Effects of Protected Conjugated Linoleic Acid Supplementation on Milk Fatty Acid in Dairy Cows

: The objective was to determine the effects of supplementation of protected conjugated linoleic acid (CLA), CLA-20 comprising 10% each of cis-9, trans-11 and trans-10, cis-12 , on milk production and fatty acid profiles in plasma and milk in lactating dairy cows. Five mid-lactation, multiparous crossbred Holstein Friesian cows with average 402 ± 20 kg BW were used in a 5 × 5 Latin square design for 21-d periods. Cows were given a total mixed ration (TMR) and supplemented with CLA-20 at 0, 20, 40, 80 and 160 g/d. The results showed that dry matter intake depression occurred in cows supplemented with CLA-20 at 160 g/d. Milk production slightly increased when CLA-20 supplementation was at 20, 40 and 80 g/d. However, 3.5% fat-corrected milk (FCM) was not affected by CLA-20 supplementation. Increased levels of CLA-20 supplementation resulted in a significantly decreased percentage of milk fat. Plasma concentrations of fatty acid were not altered by the amounts of CLA-20 supplementation except for the concentration of trans-10, cis-12 CLA. For all dietary treatments, percentages of fatty acids (C4:0, C6:0, C8:0, C13:0, C14:0 C14:1 C15:0 C15:1 C16:0, C16:1, C18:1n9t, C18:2n6t, C18:2n6c, C20:0, C18:3n6, C18:3n3, C20:1 and C20:3n6) in milk fat were similar. Concentrations of C10:0, C11:0, C12:0 and C18:1n9c were decreased cubically and C18:0 was elevated linearly (p<0.01) according to the increased amounts of CLA-20 supplemented. The linear increase was observed for cis-9, trans-11 CLA (0.62, 1.17, 1.94, 1.87 and 1.82% of total fatty acid), trans-10, cis-12 CLA (0.01, 0.63, 0.67, 0.93 and 0.95% of total fatty acid) and total CLA (0.80, 2.25, 3.16, 3.97 and 3.94% of total fatty acid) in milk fat from 0 to 160 g/d of CLA-20 supplement. In conclusion, concentration of cis-9, trans-11 CLA in milk fat was concomitantly elevated at an increasing rate with the increased amounts of CLA-20. Based on the results in this study, supplementation of CLA-20 at 80 g/d optimally enhanced total CLA in milk fat. (


INTRODUCTION
the effect of rumen protected CLA on fatty acid composition in milk fat in lactating cows.

Animals and treatments
Five, multiparous Holstein Friesian crossbred cows with average 402±20 kg BW and 70.6±4 d DIM, were randomly assigned to receive dietary treatments in a 5×5 Latin square design for 21-day periods. Cows were housed individually and provided with water and mineral block throughout the experimental period. The dietary treatments were assigned to deliver 0, 20, 40, 80 and 160 g/d of CLA in the form coated by partly hydrogenated vegetable fats containing 10±1% of cis-9, trans-11 and 10±1% of trans-10, cis-12 isomers (CLA-20; BASF Co., Inc, Italy). Total mixed ration (TMR) ( Table 1) was formulated according to their requirement (NRC, 1988) and was offered twice daily (0600 h and 1600 h) for ad libitum consumption to yield 5 to 10% feed refusal. CLA-20 was offered by top dressing daily.

Sampling and laboratory analysis
Dry matter intake (DMI, %BW) of the TMR was measured daily. TMR was sampled on the last 5 days of each period. Samples were dried in a forced-air oven at 60°C and stored in sealed containers at room temperature until analyzed. Equal amounts of samples from each period were combined to determine chemical composition and fatty acid profiles. In preparation for analyses, TMR was ground through a 2-mm screen (Thomas-Wiley Laboratory Mill, Arthur H. Thomas, Philadelphia, PA) and later ground through a 1-mm screen for crude protein (CP), ether extract (EE), ash (AOAC, 1990) and acid detergent fiber (ADF) and neutral detergent fiber (NDF) (Goering and Van Soest, 1970) and fatty acid content. Cows were milked and recorded for milk weight twice daily at 0500 h and 1500 h. Milk samples were pooled on the last 5 days of each period by equal volume and analyzed for milk fat, protein, lactose, solids-not-fat (SNF) (Milkoscan 605; Foss Electric, Hillerod, Denmark), CLA isomers and fatty acid profiles.
Blood samples were collected at 3 h post-feeding (0800 h) from the jugular vein in vacutainer tubes and centrifuged at 3,000×g for 15 min. Serum was recovered and stored at -18°C until analyzed for fatty acid profiles.
Samples of TMR, milk fat and plasma lipid fractions were methylated by in situ transterification with 0.5 N methanolic NaOH followed by 14% boron trifluoride in methanol (Loor and Herbein, 2001). Fatty acid methyl esters were separated and quantified using gas chromatography. Samples were injected by auto-sampler into a Hewlett-Packard 5890A gas chromatograph equipped

Statistical analysis
Data were subjected to analysis of variance according to a 5×5 Latin square design using the GLM procedure of SAS (1996). Significant differences were determined using Duncan's News Multiple Range Test and orthogonal polynomial contrast.

RESULTS AND DISCUSSION
Chemical and fatty acid compositions of TMR used in the experiment are presented in Table 1 and 2 whereas effect of CLA-20 supplementation on DMI, milk yield and milk composition are presented in Table 3.
DMI was linearly increased (p<0.05) by levels of CLA-20 supplementation from 0 to 80 g/d, but decreased (p<0.05) from 80 to 160 g/d. However, an increase DMI after CLA supplementation was not well understood. During short-term feeding of calcium salts of CLA, feed intake was unaffected (Hanson et al., 1998). Giesy et al. (2002) found that supplementation CLA-60 from 0 to 100 g/d did not alter DMI. Baumgard et al. (2000) demonstrated no effect of abomasal infusion of cis-9, trans-11 CLA on DMI, but a tendency for reduced DMI when trans-10, cis-12 CLA was infused.
Milk yield was significantly increased (quadratic, p<0.05) as levels of CLA-20 supplementation increased. This occurrence was due mainly to the increased DMI. In contrast, other studies reported no effect on milk production when CLA was supplemented (Hanson et al., 1998;Giesy et al., 2002). However, increasing levels of CLA-20 supplementation did not alter 3.5% FCM.
Reduced milk fat was obviously a dose-dependent of CLA-20 supplement (p<0.05). Loor and Herbein (1998) demonstrated that CLA administration dramatically decreased milk fat yield and content. Short-term studies confirmed that abomasally infusing supplemental CLA caused milk fat depression (Chouinard et al., 1999b). Baumgard et al. (2002) suggested that, trans-10, cis-12 CLA isomer is a very potent inhibitor of milk fat production. A daily dose of 13.6 g (0.08% of daily DMI) resulted in a 42% decrease in milk fat content and a 48% reduction in milk fat yield. Results demonstrated that the mechanisms by which trans-10, cis-12 CLA decreased milk fat yield and content involves many aspects of milk fat synthesis. Specifically, this CLA isomer dramatically reduced the mammary gland's lipogenic capacity (rates of acetate incorporation into fatty acids) and decreased the expression of genes encoding enzymes, involving in the uptake and transport of circulating fatty acids, de novo fatty acid synthesis, desaturation of fatty acids, and formation of triglycerides. In this experiment, supplementation of CLA-20 also tended to decrease in milk protein, SNF and TS (cubic, p<0.01), but not on milk fat yield and milk specific gravity. In contrast, other works have shown that milk protein concentration was not altered by abomasal infusion of CLA sources or during feeding of calcium salts of CLA (Chouinard et al., 1999b;Loor and Herbein, 2001;Giesy et al., 2002;Perfield et al., 2004). However, long-term supplementation of calcium salts of CLA enhanced milk protein percentage in cows raised in a pasture-based management system (Medeiros et al., 2000).
In contrast with previous studies (Chouinard et al., 1999a(Chouinard et al., , 1999bBaumgard et al., 2000), the short-to medium chain fatty acids were reduced to a greater extent by CLA infusion than longer chain fatty acids. As in the study of Baumgard et al. (2000), between 74 and 78% of the reduction (mmol basis) in milk fat that occurred during CLA infusion was accounted for by fatty acids of chain length C4:0 to C16:0.
Recently, Baumgard et al. (2001) demonstrated that at low concentrations of trans-10,cis-12 CLA (3.5 g/d), the inhibition of milk fat synthesis involved more uniform reductions in short chain, medium chain and long chain fatty acids. Although the mechanisms by which CLA inhibits milk fat synthesis are still unknown. Concentrations of cis-9,trans-11 CLA in milk fat were linearly increased (0.62, 1.17, 1.94, 1.87 and 1.82% of fatty acid) of cows supplemented with CLA-20 at 0, 20, 40 , 80 and 160 g/d, respectively (p<0.01) (Figure 1, Table 7). An increase of trans-10,cis-12 CLA was similar to cis-9,trans-11 CLA by linearly increasing in milk fat (0.01, 0.63, 0.67,  0.93 and 0.95% of fatty acid, in cows supplemented with all CLA-20 concentrations (0, 20, 40, 80 and 160 g/d, respectively, p<0.01). Most of others CLA isomers were increased, but lesser extend, by CLA-20 supplementation. However, variation of the others CLA isomers found in this experiment could be affected by CLA synthesized from mammary gland. Moreover, the concentration of total CLA in milk fat was also increased by dose of CLA-20. Apparently, the elevation of CLA in milk fat in this experiment was corresponded to previous studies (Loor et al., 2002;Loor and Herbein, 2003;Mackle et al., 2003;Perfield et al., 2004). Daily production of cis-9,trans-11 CLA were 4.01, 7.71, 11.87, 11.43 and 10.26 g/d when CLA-20 was supplemented at 0, 20, 40, 80 and 160 g/d equivalent to 0, 2, 4, 8 and 16 g cis-9,trans-11 CLA/d, respectively. The presence of cis-9,trans-11 CLA in milk fat in cows fed diet without CLA-20 supplement may be influenced by endogenous synthesis in the mammary gland from transvaccenic acid (TVA, trans-11 c18:1) by the Δ 9desaturase reaction (Griinari and Bauman, 1999). The control diet contained high concentration of linoleic acid (C18:2n6c) (Table 2), which was hydrogenated to trans-11 C18:1, then led to increase precursors for Δ 9 -desaturation in mammary gland, and resulted in an increase cis-9,trans-11 CLA content in milk fat (AbuGhazaleh et al., 2003;Loor and Herbein, 2003). Apparent transferring efficiencies of cis-9,trans-11 CLA from the supplement to milk fat was calculated according to Piperova et al. (2004) were 100, 100, 92 and 39% for 20, 40, 80 and 160 g/d of CLA-20 supplement, respectively. This observation was unexpected. Piperova et al. (2004) demonstrated that the apparent transferring efficiency of cis-9, trans-11 CLA from the supplement to milk was 11%, which was similar to Giesy et al. (2002) who reported the 9-10% of transferring efficiency for cis-9, trans-11 CLA in lactating cows by increasing doses of Ca-CLA-60. While Chouinard et al. (1999a) indicated transferring efficiency was about 33.5% during abomasal infusion of cis-9, trans-11 CLA. The different transferring efficiency between this observation and previous reports was probably due to the fact that the fat percentage in our experiment was probably higher (3.59-4.26%) than in Piperova et al. (2004) (2.54-3.39%), Giesy et al. (2002) (2.29-3.45%), and Choinard et al. (1999a) (2.40-3.34%). Incomplete protection exogenous CLA source from ruminal biohydrogenation of CLA and amount of CLA supplementation could be the main reason for the difference of apparent transferring efficiencies between the current study and those reports.

CONCLUSIONS
Supplementation of CLA as CLA-20 on milk fat content and composition were examined. DMI and milk yield was slightly increased when the cows were supplemented with CLA-20 from 20 to 80 g/d. Increased supplementation of CLA up to 160 g of CLA-20/d resulted in milk fat depression. Content of cis-9,trans-11 and trans-10,cis-12 CLA in milk fat increased at an increasing rate of CLA-20 supplementation. Based on this experiment, supplementation with CLA-20 at 80 g/d was potentially increased the concentration of cis-9,trans-11, trans-10,cis-12 CLA and total CLA in milk fat.