Effect of feeding rumen-protected dietary protein–oil supplements on fatty acid composition and α-tocopherol content of blood serum and muscle lipids of lambs

Small Ruminant Research 72 (2007) 101–110

Effect of feeding rumen-protected dietary protein–oil supplements on fatty acid composition and ␣-tocopherol content of blood serum and muscle lipids of lambs J.H. Lee a,∗ , J.C. Waller b , Y. Yilmaz b , S.L. Melton b a

Georgia Small Ruminant Research and Extension Center, Fort Valley State University, Fort Valley, GA 31030, USA b Agricultural Experimental Station, University of Tennessee, Knoxville, TN 37996, USA Received 3 January 2006; received in revised form 2 August 2006; accepted 29 August 2006 Available online 11 October 2006

Abstract This study evaluated the effect of feeding a rumen-protected, protein–oil supplement (RDPS) containing ␣-tocopheryl acetate (TA) and sunflower oil on fatty acid composition and ␣-tocopherol concentrations of blood serum and muscle in lambs. The RDPS contained 32.0% CP and 32.2% fat on a DM basis, and 465 IU Vitamin E/kg. Twenty-four, cross-bred lambs were allotted into three treatment groups with two pens of four lambs per group. Each pen of four lambs was fed one of three dietary treatments for 10 weeks: (1) control diet, 5.12 kg DM of corn basal diet (BD), 1.50 kg DM of grass hay, and 40 g of molasses as-fed basis, (2) Vitamin E diet, TA (1000 IU) plus control diet, or (3) RDPS diet, 2.0 kg as-fed basis of RDPS plus control diet (3.12 kg of DM of BD). Blood samples were collected biweekly from individual lambs over the 10-week feeding trial; longissimus dorsi (LD) and psoas major (PM) muscles were obtained from each lamb carcass to analyze ␣-tocopherol and fatty acid composition. Compared with lambs fed the control or Vitamin E diet, lambs fed the RDPS had a higher (P < 0.05) concentration of linoleic (C18:2n6) acid (23.04% or 24.93% versus 33.5%) and lower (P < 0.05) concentrations of palmitic (C16:0) and oleic (C18:1n9) acids in blood serum lipids. However, no differences (P > 0.05) were found in concentrations of C16:0, C18:1n9 or C18:2n6 in the serum lipids from lambs fed the control and Vitamin E diets. Furthermore, the RDPS diet substantially increased (P < 0.05) C18:2n6 levels in LD and PM muscle lipids but decreased (P < 0.05) C16:0 and C18:1n9 compared to the control or Vitamin E diet. Lambs fed Vitamin E or RDPS diet had higher (P < 0.05) concentrations of serum ␣-tocopherol than lambs fed the control diet, whereas serum ␣-tocopherol content in lambs fed the RDPS was 28.7% higher (P < 0.05) than lambs fed the Vitamin E. Consequently, lambs fed the RDPS had higher (P < 0.05) ␣-tocopherol concentrations in PM (7.43 ␮g/g) muscle than lambs fed the Vitamin E; however, no significant differences (P > 0.05) were found in ␣-tocopherol concentrations in LD muscle from lambs fed either Vitamin E or RDPS diet. Results indicated that the RDPS protected polyunsaturated fat and ␣-tocopheryl acetate from ruminal degradation, resulting in increased content in blood serum and deposition in muscle tissues of lambs. © 2006 Elsevier B.V. All rights reserved. Keywords: ␣-Tocopherol; Fatty acid; Lamb; Rumen-protected; Sunflower oil

1. Introduction ∗

Corresponding author at: Fort Valley State University, Agricultural Research Station, 1005 State University Drive, Fort Valley, GA 31030, USA. Tel.: +1 478 827 3077; fax: +1 478 825 6376. E-mail address: [email protected] (J.H. Lee). 0921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.08.012

Consumer awareness of the relationship of saturated fat with higher LDL-cholesterol levels in blood, which is associated with increased incidence of coronary heart disease, has resulted in decreasing consumption of

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red meat products in developed countries (Vanderveen, 1996; Putnam, 2000). Since milk and meat from ruminants contain fat that is 48–68% saturated, increasing the unsaturated fat should produce healthier, animalbased, food products. However, the microbes in the rumen hydrolyze and hydrogenate dietary lipids depending on the matrix in which they are delivered into the rumen (Gulati et al., 1997b). Feeding lipids to ruminants at levels about 4–5% of the dietary dry matter (DM) may result in depressed digestion of other components of the diet and depressed intake by the ruminant (Hartfoot and Hazlewood, 1988). Four main processes have been used to protect lipids from ruminal degradation (Gulati et al., 1997a,b): (1) encapsulation of triacylglycerols in a matrix of aldehyde-treated protein; (2) formation of calcium salts of saturated or monounsaturated fatty acids; (3) pelleting of hydrogenated and small amounts of starch to form prilled fat supplements; (4) extrusion of vegetable oil seeds. The most effective way of protecting dietary lipids (less than 13% metabolized) from the ruminal biohydrogenation was a formaldehyde-treated product (Gulati et al., 1997b). However, formaldehyde-treated, dietary lipid supplements have not been approved in the United States because of concerns that formaldehyde will be transferred to the animal products. Lee et al. (2004) investigated the possibility of using alternative generally recognized as safe (GRAS), chemical agents nearly as effective as formaldehyde. Additional research is needed to substantiate the claim that the unsaturated fatty acid content in meat lipids can be increased by feeding ruminants vegetable oil containing high levels of unsaturated fat. Modifying animal fat by feeding various lipid supplements containing unsaturated fatty acids increases the risk of developing oxidized flavor in animal products such as milk and meat (Smith et al., 1996; Focant et al., 1998). Vitamin E has been fed to ruminants to enhance immunity (Hidiroglou et al., 1995; McDowell et al., 1996), minimize off-flavors in milk and meat due to lipid oxidation (Weiss and Wyatt, 2003; Lauzurica et al., 2005), and improve color stability of meat by inhibiting oxidation of oxymyoglobin (Mitsumoto et al., 1993; Granti et al., 2005). The absorption of tocopherols is incomplete in ruminants (Schelling et al., 1995; Weiss, 1998), but in practice, tocopherol absorption in ruminants might be increased by feeding an emulsified form of Vitamin E (Ochoa et al., 1992). Successful incorporation of Vitamin E and unsaturated fat in animal products by feeding rumen-protected, protein–oil supplements containing ␣-tocopheryl acetate and sunflower oil may provide healthier, animal-based,

food products for humans and extend the shelf life of products. The objectives of this research were to produce emulsified ␣-tocopherol acetate in protein–oil supplements consisting of sunflower oil combined with defatted soy flour by the GRAS chemical (diacetyl) and to determine the efficacy of the protein–oil supplement to incorporate ␣-tocopherol and polyunsaturated fat in the blood serum and edible tissues of lambs. 2. Materials and methods 2.1. Preparing rumen-protected dietary protein–oil supplements Rumen-protected, protein–oil supplements (RDPS) were prepared using the following procedure as described by Lee et al. (2004). Sodium caseinate (597.4 g) was dispersed in 0.1% (w/w) NaOH solution in a Groen steam kettle (DI Foodservice Co. Jackson, MS, USA). When the ingredients in the kettle were agitated and heated to 85 ◦ C, a 7.8 g of tocopheryl acetate (TA) was dissolved in 1478 g of sunflower oil (SFO) in a stainless steel container. The heated solution was then transferred to a Stephan Universal Schnell Cutter (Cryovac Inc., Duncan, SC, USA). Defatted soy flour (2343 g) and SFO containing TA were added to the cutter and mixed with 454.5 g of diacetyl. After the slurry of mixtures congealed, the gel was stored in a refrigerator at 4 ◦ C overnight (∼8 h). The SFO-gel was ground through a 0.625 cm screen in a Hobart Grinder (Hobart Co., Troy, OH, USA), packaged in closed plastic bags and stored at 4 ◦ C until fed. 2.2. Animals, feeding, and sampling Twenty-four, cross-bred (Dorset × Suffolk) feeder lambs obtained from The University of Tennessee Research Flock, were assigned in a completely randomized design to a 10-week finishing trial consisting of three dietary treatments: (1) control diet, 5.12 kg DM of cracked corn and pelleted alfalfa basal diet (BD), 1.50 kg DM of grass hay, and 40 g of molasses as-fed basis; (2) Vitamin E diet, TA (1000 IU) plus control diet; or (3) RDPS diet, 2.0 kg as-fed basis of SFO-gel plus adjusted the control diet by reducing BD to 3.12 kg DM (Table 1). Supplemental Vitamin E (Vitamin E and RDPS) diets offered equal amounts of Vitamin E (250 IU/day) to individual lambs. Each treatment was replicated in two pens, containing four lambs per pen. Pens were in a closed barn, and each lamb was provided 1.56 m2 of floor space with free access to water. A 14-day adjustment period was used to adapt lambs to the new feeding facilities and to allow lambs to recover from stresses associated with weaning. Each pen was fed one of three dietary treatments for 10 weeks. Blood samples were taken from each lamb biweekly during the feeding trial prior to slaughter. Samples were collected via jugular vein puncture into 20-mL vacuum tubes

J.H. Lee et al. / Small Ruminant Research 72 (2007) 101–110 Table 1 Ingredient composition of the three different diets fed to lambs Ingredient component (%)

Dieta Control

Coarse ground corn Alfalfa pellets Soybean mealb Rolled oats Trace mineral mix Limestone Antibiotic Ammonium chloride Sunflower oil-gelc ␣-Tocopheryl acetate (IU)

63.8 19.9 10.0 5.0 0.5 0.3 0.2 0.3

Vitamin E 63.8 19.9 10.0 5.0 0.5 0.3 0.2 0.3 250

RDPS 42.5 13.3 6.7 3.3 0.3 0.2 0.1 0.2 33.3 250

Control: no supplemental Vitamin E; Vitamin E: supplemented ␣tocopheryl acetate; RDPS: diacetyl-treated protein–lipid supplement. b 48% crude protein. c Ground diacetyl-treated dietary supplement containing sunflower oil and ␣-tocopheryl acetate. a

and immediately placed in an ice bath. Serum was separated from the blood sample by centrifugation at 2000 × g for 20 min using a Sorvall Superspeed Model 5RC2-B Automatic Refrigerated Centrifuge (Ivan Sorvall Inc., Newton, CT, USA) and stored in 10-mL vials at −18 ◦ C until analysis. At the conclusion of the finishing trial, longissimus dorsi (LD) and psoas major (PM) muscles were obtained from the carcass of each lamb, frozen, powdered under liquid nitrogen, packaged in polyethylene bags (NASCO Inc., Fort Atkinson, WI, USA), sealed, and stored at −18 ◦ C until analyzed for ␣-tocopherol and fatty acid composition. 2.3. Chemical analysis of blood serum and muscle samples Total lipids from blood serums were extracted by a modified procedure of Bright et al. (1994), where butylated hydroxytoluene (BHT) was added to chloroform (CHCl3 ) for the extraction. Total lipids from muscle samples were extracted by a modified method of Melton et al. (1979). The modification included a reduction in sample size was 5.0–0.2 g of fat with solvents and other chemicals reduced accordingly. Fatty acid composition of total lipids extracted from blood serum and muscle of each animal was analyzed by preparing fatty acid methyl esters (FAME). The FAME were prepared according to AOCS procedures (1993) and analyzed with a Shimadzu GC-9A gas chromatograph equipped with a Shimadzu AOC-9 automatic injection system (Shimadzu Scientific Instruments Inc., Columbia, MD, USA). A 0.25-mm i.d., 30-m long fused silica SP-2330 capillary column (Supelco Inc., Bellefonte, PA, USA) was used to separate the methyl esters, which were detected with a flame ionization detector. An injection temperature of 250 ◦ C was used, and the column temperature was programmed from 130 to 230 ◦ C at 2 ◦ C/min with 20 min holding at 230 ◦ C. Helium was used as the carrier gas with a flow

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rate of 50 mL/min and a split ratio of 30:1. Identification of individual FAME in the samples was achieved by matching the retention time of the sample peak with that of the standard peak (Alltech Associates Inc., Deerfield, IL, USA). The relative weight percents of individual FAME in each sample were calculated using their corrected areas. Concentrations of ␣-tocopherol in blood serum and muscle samples were determined according to procedures adapted from Liu et al. (1996). An aliquot (1 mL of serum or 1.0 g of muscle) of each sample was delivered into a 15-mL test tube and mixed with a 0.25 g of ascorbic acid as well as 7.3 mL of 8.04% NaOH solution (8.04 g NaOH/41.4 mL ddH2 O + 50.1 mL ethanol). The mixture was briefly vortexed until the ascorbic acid dissolved. The content was saponificated for 15 min in an 80 ◦ C shaking water bath and cooled in an ice bath. Approximately 4.0 mL of isooctane were added into the ice-cold tube and vortexed for 2 min. The tube was allowed to stand at room temperature to separate phases. An aliquot of the upper layer was filtered through a nylon 0.45 ␮m disk using a disposable syringe (Becaton Dickinson & Co., Rutherford, NJ, USA). The separation of ␣-tocopherol in filtered isooctane phases was performed by a Water’s HPLC, equipped with a U6K injector, a Model 510 pump, and a Shimadzu RF-530 Fluorescence detector (Shimadzu Corporation, Columbia, MD, USA). A 3.9 mm × 300 mm Bondclone (10 ␮m) C-18 column (Phenomenex Inc., Torrance, CA, USA) with a mobile phase of 5% water in methanol at a flow rate of 1.0 mL/min was used to separate ␣-tocopherol and detected at an excitation wavelength of 295 nm and emission wavelength of 330 nm. 2.4. Statistical analysis Blood serum data were analyzed as a completely randomized, repeated measures design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA) with individual lambs as the experimental unit. The effects of dietary treatment, length of supplementation, and their interaction were considered fixed and animals within dietary treatment were used as a random error term for dietary treatment. Muscle data were analyzed with a completely randomized design using the GLM procedure of SAS. The Pearson correlation analysis was used to determine the relationship of ␣-tocopherol and individual fatty acids by the CORR procedure of SAS (Steel et al., 1996). Least squares means were generated and separated using the PDIFF option of SAS for main or interactive effects. Significance was determined at P < 0.05, but difference of 0.05 ≤ P < 0.1 were considered trends.

3. Results and discussion 3.1. Dietary protein–oil supplement and feeding performance The sunflower oil (SFO)-gel (RDPS) contained 52.8%, 15.2%, and 15.1% of moisture, fat, and pro-

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tein, respectively, as well as 465 IU of Vitamin E/kg. Major fatty acids of the SFO-gel were linoleic (C18:2n6) and oleic (C18:1n9) acids, which consisted of 71.4% and 17.2% of total fat, respectively. Each lamb on control, Vitamin E, and rumen-protected dietary supplement (RDPS) diets ingested an average of 1061, 1072, and 897 g, with lambs consumed an average of 250 IU (Vitamin E diet) and 240 IU (RDPS diet) of ␣-tocopheryl acetate per day. Average daily fat intakes were 38, 38, and 103 g in control, Vitamin E, and RDPS diets, respectively. Initial body weight of feeder lambs (n = 24) ranged from 28.6 to 39.5 kg at the beginning of the feeding trial and ranged from 40.0 to 54.0 kg at the end of the feeding trial before slaughtering. 3.2. Fatty acid composition of blood serum and muscle lipids The percentage of fatty acids in blood serum lipids from the lambs fed three different dietary treatments (control, Vitamin E, and RDPS diets) across the 10-week feeding period are presented in Table 2. Significant differences (P < 0.05) were found in weight percentages of palmitic (C16:0), heptadecanoic (C17:0), heptadecenoic (C17:1n8), oleic (C18:1n9), and linoleic (C18:2n6) acids in serum lipids across treatments; however, no significant differences (P > 0.05) were found in levels of these five fatty acids in serum lipids from lambs fed control and Vitamin E diets. Lambs fed the RPDS decreased (P < 0.05) the serum lipid proportion of palmitic (C16:0) and oleic (C18:1n9) acids compared to lambs fed either the control or Vitamin E diet; the portions of heptadecanoic (C17:0) and heptadecenoic (C17:1n8) acids were also significantly decreased (P < 0.05) compared to control and Vitamin E diets. However, the RDPS diet increased (P < 0.05) the serum content of linoleic (C18:2n6) acid from 24.0% to 33.5%. In the present study, the weight percentages of fatty acids from serum of lambs fed either control or Vitamin E diet were not different (P > 0.05) across the feeding period (Table 2). However, an increase in polyunsaturated fatty acid (PUFA) concentrations (e.g. C18:2n6) is expected in the serum lipids of lambs fed the Vitamin E diet because supplementation of Vitamin E could protect the PUFA against lipid oxidation and increase the concentration of the PUFA in the serum lipids of lambs fed the Vitamin E diet (Machlin, 1991). The protection of the PUFA in the SFO-gel supplement (RDPS) against ruminal biohydrogenation increased linoleic (C18:2n6) acid in serum lipids which was accompanied by decreasing palmitic (C16:0) and oleic (C18:1n9) as well as heptadecanoic (C17:0) and heptadecenoic (C17:1n8) acids.

Table 2 Fatty acid compositiona,b of serum lipids of lambs fed the three different experimental diets across feeding times Fatty acid (%)

C14:0 C14:1n5 C16:0 C16:1n7 trans C16:1n7 C17:0 C17:1n8 C18:0 C18:1n9 C18:2n6 C18:2n6 trans C18:3n3 C18:3n6 C20:0 C20:1n9 C20:2n6 C20:3n6 C20:4n6 C20:5n3 C22:5n3 C22:6n3 C24:0

Dietc

S.E.M.

Control

Vitamin E

RDPS

0.04 0.08 16.66 a 0.43 0.80 1.19 a 0.65 a 18.91 23.91 a 23.04 a 0.25 0.76 0.61 0.02 0.34 0.11 0.36 4.27 0.73 1.41 4.54 0.58

0.07 0.08 16.40 a 0.37 1.01 1.33 a 0.77 a 17.48 23.41 a 24.93 a 0.20 0.37 0.73 0.02 0.34 0.13 0.39 4.30 0.58 1.09 4.22 0.64

0.04 0.02 12.36 b 0.37 0.58 0.72 b 0.25 b 22.94 16.98 b 33.47 b 0.26 0.39 0.39 0.01 0.30 0.05 0.48 4.59 0.39 0.74 3.83 0.54

0.04 0.03 0.44 0.07 0.49 0.08 0.06 1.76 0.93 0.94 0.03 0.16 0.19 0.01 0.03 0.04 0.06 0.72 0.24 0.23 0.72 0.05

a

Wt.% of fatty acid methyl esters. Least-square means in a row with a common letter are not significant at the 5% level. c Control: no supplemental Vitamin E; Vitamin E: supplemented ␣tocopheryl acetate; RDPS: diacetyl-treated protein–lipid supplement. b

In a previous study, Dje (1994) used acetaldehyde and diacetyl as alternative chemical agents for formaldehyde to produce rumen-protected dietary polyunsaturated fat. In this study, lactating ewes fed a ground full-fat soybean supplement containing 16% fat, consisted of 53.5% linoleic (C18:2n6) acid. The serum linoleic (C18:2n6) acid content of lactating ewes were increased (P < 0.05) from 28.9% to 33.6% by feeding acetaldehyde-treated soybean supplement compared to that of ewes fed untreated soybean supplement. Furthermore, feeding diacetyl-treated supplement to the ewes increased serum linoleic (C18:2n6) acid content (30.0%) compared to those fed the untreated supplement, but less than those fed the acetaldehyde-treated supplement. Consequently, without protection from ruminal biohydrogenation, linoleic (C18:2n6) acid hydrogenated to trans-oleic acid as an intermediate metabolite and further hydrogenated to stearic (C18:0) acid in the rumen which is the rate-limiting step in the conversion of PUFA to saturated fatty acid (Byers and Schelling, 1993; Mosley et al., 2002). Therefore, feeding unprotected oil increased mainly stearic (C18:0) and oleic (C18:1n9) acids. The

J.H. Lee et al. / Small Ruminant Research 72 (2007) 101–110

increase of the latter was probably due, to a large extent, to unidentified trans isomers of oleic acid (Hartfoot and Hazlewood, 1988; Byers and Schelling, 1993; Mosley et al., 2002). This illustrates that both total and partial hydrogenation of unsaturated fatty acids takes place in the rumen. The daily intake of dietary lipids from the experimental diets might also influence the fatty acid composition of serum of lambs. Lambs fed control and Vitamin E diets provided 38.0 and 38.5 g of dietary lipids, mainly from corn, during the 10-week feeding period. Conversely, lambs fed the RDPS diet received an average of 103.0 g of dietary lipids per head daily, which was 2.7 times more lipids than the lambs in the other two diets. This is another indication of protecting polyunsaturated fatty acid in the RDPS because feeding lipids to ruminants at levels about 4–5% of the dietary dry matter (DM) may result in depressed intake and digestion of other components of the diet (Hartfoot and Hazlewood, 1988). There were significant increments (P < 0.05) in the weight percentages of all fatty acids in serum lipids of lambs after 2 weeks of feeding across treatments, except for trans-7-hexadeanoic (C16:1n7, trans), palmitoleic (C16:1n7), heptadecanoic (C17:0), and heptadecenoic (C17:1n8) acids (Table 3). However, the percentages of fatty acids in serum lipids of lambs across treatments neither continuously increased nor decreased over the 10-week feeding trial. Palmitic (C16:0) acid content of serum lipids across the treatments decreased (P < 0.05) from 17.7% to 14.5% over the feeding trial. Furthermore, oleic (C18:1n9) acid proportion of serum lipids of lambs decreased (P < 0.05) from 23.8% to 19.7% during the first four 2-week periods, then increased (P < 0.05) to 21.9% on the 10th week. On the other hand, linoleic (C18:2n6) acid levels increased (P < 0.05) from 18.0% to 30.3% during the first 4 weeks of the feeding trial and did not change (P > 0.05) significantly until the 10th week of the trial. The dietary treatments and time on feeding interaction was found (P < 0.05) in the percentages of palmitic (C16:0), palmitoleic (C16:1n7), oleic (C18:1n9), linoleic (C18:2n6), and linoelaidic (C18:2n6, trans) acids in serum lipids of lambs (Fig. 1). Within the feeding time, the RDPS diet decreased (P < 0.05) the content of palmitic (C16:0) acid in serum lipids compared to the control diet with the exception of at the beginning of the feeding time; and this pattern was also found for the concentration of oleic (C18:1n9) acid. The level of linoleic (C18:2n6) acid was increased (P < 0.05) in blood serum of lambs fed the RDPS diet compared with that from lambs fed the other two diets. The protection of dietary lipids of SFO-gel (RDPS) from biohydrogenation in rumen significantly changed

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Fig. 1. Effect of time on feed and the experimental diets on serum fatty acid levels of lambs over a 10-week feeding trial: palmitic (C16:0, S.E.M. = 0.84), palmitoleic (C16:1n7, S.E.M. = 0.18), oleic (C18:1n9, S.E.M. = 1.75), linoleic (C18:2n6, S.E.M. = 1.79), and linoelaidic (C18:2 trans, S.E.M. = 0.09). Points with no common letter are different (P < 0.05).

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Fig. 1. (Continued ).

(P < 0.05) the relative weight concentrations of palmitic (C16:0), oleic (C18:1n9), and linoleic (C18:2n6) acids in muscle lipids of lambs (Fig. 2). The SFO-gel supplement (RDPS diet) caused substantial increases (P < 0.05) in the proportion of linoleic (C18:2n6) acid with decreases (P < 0.05) in the proportion of oleic (C18:1n9) and palmitic (C16:0) acids in the longissimus dorsi and psoas major muscle lipids. The proportion of stearic acid (C18:0) in muscle lipids was not markedly altered

by feeding the SFO-gel supplement (RDPS diet). Cook et al. (1972) reported that fat deposition of sheep was altered by feeding supplements of formaldehyde-treated particles containing safflower oil and casein for a period of 6 weeks immediately prior to slaughter. In that study, the proportion of linoleic (C18:2n6) acid increased from 2–3% to 28–29% in perirenal and subcutaneous fat and there were corresponding decreases in the proportions of palmitic (C16:0), stearic (C18:0), and oleic (C18:1n9) acids. In the present study, the level of linoleic (C18:2n6) acid increased from 4.5% to 10.4% in psoas major muscle lipids and from 7.2% to 13.0% in longissimus dorsi muscle lipids. The diacetyl-treated supplements containing defatted soy flour and sunflower oil might protect linoleic (C18:2n6) acid from ruminal biohydrogenation and readily incorporate into muscle tissues. Correlation coefficients between the fatty acid composition of blood serum lipids from the 10th week and longissimus dorsi as well as psoas major muscles are shown in Table 4. The correlations between the fatty acid composition of the serum lipids and two different types of muscles from lambs fed the Vitamin E diet were not significant (P > 0.05), except the correlation

Table 3 Fatty acid compositiona,b of serum lipids across the experimental diets in lambs Fatty acid (%)

C14:0 C14:1n5 C16:0 C16:1 trans C16:1n7 C17:0 C17:1n8 C18:0 C18:1n9 C18:2n6 C18:2 trans C18:3n3 C18:3n6 C20:0 C20:1n9 C20:2n6 C20:3n6 C20:4n6 C20:5n3 C22:5n3 C22:6n3 C24:0 a b

Feeding period

S.E.M.

0-week

2-week

4-week

6-week

8-week

10-week

0.245 a 0.142 a 17.694 a 1.178 0.588 1.291 0.726 19.954 a,b,c 23.786 a 18.020 d 0.138 b,c 0.654 a,b 0.445 a 0.034 b 0.608 a 0.230 a 0.487 a 4.564 b 1.213 a 1.975 a 5.329 a 0.366 c

0.042 b,c 0.114 a 15.039 b,c 1.000 0.511 1.263 0.510 21.463 a 21.655 a,b 25.817 b 0.326 a 0.303 c 0.895 a 0.048 a 0.433 b 0.226 a 0.587 a 3.698 c,d 0.568 b 1.267 a,b 3.666 b,c 0.179 c

0.065 a,b 0.112 a 14.530 c,d 0.915 0.476 1.195 0.524 18.015 c 20.153 b 30.349 a 0.322 a 0.265 c 1.010 a 0.026 b 0.478 b 0.213 a 0.595 a 4.298 b,c 0.495 b,c 1.188 a,b,c 4.076 b 0.172 c

0.000 c 0.003 b 15.844 b 0.449 0.334 0.983 0.409 18.719 c 19.703 b 32.406 a 0.320 a,b 0.632 b 0.386 b 0.00 c 0.106 c 0.001 b 0.241 c 3.489 d 0.355 c 0.676 e 3.821 b,c 0.993 a,b

0.000 c 0.031 b 14.621 b,c,d 0.468 0.377 0.915 0.469 18.650 b,c 19.754 b 31.950 a 0.058 c 0.725 a,b 0.324 b 0.012 c 0.134 c 0.000 b 0.187 b,c 4.678 b 0.427 b,c 0.933 b,c,d 4.260 b 0.740 b

0.000c 0.000b 14.541 b,c,d 0.779 0.563 0.989 0.599 20.691 a,b 21.930 a,b 29.768 a 0.132 c 0.848 a 0.398 b 0.002 c 0.165 c 0.000 b 0.416 a,b 3.619 c,d 0.338 c 0.699 d,e 2.773 c 0.420 c

Wt.% of fatty acid methyl esters. Least-square means in a row with a common letter are not significant at the 5% level.

0.046 0.026 0.493 0.101 0.072 0.145 0.064 0.984 1.026 1.066 0.056 0.083 0.098 0.011 0.044 0.022 0.096 0.259 0.066 0.097 0.370 0.097

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Table 4 Correlation coefficients of percentages of fatty acids in blood seruma with longissimus dorsi and psoas major muscles in lambs fed with Vitamin E supplemented dietsb Fatty acid

C16:0 C16:1n7 trans C16:1n7 C17:0 C17:1n8 C18:0 C18:1n9 C18:2n6 C18:3n3 C20:1n9 C20:3n6 C20:4n6 C20:5n3 C22:5n3 C24:0

Longissimus dorsi

Psoas major

Vitamin E

RDPS

Vitamin E

RDPS

0.33 −0.29 −0.20 −0.04 0.19 0.26 −0.02 −0.00 0.49 0.45 0.29 −0.28

0.59 −0.21 −0.21 −0.41 0.54 0.23 0.88* 0.83* −0.43 −0.08 −0.46 0.60 −0.27

0.53 0.42 0.04 0.98 0.61 −0.16 −0.37 −0.20 −0.49 −0.60 0.95* 0.93 −0.33 0.91 0.37

0.65 0.33 −0.98 −0.43 −0.96 −0.39 0.77 0.94 0.57 −1.00*

−0.60

0.36

−0.71 0.93 −0.99*

a

Fig. 2. Percentages of three major fatty acids, palmitic (C16:0), oleic (C18:1n9), and linoleic (C18:2n6) acids, in longissimus dorsi and psoas major muscles of lambs fed different diets. For any one acid, bars bearing unlike letters are different (P < 0.05).

of cis-8,11,14-eicosadienoic (C20:3n6) acid. The C20:3n6 in serum lipids of lambs fed the Vitamin E diet was positively correlated (P < 0.05) to that in psoas major muscles. There was no significant correlation between the predominant fatty acids, palmitic (C16:0), stearic (C18:0), oleic (C18:1n9), and linoleic (C18:2n6), of serum lipids and two different muscles from lambs fed the Vitamin E diet. Oleic (C18:1n9) and linoleic (C18:2n6) acids in serum lipids of lambs fed the RDPS was positively correlated (P < 0.05) to those in longissimus dorsi muscles. Dryden and Marchello (1973) reported that diets with sunflower oil increased the level of unsaturated fatty acids in longissimus muscle tissues of steers, whereas animal fat supplementation of diet increased the saturation of intermuscular fat. That study also reported that changes in dietary lipids in steer diets were related to changes in the free fatty acid and glyceride proportions of the serum lipids. In the present study, there was a positive correlation between linoleic (C18:2n6) acid levels in serum lipids and longissimus dorsi muscle lipids of the lambs on the RDPS diet; therefore, increasing serum level of this fatty acid increased the

Blood serum of lambs fed Vitamin E supplemented diets (Vitamin E and RDPS diets) at the 10th week of the feeding trial rail. b Vitamin E diet supplemented ␣-tocopheryl acetate (250 IU/day); RDPS diet supplemented sunflower oil gel (500 IU of ␣-tocopheryl acetate/kg). * P < 0.05.

level of linoleic (C18:2n6) acid in the longissimus dorsi muscle. 3.3. α-Tocopherol in blood serum and muscle tissues Effect of feeding Vitamin E in dietary fat supplements to lambs on blood serum ␣-tocopherol concentrations is presented in Table 5. Dietary Vitamin E supplements (Vitamin E and RDPS diets) increased (P < 0.05) serum ␣-tocopherol concentrations compared with the control diet. Increased levels of ␣-tocopherol were expected Table 5 ␣-Tocopherol concentrationsa in blood serum and muscles tissues from lambs fed different diets Component

Dietb

S.E.

Control

Vitamin E

RDPS

Blood serum (␮g/mL)

0.27 c

1.64 b

2.11 a

0.12

Muscle (␮g/g) Longissimus dorsi Psoas major

1.46 b 2.16 c

3.20 a 6.02 b

4.21 a 7.43 a

0.36 0.31

a

Least-square means in a row with a common letter are not significant at the 5% level. b Control: no supplemental Vitamin E; Vitamin E: supplemented ␣tocopheryl acetate; RDPS: diacetyl-treated protein–lipid supplement.

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Fig. 3. Effect of time on feed and the experimental diets on lamb serum ␣-tocopherol contents over a 10-weeks feeding trial (S.E.M. = 0.26). Points with no common letter are different (P < 0.05).

in the serum from lambs fed Vitamin E diet, but the increment was not over that of lambs fed the RDPS diet. Levels of serum ␣-tocopherol in lambs fed the RDPS were 28.7% higher than those fed the Vitamin E diet, even though Vitamin E and RDPS diets provided equal amounts of Vitamin E (250 IU/day) to each lamb. This suggests that RDPS enhanced the absorption of ␣tocopheryl acetate in the small intestine of ruminants. Extending time on feed across experimental diets significantly affected (P < 0.05) serum ␣-tocopherol levels in lambs (Fig. 3). Application of ␣-tocopheryl acetate increased (P < 0.05) serum ␣-tocopherol content continuously in lambs fed Vitamin E supplemented diets (Fig. 3), with some deviation during the 10-week feeding trial. However, serum ␣-tocopherol level was almost constant in lambs fed the control diet. Feeding of ␣tocopheryl acetate supplement and time on feed significantly influenced (P < 0.05) the serum levels of ␣tocopherol in lambs fed the Vitamin E supplemented (Vitamin E and RDPS diets) diets (Fig. 3). After the first 2 weeks, the Vitamin E supplemented diets had serum ␣-tocopherol concentrations approximately twofold (Vitamin E diet) or five-fold (RDPS diet) higher than control lambs (0.31 ␮g/mL). Lambs fed the RDPS diet had the highest concentration of ␣-tocopherol in serum at 2 weeks. This result may be due to the emulsified ␣-tocopheryl acetate in the RDPS, enhancing the absorption of Vitamin E in the small intestine; moreover, time on feed also affected the increment of serum ␣-tocopherol. There was no further increase of serum ␣-tocopherol levels in lambs fed the Vitamin E supplemented diets until week 6. This pattern might indicate that a saturation level of serum ␣-tocopherol was reached after 2 weeks of Vitamin E supplementation. It also suggests that after blood was saturated with ␣-tocopherol at week 2, tissue deposits of ␣-tocopherol began. The

trend for elevation of plasma ␣-tocopherol and saturation in the levels of dietary Vitamin E supplementation has been reported previously (Arnold et al., 1993; O’Grady et al., 2001). However, further increase of serum ␣tocopherol in lambs fed the Vitamin E supplemented diets was shown in the present study. The Vitamin E supplemented diets significantly increased (P < 0.05) the serum content of ␣-tocopherol in lambs after week 8 (RDPS) or 10 (Vitamin E) in the feeding trial (Fig. 3). Lambs fed the RDPS had 25.8% higher levels of serum ␣-tocopherol than those fed TA diet at week 10. Based on these findings, ␣-tocopheryl acetate emulsified in the sunflower oil and protein matrix was more effective in raising the circulating serum levels of ␣-tocopherol than mixing ␣-tocopheryl acetate with molasses. In general, serum levels of ␣-tocopherol are a reliable indicator of nutritional Vitamin E status, which are correlated with intake of Vitamin E (Njeru et al., 1994; McDowell et al., 1996). The ␣-tocopherol concentrations in muscles of lambs fed the control, Vitamin E, or RDPS diet are reported in Table 5. Lambs supplemented with Vitamin E (Vitamin E and RDPS diets) had higher (P < 0.05) levels of ␣-tocopherol in all muscle samples than did lambs not supplemented (control diet). Feeding lambs with the RDPS further increased (P < 0.05) ␣-tocopherol concentration in longissimus dorsi (LD) muscle compared with those of lambs fed the Vitamin E diet but no significant differences (P > 0.05) were found in ␣-tocopherol concentrations of LD muscle from lambs fed two supplemented Vitamin E diets. The lambs fed the RDPS had higher (P = 0.07) levels of ␣-tocopherol in LD muscles than did lambs fed the Vitamin E diet. Meat containing ␣-tocopherol concentrations of less than 3.0 ␮g/g has shorter case life in terms of reduced metmyoglobin accumulation or lipid oxidation; however, scientists have shown that case life of beef retail products can be improved if cattle achieve muscle concentration of Vitamin E in excess of 3–4 ␮g/g muscle tissue (Smith et al., 1996; Roeber et al., 2001; Turner et al., 2002). In the present study, supplementation of Vitamin E (Vitamin E and RDPS diets) achieved critical concentration of ␣-tocopherol in the longissimus dorsi and psoas major muscle tissues of lambs. 3.4. Correlation between fatty acid composition and α-tocopherol in blood serum lipids The correlation coefficients between serum ␣tocopherol content and fatty acid percentages of serum lipids in lambs across the feeding trial are presented in Table 6. There was a positive correlation (P < 0.05)

J.H. Lee et al. / Small Ruminant Research 72 (2007) 101–110 Table 6 Correlation coefficients of percentages of fatty acids with Vitamin E levels in blood seruma from lambs fed with Vitamin E supplemented dietsb Fatty acid

C14:0 C14:1 C16:0 C16:1 trans C16:1n7 C17:0 C17:1n8 C18:0 C18:1n9 C18:2n6 C18:2 trans C18:3n3 C18:3n6 C20:0 C20:1n9 C20:2n6 C20:3n6 C20:4n6 C20:5n3 C22:5n3 C22:6n3 C24:0 C24:1

Supplemented Vitamin E diet Vitamin E

RDPS

−0.42* −0.27 0.14 −0.33 −0.27 0.25 0.32 0.13 −0.02 0.40 0.10 0.14 0.12 −0.26 −0.36 −0.28 −0.19 −0.50* −0.56* −0.75* −0.37 −0.33 −0.16

−0.22 −0.36 −0.74* −0.29 −0.57* −0.37 −0.65* 0.14 −0.71* 0.63* 0.05 −0.07 −0.03 −0.36 −0.75* −0.53* −0.13 0.06 −0.72* −0.36 −0.76* −0.02 0.41

a

Blood serum of lambs fed Vitamin E supplemented diets (Vitamin E and RDPS diets) across the 10-week feeding trial. b Vitamin E diet supplemented ␣-tocopheryl acetate (250 IU/day); RDPS diet supplemented sunflower oil gel (500 IU of ␣-tocopheryl acetate/kg). * P < 0.05.

between the ␣-tocopherol content and linoleic (C18:2n6) acid proportion of serum lipids in lambs fed the RDPS diet, but the proportion of major fatty acids, such as palmitic (C16:0) and oleic (C18:1n9) acids, were negatively correlated (P < 0.05) to ␣-tocopherol content of blood serum in lambs fed the RDPS diet. There was no correlation (P > 0.05) between the ␣-tocopherol content and palmitic (C16:0) and oleic (C18:1n9) acids in serum lipids from lambs fed the Vitamin E diet (Table 6). This relation was expected, because feeding ␣-tocopheryl acetate alone (Vitamin E diet) increased (P < 0.05) the content level of ␣-tocopherol in serum over that of lambs fed the control diet (Table 5), while not greatly affecting the concentrations of palmitic (C16:0) and oleic (C18:1n9) acids in the blood serum lipids (Table 2). According to Combs (1991) and other scientists (Hidiroglou et al., 1992; Roquet et al., 1992), rumen function facilitates emulsification of fat-soluble vitamins, and Vitamin E becomes dissolved in the emul-

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sion upon entering the duodenum. Chylomicrons are broken down in the small intestine, and this process depends on the release of lipase enzymes and bile from the gall bladder. The products of the hydrolysis of these chylomicrons form small particles called micelles. The hydrophobic region of these micelles attracts non-polar substances such as cholesterol and Vitamin E. Micelles serve as a carrier for Vitamin E to enhance the absorption of Vitamin E in the small intestine. Since linoleic (C18:2n6) acid made up 71.4% of the total lipids in the RDPS diet, it might be considered a major carrier for Vitamin E in the diet. 4. Conclusion The implications of this research are that a rumenprotected dietary protein–lipid supplement made with a safe chemical agent and containing polyunsaturated fat and Vitamin E supplement can change the fatty acid composition of serum and meat lipids when fed to lambs. This supplement can also provide additional antioxidant activity to increased levels of polyunsaturated fatty acids in the serum and muscle. In addition, significant relationships in the major fatty acid levels in the serum and in muscle lipids in lambs fed the rumen-protected dietary protein–lipid supplement might indicate that serum fatty acid levels could predict muscle fatty acid levels. Feeding lambs the rumen-protected dietary protein–lipid supplement containing sunflower oil and ␣-tocopheryl acetate increased linoleic acid content of blood serum and meat lipids with increasing levels of Vitamin E as an antioxidant. Therefore, this supplement made with a generally recognized as safe agent may have a significant impact in increasing polyunsaturated fat intake from animal products for consumers in the United States. References AOCS, 1993. Official Methods and Recommended Practices of the American Oil Chemists’ Society, 4 ed. American Oil Chemists’ Society, Champaign, IL. Arnold, R.N., Arp, S.C., Scheller, K.K., Williams, S.N., Schaefer, D.M., 1993. Tissue equilibration and subcellular distribution of Vitamin E relative to myoglobin and lipid oxidation in displayed beef. J. Anim. Sci. (71), 105–118. Bright, J.M., Sullivan, P.S., Melton, S.L., Schneider, J.F., McDonald, T.P., 1994. The effects of n − 3 fatty acid supplementation on bleeding time, plasma fatty acid composition, and in vitro platelet aggregation in cats. J. Vet. Intern. Med. 8 (4), 247–252. Byers, F.M., Schelling, G.T., 1993. Lipids in ruminant nutrition. In: Church, D. (Ed.), The Ruminant Animal Digestive Physiology and Nutrition. Prentice Hall, New Jersey, pp. 298–322. Combs, G.F., 1991. Mechanism of absorption, transport and tissue uptake of Vitamin E. In: Coelho, M.B. (Ed.), Vitamin

110

J.H. Lee et al. / Small Ruminant Research 72 (2007) 101–110

E in Animal Nutrition and Management. BASF Co., NJ, pp. 11–18. Cook, L.J., Scott, T.W., Faichney, G.J., 1972. Fatty acid interrelationships in plasma, liver, muscle and adipose tissue of cattle fed safflower oil protected from ruminal hydrogenation. Lipids (7), 83–89. Dje, Y., 1994. The effect of feeding protected dietary lipid supplements on fatty acids composition of the blood serum and the milk fats of lactating ewes. Ph.D. dissertation, University of Tennessee, Knoxville. Dryden, F.D., Marchello, J.A., 1973. Influence of dietary fats upon carcass lipid composition in the bovine. J. Anim. Sci. (37), 33–39. Focant, M., Mignolet, E., Marique, M., Clabots, F., Breyne, T., Dalemans, D., Larondelle, Y., 1998. The effect of Vitamin E supplementation of cow diets containing rapeseed and linseed on the prevention of milk fat oxidation. J. Dairy Sci. (81), 1095– 1101. Granti, S., Angel, S., Akiri, B., Holzer, Z., Aharoni, Y., Orlov, A., Kanner, J., 2005. Effects of Vitamin E supplementation on lipid peroxidation and color retention of salted calf muscle from a diet rich in polyunsaturated fatty acids. J. Agric. Food Chem. (49), 5951–5956. Gulati, S.K., Byers, E.B., Byers, Y.G., Ashes, J.R., Scott, T.W., 1997a. Effect of feeding different fat supplements on the fatty acid composition of goat milk. Anim. Feed Sci. Technol. (66), 159–164. Gulati, S.K., Scott, T.W., Ashes, J.R., 1997b. In-vitro assessment of fat supplements for ruminants. Anim. Feed Sci. Technol. (64), 127–132. Hartfoot, C.G., Hazlewood, G.P., 1988. Lipid metabolism in rumen. In: Hobson, P.N. (Ed.), The Rumen Microbial Ecosystem. Elsevier Publishing Co., New York, pp. 285–322. Hidiroglou, N., McDowell, L.R., Papas, A.M., Antapli, M., Wilkinson, N.S., 1992. Bioavailability of Vitamin E compounds in lambs. J. Anim. Sci. (70), 2556–2561. Hidiroglou, M., Batra, T.R., Ivan, M., 1995. Effects of supplement Vitamins E and C on the immune responses of calves. J. Dairy Sci. (78), 1578–1583. Lauzurica, S., Fuente, J., Diaz, M.T., Alvarez, I., Perez, C., Caneque, V., 2005. Effect of dietary supplementation of Vitamin E on characteristics of lamb meat packed under modified atmosphere. Meat Sci. (70), 639–646. Lee, J.H., Waller, J.C., Melton, S.L., Saxton, A.M., Pordesimo, L.O., 2004. Feeding encapsulated ground full-fat soybeans to increase polyunsaturated fat concentrations and effects on flavor volatiles in fresh lamb. J. Anim. Sci. (82), 2734–2741. Liu, Q., Scheller, K.K., Schaefer, D.M., 1996. Technical note: a simplified procedure for Vitamin E determination in beef muscle. J. Anim. Sci. (74), 2406–2410. Machlin, L.J., 1991. Handbook of Vitamin, 2nd ed. Marcel Dekker, New York, p. 595. McDowell, L.R., Williams, S.N., Hidiroglou, D., Njeru, C.A., Hill, G.M., Ochoa, L., Wilkinson, N.S., 1996. Vitamin E supplemen-

tation for the ruminant. Anim. Feed Sci. Technol. (60), 273– 296. Melton, S.L., Moyers, R.E., Playford, C.G., 1979. Lipid extracted from soy products by different procedures. J. Am. Oil Chem. Soc. (56), 489–497. Mitsumoto, M., Arnold, R.D., Schaefer, D.M., Cassens, R.G., 1993. Dietary versus postmortem supplementation of Vitamin E on pigment and lipid stability in ground beef. J Anim. Sci. (71), 1812–1816. Mosley, E.E., Powell, G.L., Riley, M.B., Jenkins, T.C., 2002. Microbial biohydrogenation of oleic acid to trans isomers in vitro. J. Lipid Res. 43 (2), 290–296. Njeru, C.A., McDowell, L.R., Wilkinson, N.S., Williams, S.N., 1994. Assessment of Vitamin E nutritional status in sheep. J. Anim. Sci. (72), 3207–3212. O’Grady, M.N., Monahan, F.J., Fallon, R.J., Allen, P., 2001. Effects of dietary supplementation with Vitamin E and organic selenium on the oxidative stability of beef. J. Anim. Sci. (79), 2827–2834. Ochoa, L., McDowell, L.R., Williams, S.N., Wilkinson, N., Boucher, J., 1992. ␣-Tocopehrol concentrations in serum and tissues of sheep fed different sources of Vitamin E. J. Anim. Sci. (70), 2568–2573. Putnam, J., 2000. Major trends in U.S. food supply, 1909–1999. Food Rev. (23), 8–15. Roeber, D.L., Belk, K.E., Tatum, J.D., Wilson, J.W., Smith, G.C., 2001. Effects of three levels of ␣-tocopheryl acetate supplementation to feedlot cattle on performance of beef cuts during retail display. J. Anim. Sci. (79), 1814–1820. Roquet, J., Nockels, C.F., Papas, A.M., 1992. Cattle blood plasma and red blood cell ␣-tocopherol levels in response to different chemical forms and routes of administration of Vitamin E. J. Anim. Sci. (70), 2542–2550. Schelling, G.T., Roeder, R.A., Garber, M.J., Pumfrey, W.M., 1995. Bioavailability and interaction of Vitamin A and Vitamin E in ruminants. J. Nutr. (125), 1799S–17803S. Smith, G.C., Morgan, J.C., Sofos, J.N., Tatum, J.D., 1996. Supplemental Vitamin E in beef cattle diets to improve shelf-life of beef. Anim. Feed Sci. Technol. (59), 207–214. Steel, R.G.D., Torrie, J.H., Dickey, D.A., 1996. Principles and Procedures of Statistics: A Biometrical Approach, 3rd ed. McGraw–Hill Book Co., New York, p. 672. Turner, K.E., McClure, K.E., Weiss, W.P., Borton, R.J., Foster, J.G., 2002. Alpha-tocopherol concentrations and case life of lamb muscle as influenced by concentrate or pasture finishing. J. Anim. Sci. (80), 2513–2521. Vanderveen, J.E., 1996. Dietary recommendations for lipids and measures designed to facilitate implementation. In: McDonald, R.E., Min, D.B. (Eds.), Food Lipids and Health. Marcel Dekker, New York, pp. 1–18. Weiss, W.P., 1998. Requirements of fat-soluble vitamins for dairy cows: a review. J. Dairy Sci. (81), 2493–2501. Weiss, W.P., Wyatt, D.J., 2003. Effect of dietary fat and Vitamin E on ␣tocopherol in milk from dairy cows. J. Dairy Sci. (86), 3582–3591.