Effects of different fat mixtures on milk fatty acid composition and oxidative stability of milk fat

Animal Feed Science and Technology 185 (2013) 35–42

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Effects of different fat mixtures on milk fatty acid composition and oxidative stability of milk fat Xiaowei Zhao a,b , Jiaqi Wang a,∗ , Yongxin Yang b , Dengpan Bu a,∗ , Hai Cui a , Yan Sun a , Xiaoyan Xu a , Lingyun Zhou a a State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China b Institute of Animal Science and Veterinary Medicine, Anhui Academy of Agricultural Sciences, Hefei 230031, PR China

a r t i c l e

i n f o

Article history: Received 6 March 2012 Received in revised form 13 June 2013 Accepted 22 June 2013

Keywords: Fatty acids mixture Milk fatty acids composition Oxidation changes Milk fat

a b s t r a c t The experiment was carried out to determine effects of different fatty acids (FA) mixtures on milk FA composition and milk fat oxidation change. Thirty-six dairy cows were used in a completed design experiment for 8 weeks. Cows were fed one of three lipid supplements (1) short- and medium-chain fatty acids (SMCFA), (2) butterfat, and (3) long-chain fatty acids (LCFA). Data were analyzed using MIXED procedure of SAS. Daily dry matter intake (DMI), milk yield, and milk protein, total solids (TS), lactose, free fatty acids (FFA) content were unaffected among 3 treatments, whereas milk fat concentration was decreased (P<0.05) as carbon chain length increased in supplement lipid. Relative to SMCFA, 12:0, 14:0 and 16:0 concentrations in milk fat from cows on butterfat and LCFA treatment were decreased (P<0.05). Proportions of cis9-18:1, trans9-C18:1, and cis9, trans11-conjugated linoleic acid (CLA) were increased by 15.5, 74.3, and 20.3% (P<0.05) in milk fat from cows on LCFA compared with cows on SMCFA. Similar increases in trans9-18:1, trans11-18:1, cis9, trans11-CLA, 18:2n-6 and 18:3n-3 were 34.2, 31.5, 21.4, 11.0, and 15.4% (P<0.05) in butterfat treatments, respectively. Furthermore, activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were decreased (P<0.05) when increased longchain FA in diets, while concentration of malondialdehyde (MDA) was increased (P<0.01) in bovine milk. Our results indicated that long-chain FA exhibit positive effects on milk FA composition, but may decrease oxidative stability of milk fat. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Milk fat is an important component of milk, and accounts for many of the physical properties, manufacturing characteristics and organoleptic qualities of milk and dairy products (Jensen et al., 1991; German et al., 1997). Common bovine milk fat contains on average 70% saturated fatty acids (SFA), 25% monounsaturated fatty acids (MUFA) and 5% polyunsaturated fatty

Abbreviations: ADF, acid detergent fiber; CAT, catalase; CLA, conjugated linoleic acid; CP, crude protein; DIM, days in milk; DM, dry matter; DMI, dry matter intake; FA, fatty acids; FFA, free fatty acids; GSH-Px, glutathione peroxidase; INT, 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phe-nyltetrazolium chloride; LCFA, long-chain fatty acids; MDA, malondialdehyde; MUFA, monounsaturated fatty acids; NDF, neutral detergent fiber; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; SMCFA, short- and medium-chain fatty acids; SOD, superoxide dismutase; SOF, spontaneous oxidized off-flavor; T-AOC, total antioxidant capacity; TMR, total mixed ration; TS, total solids; UFA, unsaturated fatty acids. ∗ Corresponding authors at: State Key Laboratory of Animal Nutrition, Ruminant Nutrition Lab, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China. Tel.: +86 10 6289 0458; fax: +86 10 6289 7587. E-mail addresses: [email protected] (J. Wang), [email protected] (D. Bu). 0377-8401/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anifeedsci.2013.06.009

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acids (PUFA) (Grummer, 1991). However, intake of dietary lipids by humans with a high ratio of SFA to unsaturated fatty acids (UFA) was associated with an increased risk for atherosclerosis and cardiovascular disease (Kromhout et al., 2002). Due to the potential harmful effects of SFA, many measures have been applied to improve fatty acids (FA) composition, especially enhance long-chain UFA content in milk (Bu et al., 2007; Relling and Reynold, 2007; Khas-Erdene et al., 2010). However, previous research demonstrated that the susceptibility of milk fat to develop spontaneous oxidized flavor (SOF) increases with elevated concentration of long-chain unsaturated FA in milk (Timmons et al., 2001; Huang et al., 2004). Oxidative process in milk fat, resulting in SOF, received much attention only in limited countries. Including China, many countries failed to understand and solve this phenomenon of SOF, primarily due to lack of related knowledge of the process and failure to monitor the milk. Oxidation of milk fat largely results from the presence of pro-oxidants and anti-oxidation system and their substrate which is UFA. The PUFA in milk are considered to make it more prone to oxidation, which results in shorter shelf life of dairy products and a reduction in consumer acceptance of these products. There are also numerous studies that showed a relationship among pro-oxidants (Timmons et al., 2001; Juhlin et al., 2010), UFA (Timmons et al., 2001; Kristensen et al., 2004), and antioxidants (Granelli et al., 1998; Al-Mabruk et al., 2004) have a strong relationship with oxidative stability of milk fat. However, little research evaluated the relationship with enzymatic radical scavenging systems, UFA, and oxidative stability of milk fat. Due to the lack of studies that evaluated relationship with antioxidant enzyme, UFA, and oxidative stability of milk fat, the main objectives of this paper were to confirm effect of different FA mixtures on milk FA composition and to assess effect of milk FA composition and antioxidant enzyme on oxidative stability of milk fat. We hypothesis that cows supplemented with lipids containing high ratio of long-chain FA would improve milk FA profile (more UFA and less SFA), but would increase incidence of SOF of milk fat. 2. Materials and methods All animal care and procedures were approved and conducted under established standards of Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China. 2.1. Animals, experimental design and diets Thirty-six multiparous Chinese Holstein dairy cows (183 ± 40 days in milk, DIM), in mid to late lactation were randomly assigned to three treatments, balanced for milk yield, DIM and parity, and twelve cows per treatment. Cows fed corn silage based total mixed ration (TMR) were supplemented with 1 of 3 dietary lipids supplements (400 g/d). Ingredients and chemical composition of TMR diets are given in Table 1. Dietary treatments were formulated to meet China NY/t34 (2004) nutrient requirements and consisted of FA mixture supplements containing mixtures of different types of chain length FA: (1) shortand medium-chain FA (SMCFA) (6: 0–6.0%, 8: 0–4.0%, 10: 0–9.0%, 12: 0–10.0%, 14: 0–32%, and 16: 0–39%), (2) butterfat, and (3) long-chain FA (LCFA) (59% cocoa butter, 16% olive oil, and 25% palm oil). 2.2. Measurements and sample collection Experimental periods were conducted for 8 weeks. Initial 4 weeks acted as preliminary period for unpleasant scent of short-chain fatty acids (such as caproic acid and caprylic acid), and some cows in SMCFA treatment refused to eat. Therefore, first 4 weeks allowed cows adapt to diets. Cows were milked three times at 08:00 h, 14:00 h, and 20:00 h and fed individually equally sized portions at 08:30 h, 14:30 h, and 20:30 h. Following milking, cows were offered their diets ad libitum. FA mixtures supplemented for each cow were mixed with a smaller amount of TMR at 08:30 h, when cows finished the supplement lipid mixtures, then given remaining TMR. Samples of TMR and orts daily were recorded for individual cows to determine dry matter intake (DMI) on the last 4 weeks. Orts were restricted to 10% of intake on an as-fed basis. Milk samples were collected from the 3 times daily milking (1 day) of each week. Milk sample from each cow was composited according to milk yield. An aliquot of composite sample was stored at 4 ◦ C, and was determined by near mid infrared procedures using a Foss-Milkoscan TM Minor (MilkoScan FT120, Foss Electric A/S, Hillerod, Denmark) for milk composition. A second set of milk samples from individual cows was stored at −20 ◦ C for further analysis of FA profile and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and concentration of malondialdehyde (MDA). 2.3. Feed assay TMR and orts were dried in 65 ◦ C forced-air oven for 72 h. All samples were ground with a 1-mm screen mill (SM100, Retsch, Haan, Germany). Orts were analyzed in duplicate for dry matter (DM) (AOAC, 1990; method no. 930.15). Diet ingredients were analyzed in duplicate for DM, neutral detergent fiber (NDF), acid detergent fiber (ADF) (AOAC, 1990; method no. 973.18), and crude protein (CP) (AOAC, 1990; method no. 948.13). NDF was determined according to Van Soest et al. (1991) using heat-stable ␣-amylase without the use of sodium sulfite and sodium sulfite.

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Table 1 Ingredients and chemical composition of the total mixed ration (TMR) diet. g/kg of DM Ingredient Alfalfa hay Chinese wildrye Corn silage Dry distillers grains Oligose soybean hull Corn grain, ground Wheat bran Soybean meal Cottonseed meal Rapeseed meal Sunflower Seeds meal Yeast Sodium bicarbonate Calcium bicarbonate Limestone Sodium chloride Calcium carbonate precipitated light Magnesium oxide Mineral-vitamin premixa Chemical composition,b % NEL (MJ/kg DM) DM CP NDF ADF EE Ca P

107.90 107.90 217.70 55.20 12.00 233.40 52.90 73.40 16.80 16.80 12.00 52.90 10.80 4.80 8.40 6.00 4.80 1.20 4.80 6.20 50.00 16.07 39.78 22.39 3.52 0.90 0.42

a Mineral-vitamin mix combined (per kg of mix, DM) 400 mg of Fe, 540 mg of Cu, 2100 mg of Zn, 560 mg of Mn, 15 mg of Se, 35 mg of I, 68 mg of Co, 250,000 IU of Vitamin A, 65,000 IU of Vitamin D, and 2100 IU of Vitamin E. b NEL = Calculated value; DM = Dry matter; CP = Crude protein; NDF = Neutral detergent fiber; ADF = Acid detergent fiber; EE = Ether extract; Ca = Calcium; P = Phosphorus.

2.4. Milk fatty acid assay For milk FA analysis, frozen milk samples from each cow were thawed in a 4 ◦ C refrigerator and centrifuged at 17,800×g for 20 min at 8 ◦ C to separate fat. Fat cake (1 g) was transferred to a 5 ml tube, and 20 mg of fat was esterified using method described by Kramer et al. (1997). Separation of fatty acid was achieved by gas chromatography (Model 6890 Series II, Agilent Technologies, Hewlett Packard CO, Santa Clara, USA) fitted with a flame-ionization detector. Samples containing methyl esters in hexane (1 ␮l) were injected through the split injection port (100:1) onto an SP-2560 fused silica 100 m×0.25 mm column with a 0.20 um film (Supelco Inc., Bellefonte, PA). Oven temperature was initially 170 ◦ C for 30 min, and was then increased to 200 ◦ C at 1.5 ◦ /min, and held at that temperature for 20 min. Temperature was then increased at 5 ◦ /min to 220 ◦ C and held for 20 min. Injector and detector temperatures were maintained at 240 ◦ C, and total run time was 94 min. Heptadecanoic acid was used as a qualitative internal standard. Each peak was identified using known standards of FA and FA methyl esters. Percentage of each FA was calculated by dividing area under FA peak (minus peak area for heptadecanoic acid) by sum of the peak areas for all reported FA, and values were presented as grams per kilogram of total fatty acid methyl esters.

2.5. SOD, GSH-Px, and CAT assay For milk SOD, GSH-Px, and CAT activity assays, milk samples were thawed and centrifuged for 30 min at 13,700×g at 4 ◦ C. Supernatant was collected, used to precipitate casein in milk with 4% acetic acid, and then centrifuged at 13,700×g at 4 ◦ C for 30 min. Then supernatant was collected for level of enzyme activity determinations. All samples and reagents were maintained at 4 ◦ C throughout preparatory steps until analysis for above-mentioned indexes. All kits for determining SOD, GSH-Px, and CAT activities in whey were bought from Nanjing Jiancheng Bioengineer Institute, China. Measurement of milk SOD enzyme activity was based on generation of superoxide radicals produced by xanthine and xanthine oxidase, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a red formazan dye (McCord and Fridovich, 1969). CAT activity was measured according to ammonium molybdate spectrophotometric method, as based on ammonium molybdate could rapidly terminate H2 O2 degradation reaction catalyzed by CAT and reacted the residual H2 O2 to generate a yellow complex which could be monitored by absorbance at 405 nm

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Table 2 Fatty acid composition (g/kg total fatty acids) of three kinds of fatty acids mixture. Fatty acida

4:0 6:0 8:0 10:0 12:0 14:0 14:1 16:0 16:1 17:0 18:0 cis9-C18:1 trans11-C18:1 CLA 18:2n-6 18:3n-3 20:0 others a b

Treatmentb SMCFA

Butterfat

LCFA

0 65.38 40.11 90.73 106.04 307.15 0 386.63 0 0 0 0 0 0 0 0 0 3.96

42.84 24.13 13.93 33.31 37.83 110.94 15.32 286.73 12.85 5.75 107.45 207.73 33.24 17.04 25.15 8.74 2.54 13.65

0 0 0 0 0 0 0 289.09 2.24 1.83 227.34 394.84 0.44 1.42 60.93 3.34 4.64 13.75

CLA = Conjugated linoleic acids. SMCFA = Short- and medium chain fatty acids; LCFA = Long chain fatty acids.

(Goth, 1991). The GSH-Px activity was measured quantifying rate of H2 O2 -induced oxidation of GSH to oxidized glutathione, catalyzed by GPH-Px (Paglia and Valentine, 1967). Values were all presented as U/ml. 2.6. Lipid peroxidation (MDA) assay Lipid peroxidation (as MDA) level in milk were measured based on reaction between malondialdehyde and thiobarbituric acid by method of Conti et al. (1991), which could be monitored by absorbance at 532 nm, and values were presented as U/ml. 2.7. Statistical analysis The data for feed intake, lactation performance, milk FA composition and oxidation stability of milk fat were analyzed as a completely randomized design with repeated measures using PROC MIXED of SAS 9.0 (SAS Institute, Cary, NC). The variation in model is dietary treatment, and cow was designated as a random effect in model. Least squares means were calculated and are presented with their standard errors throughout. Significance was declared at P<0.05. 3. Results 3.1. Feed intake and lactation performance FA profile of different lipid mixtures are presented in Table 2. Cattle supplemental lipid mixture in SMCFA treatment provided primarily C<16 FA and palmitic acid, LCFA treatment mainly provided long chain FA (C≥16), and butterfat contained C<16 FA, in addition to palmitic acid and C>16 LCFA. Average daily DMI, milk yield, and milk profile responses to different lipid mixtures are presented in Table 3. No changes were observed with DMI, milk yield, and milk protein, total solids (TS), lactose and free fatty acids (FFA) concentration in bovine milk between three treatments. However, milk fat concentration was decreased (P<0.05) in bovine milk with lipid mixture as carbon chain length and degree of unsaturation increased. 3.2. Fatty acid composition Simple statistics describing milk fatty acid composition variables are shown in Table 4. Total fatty acid content of C>16 was increased (P<0.01) and C≤16:0 was deceased (P<0.01) in milk fat with proportion of average carbon chain length and degree of unsaturation increased in lipid supplements. This increase (P0.05) was predominantly due to greater proportion of 18:0, cis9-C18:1, trans9-18:1, trans11-18:1, cis9, trans11-conjugated linoleic acid (CLA), 18:2n-6 and 18:3n-3. Similarly decrease (P<0.01) was predominantly due to greater proportion of 12:0, 14:0, and 16:0. Furthermore, increasing long-chain FA in diets increased total MUFA (P<0.05) and decreased total SFA content in milk fat (P<0.05). Butterfat and LCFA treatments tended to increase total PUFA content in milk fat, and the PUFA concentration in butterfat treatment were higher than others (P=0.054).

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Table 3 Performance of cows fed different fatty acids mixtures. Item1

DMI, kg/d Milk, kg/d Milk composition, g/kg Fat Protein TS Lactose FFA

Treatment2

SEM

P-value

SMCFA

Butterfat

16.92 22.19

16.46 23.61

LCFA 16.74 24.27

0.17 1.18

0.18 0.45

45.69a 35.44 137.21 46.10 51.44

42.30ab 33.58 132.21 46.34 51.73

39.37b 33.00 130.51 47.68 54.36

1.44 1.01 2.34 0.62 2.78

0.02 0.21 0.10 0.18 0.73

a, b

Means within a row with different superscripts differ (P<0.05). DMI = Dry matter intake; TS = Total solid; FFA = Free fatty acid. 2 SMCFA = Short- and medium chain fatty acids; LCFA = Long chain fatty acids. 1

Table 4 Fatty acids profile (g/kg total fatty acids) in milk for cows fed different fatty acids mixtures. Fatty acid1

4:0 6:0 8:0 10:0 12:0 14:0 14:1 15:0 16:0 16:1 18:0 cis9-18:1 trans11-18:1 trans9-18:1 cis9, trans11-CLA 18:2n-6 18:3n-3 20:0 22:0 Other Summation ≤C16:0 >C16:0 SFA MUFA PUFA

Treatment2

SEM

P-value

SMCFA

Butterfat

LCFA

39.03 23.13 13.23 28.18 36.10a 129.52a 13.43a 9.19b 326.93a 7.06 98.62c 205.02c 11.92b 7.25c 5.47c 29.74b 4.35b 0.29 1.59 12.06

42.32 23.65 12.81 25.60 29.40bc 108.02b 11.66ab 11.24a 314.87b 7.39 113.11b 216.28bc 15.67a 9.73b 6.40ab 33.01a 5.02a 0.32 1.36 12.21

44.77 24.42 13.01 25.50 25.97c 93.76c 8.96c 8.97b 300.87c 6.55 126.78a 236.88 a 15.47ab 12.64a 6.58a 31.75ab 4.62ab 0.39 1.31 11.19

2.02 0.89 0.48 1.18 1.22 1.96 0.72 0.23 3.72 0.33 3.72 5.60 0.78 0.25 0.27 0.96 0.17 0.04 0.11 0.77

0.16 0.61 0.82 0.19 <0.01 <0.01 <0.01 <0.01 <0.01 0.20 <0.01 <0.01 <0.01 <0.01 0.01 0.05 0.02 0.24 0.16 0.62

618.15a 381.85c 709.73a 245.67b 44.63b

581.10b 419.00b 689.05b 261.54ab 49.53a

547.03c 452.97a 670.56b 281.61a 47.81ab

6.37 6.36 6.79 6.17 1.44

<0.01 <0.01 <0.01 <0.01 0.054

a–c

Means within a row with different superscripts differ (P<0.05). CLA = Conjugated linoleic acids; SFA = Saturated fatty acids; MUFA = Monounsaturated fatty acids; PUFA = Polyunsaturated fatty acids. 2 SMCFA = Short- and medium chain fatty acids; LCFA = Long chain fatty acids. 1

3.3. Oxidation stability of milk fat The SOD activity (Table 5) was decreased with proportion of average carbon chain length and degree of unsaturation increased in lipid supplements (P<0.05), and reached the lowest level in LCFA treatment. GSH-Px and CAT activities were found to be higher in butterfat than SMCFA and LCFA treatments (P<0.05). There were no differences in T-AOC between three treatments. Besides, our research results found that the MDA concentration in butterfat and LCFA groups were higher than SMCFA (P<0.05), and reached the highest level in butterfat treatment. 4. Discussion 4.1. Feed intake and lactation performance Our hypothesis that cows fed different lipid mixtures would not affect DMI, milk production and some milk composition index except milk fat percentage was not always upheld. It has been demonstrated that cows abomasal infusion (Kadegowda et al., 2008) or directly (Hristov et al., 2009) fed different lipids had no effect on DMI, which is in accordance with our results.

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Table 5 Concentrations of enzymatic radical scavenging and lipid oxidation products in milk response to different fatty acids mixtures. Item1

SOD, U/ml GSH-Px, U/ml CAT, U/ml T-AOC, U/ml MDA, U/ml

Treatment2 SMCFA

Butterfat

LCFA

5.26a 430.17a 36.51a 1.58 1.03b

4.58ab 395.74bc 33.40b 1.62 1.15a

4.22b 408.13b 35.52a 1.42 1.14a

SEM

P-value

0.25 5.50 0.68 0.08 0.03

0.02 <0.01 0.01 0.19 0.01

a–c

Means within a row with different superscripts differ (P<0.05). SOD = Superoxide dismutase; GSH-Px = Glutathione peroxidase; CAT = Catalase; T-AOC = Total antioxidation; MDA = Malondialdehyde. 2 SMCFA = Short- and medium chain fatty acids; LCFA = Long chain fatty acids. 1

Previously, researchers demonstrated that milk yield, milk fat and FFA content were not differences for cows fed different types of FA (Relling and Reynold, 2007) which is in accordance with our study. In our research, milk fat concentration was highly affected among three treatments (P<0.05), and milk fat percentage was maximized in SMCFA and minimized in LCFA treatment. Hansen and Knudsen (1987) confirmed that exogenous stearic, oleic and linoleic acids inhibited de novo FA synthesis and incorporation of FA synthesized de novo into triacylglycerols, similar results with He et al. (2012). Kadegowda et al. (2008) and Vyas et al. (2012) suggest that short- and medium-chain FA may have positive effect on FA de novo synthesis in mammary gland. These studies are in accordance with our study. However, some earlier studies shown that cows supplemented saturated long-chain FA tended to increase milk fat content (Steele and Moore, 1968; Drackley et al., 1992). The different effects of long-chain FA on milk fat concentration might because the saturation of long-chain FA was different in these studies. 4.2. Fatty acid composition In this study, increased long-chain FA in diets exhibit positive effects on some long-chain FA content in milk fat (such as 18:0, cis9-18:1, trans11-18:1, 18:2n-6 and 18:3n-3), and this increase was countered by a general decrease in weight percentage of many other FA (some medium-chain FA). These results were qualitatively similar to that of Enjalbert et al. (1998), who infused different types of FA (control, palmitic, and oleic) and found that oleic increased total 18C FA, and reduced 14:0 and 16:0 content in milk fat. However, cows supplemented with short- and medium-chain FA tended to have opposite effects on total 18C FA compared with LCFA treatment (Odongo et al., 2007). The fatty acids of bovine milk fat arise from two sources with short- and medium-chain FA de novo synthesized in mammary gland (4:0 to 14:0, and 50% of 16:0) while long-chain FA (50% of 16:0 and all 18-carbon) are extracted from arterial blood (Palmquist and Jenkins, 1980). Furthermore, synthesized FA or extracted are incorporated into milk triglycerides. Our results showed that cows supplemented with short- and medium-chain FA would have increased content of FA with C≤16 (mostly short and medium chain FA), which were also shown by Odongo et al. (2007). This means that short- and mediumchain FA may have positive effects on FA de novo synthesis in mammary gland. Harvatine and Allen (2006) demonstrated that increasing long-chain FA decreased total concentration of short- and medium-chain FA and increased concentration of some long-chain FA, which is in accordance with our study. Similar milk FA results by Kadegowda et al. (2008) suggest that long-chain FA may have negative effect on FA de novo synthesis in mammary gland. Our results also show that cows supplement butterfat and LCFA which are high in long-chain FA could reduce total SFA (12:0, 14:0, and 16:0) and enhance total UFA (18:0, 18:1, 18:2n-6, and 18:3n-3) concentration in bovine milk. Earlier studies have shown that 12:0, 14:0 and 16:0 are three unhealthy FA, and consuming high concentration of these FA in bovine milk will affect people’s healthy (Mensink et al., 2003; Woodside and Kromhout, 2005), for example, may lead to the occurrence of atherosclerosis and cardiovascular disease (Kromhout et al., 2002). Therefore, an increase in the proportions of UFA in milk fat is desirable because of the potential health benefits when included in human diet (Blaxter and Webster, 1991; KrisEtherton et al., 1999; Owen et al., 2000). Our milk FA composition results confirm that long-chain FA would have positive effects on milk FA profile and milk quality. 4.3. Oxidation stability of milk fat Lipid oxidation is one of the most basic chemical reactions that occur in food, generally resulting in deterioration in sensory and nutritional quality. Fundamental principles of lipid oxidation process were elucidated by work of Farmer et al. (1942), Bolland and Gee (1946), and Bateman et al. (1953). In the process of lipid oxidation, an initial free radical was formed from UFA which will start oxidation reaction. Meanwhile, some lipid oxidation products will be formed, and will affect milk quality and flavor. Many studies have reported that antioxidant enzymes, such as SOD, GSH-Px, and CAT, have ability to clean up free radicals (Halliwell et al., 1995; Vanderlelie et al., 2005). Michiels et al. (1994) reported process of antioxidant enzyme scavenged free radical, SOD, as first line of defense system. This can catalyzes the dismutation of superoxide anion into hydrogen peroxide, and then CAT and GSH-Px detoxify hydrolytic hydrogen peroxide into non-toxic alcohols (Czernichow and Hercberg, 2001).

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In our research, cows supplement lipid high in long-chain FA had negative effects on SOD, GSH-px, and CAT concentrations in milk. Enzymatic radical scavenging systems obviously were not high enough to prevent oxidation of UFA in butterfat and LCFA treatments compared with SMCFA treatment, because the content of MDA was at higher levels in butterfat and LCFA treatment than SMCFA. Our results showed that cows fed butterfat and LCFA produced higher concentration of PUFA, especially cis9, trans11-CLA, 18:2n-6 and 18:3n-3 in milk fat than SMCFA. Meanwhile, many studies have shown that increasing contents of UFA (especially 18:2 and 18:3) in milk fat will increase susceptibility of milk to oxidation (Timmons et al., 2001; Granelli et al., 1998; Kristensen et al., 2004). This could provide an explanation for higher concentration of MDA in butterfat and LCFA treatments. Milk fat varies considerably in its susceptibility to oxidation and some researchers speculated that milk fat oxidation results from joint action of several contributing factors, rather than a single one. Most important is milk FA profile, especially proportion of PUFA in milk fat (Barrefors et al., 1995). Timmons et al. (2001) surmised that cows feeding roasted soybeans or other fat sources that could increase proportions of 18:2 and 18:3 in milk would increase appearance of SOF in milk fat. Liu et al. (2010) found that cows infused duodenally with long-chain UFA increased total PUFA in milk fat, the relevant antioxidant enzyme activity tended to decrease when PUFA increased in milk fat, and the MDA content increased in milk. Our results agree with Timmons et al. (2001) and Liu et al. (2010) that cows supplement lipids containing long-chain FA increasing C18:2 and C18:3concentrations in milk fat with an accompanying increase in MDA content in milk. However, previous research proposed that animals supplement lipid increased UFA in milk fat would also result in a compensatory increase in antioxidant enzyme activity (Venkatraman and Pinnavaia, 1998; Renerre et al., 1999) which differed from findings of our study. Exact reason why we observed a reduction in antioxidant enzyme activity in milk is unknown, but many factors affect activity of antioxidant enzymes including PUFA, antioxidant and, metal ion. Therefore, further investigations should be conducted to explain this phenomenon, for example, evaluated copper ion, and ␤-carotene level in milk. 5. Conclusion Cows feeding lipids with a high content of long-chain FA and UFA would increased cis9, trans11-CLA, 18:2n-6, 18:3n-3, and decreased 12:0, 14:0, 16:0 concentration in milk fat. While the relevant antioxidant enzymes activity decreased and the lipid peroxidation increased with high proportion of 18:2, 18:3n-3 in milk fat. 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