of milk fat. Canola oil when either added to the diet or infused into the rumen increased the C18:0 and C18:1 and decreased the C16:0 contents of milk fat. Infusing canola oil into the abomasum to prevent ruminal biohydrogenation of unsaturated fatty acids increased the C18:1, C18:2, and C18:3 and decreased the C14:0 and C16:0 contents of milk fat compared with NO. Changes in the fatty acid esterified at the sn-2 position of milk fat in response to canola oil supplementation of the diet reflect changes in total fatty acid composition. Triglyceride composition of milk fat was affected by diet. Triglycerides in the C32, C34, C36, C42, and C44 groups decreased and C40, C50, and C52 groups increased with canola oil supplementation. Although some triglycerides were not affected by treatment, within a specific triglyceride, acyl-C groups were different. The study of triglyceride composition of milk fat from cattle, and possibly other ruminants, must consider dietary differences. The impact of changes in fatty acid and triglyceride compositions on the nutritional and manufacturing studies of milk fat are not known and require further study. ACKNOWLEDGMENTS The authors thank Susannah Taylor, Lisa Crocker, and Megan Osborne for their technical assistance, B. Kreuscher, F. Stewart, and W. Paroczai for care of the cows, and L. George, School of Veterinary Medicine, for the surgical preparation of the animals. Scott Essex expresses appreciation to the American Registry of Professional Animal Scientists, California Chapter, for their financial support through the Dr. Antionio A. Jimenez Memorial Award. Financial support was provided by the California Dairy Research Foundation (Davis), the California Agricultural Experiment Station (Davis), and NuAg Systems (Middletown, CA). The research is a contribution of Regional Research Project W-181. REFERENCES Aguado, B., and R. D. Campbell. 1998. Characterization of a human lysophosphatidic acid acyltransferease that is encoded by a gene
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