Rumen protected fat reverses the conjugated linoleic acid induced low milk fat content in dairy cows

Transcription

1 Rumen protected fat reverses the conjugated linoleic acid induced low milk fat content in dairy cows S. K. Gulati 1, S. McGrath 2, P. C. Wynn 2, P. C. Thomson 2, and T. W. Scott 1 1 Faculty of Veterinary Science (B19), University of Sydney, NSW 2006, Australia ( sureshg@vetsci.usyd.edu.au); 2 Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, PMB 3, Camden, NSW 2570, Australia. Received 27 April 2005, accepted 26 October Gulati, S. K., McGrath, S., Wynn, P. C., Thomson, P. C. and Scott, T. W Rumen protected fat reverses the conjugated linoleic acid induced low milk fat content in dairy cows. Can. J. Anim. Sci. 86: Conjugated linoleic acids (CLAs) were protected by encapsulation in a matrix of formaldehyde-treated protein; these rumen protected (RP-CLAs) were fed in two separate trials using Holstein cows grazing pasture to examine their effects on milk fat content, yield and fatty acid composition. In trial 1, three groups of nine cows were fed pasture alone, RP-CLA (61.5 g d 1 equivalent to 10.3 g of trans-10, cis-12 isomer) and rumen protected canola/soybean (RP-C/SB) (65.6 g d 1 to have the equivalent level of fat containing no CLA). RP-CLA reduced milk fat yield by 27, 29 and by 38%, respectively, after 4, 7 and 10 d of supplementation. Milk fat content was also reduced and after 10 d of CLA supplementation declined from 3.7 to 2.3%. RP-CLA supplementation resulted in a gradual decline in the yield of C 10 -C 14, C 16 and C 18 fatty acids in milk during the 10-d feeding period. In trial 2, feeding RP-CLA for 4 d reduced milk fat content (pasture 3.4%, RP-CLA 2.4%) and yield (pasture 826 g d 1, RP-CLA 594 g d 1 ) by the same magnitude of 29%. Milk fat content and yield was restored by feeding RP-CLA together with RP-C/SB supplement (1 2 kg d 1 ), which provided an additional 328 g or 656 g of fat per day; this suggests that CLA preferentially inhibits mammary gland lipogenesis. Key words: Rumen protected conjugated linoleic acid, rumen protected fat supplements, milk, fat, fatty acids Gulati, S. K., McGrath, S., Wynn, P. C., Thomson, P. C. et Scott, T. W La protection des lipides contre la dégradation dans le rumen rétablit la chute de concentration de la matière grasse induite par les acides linoléiques conjugués dans le lait des vaches laitières. Can. J. Anim. Sci. 86: Les auteurs ont protégé les acides linoléiques conjugués (ALC) en les encapsulant dans des protéines conditionnées au formaldéhyde. Ces ALC protégés contre la dégradation dans le rumen (ALC-PR) ont ensuite été servis à des vaches Holstein mises à l herbe dans le cadre de deux expériences, l idée étant d établir leur incidence sur la teneur en matière grasse du lait, le rendement laitier et la composition du lait en acides gras. Dans la première expérience, 3 groupes de 9 vaches ont reçu uniquement des herbages, des ALC-PR (61,5 g par jour, l équivalent de 103 g d isomère trans-10, cis-12) ou du canola/soja protégé contre la dégradation dans le rumen (C/S-PR) (65,6 g par jour, la concentration équivalente de matière grasse sans ALC). L ALC-PR réduit le rendement du lait en matière grasse de 27 %, de 29 % et de 38 % après administration du supplément pendant 4, 7 et 10 jours, respectivement. La teneur en matière grasse du lait diminue elle aussi : après administration du supplément pendant 10 jours, elle passe de 3,7 % à 2,3 %. Le supplément d ALC-PR entraîne une baisse progressive du rendement en acides gras C 10 -C 14, C 16 et C 18 du lait au cours des 10 journées de la période expérimentale. Lors de la deuxième expérience, l administration d ALC-PR pendant quatre jours a réduit la teneur en matière grasse du lait (paissance, 3,4 %; ALC-PR, 2,4 %) et le rendement (paissance, 826 g par jour, ALC-PR, 594 g par jour) d une amplitude équivalente (29 %.) La teneur en matière grasse et le rendement se sont rétablis lorsqu on a donné les ALC-PR avec un supplément de C/S-PR (1-2 kg par jour), de manière à procurer quotidiennement aux animaux 328 g ou 656 g de matière grasse supplémentaire. Les résultats laissent croire que les ALC inhibent surtout la lipogenèse dans les glandes mammaires. Mots clés: Acide linoléique conjugué protégé contre la dégradation dans le rumen, supplément de matière grasse protégé contre la dégradation dans le rumen, lait, matière grasse, acides gras 63 Conjugated linoleic acids (CLA) refer to geometrical and positional isomers of linoleic acid. They are formed either by partial biohydrogenation of C 18 di unsaturated fatty acids or can be synthesized de novo by the 9 desaturase enzyme by desaturation of C18:1 trans-11, a biohydrogenation intermediate (Griinari et al. 2000; Corl et al. 2001). The principal CLA isomer occurring in milk and ruminant tissue is cis-9, trans-11 that has anti-carcinogenic (Parodi 1997; Belury 2002) and anti-atherogenicity properties (Kritchevsky et al. 2000). CLAs have also been shown to modulate immune function (Bassaganya-Riera et al. 2002) and reduce human adiposity and insulin sensitivity (Brown and McIntosh 2003). Several studies have demonstrated that mixtures of CLA isomers reduce lipid synthesis, 9 desaturase activity and milk fat secretion (Chouinard et al. 1999a; Medeiros et al. 2000). Recently, Shingfield et al. (2004) have demonstrated that feeding dietary supplements of CLA isomers protected by encapsulation with formaldehydetreated protein (Gulati et al. 2000a) also reduced milk fat content, increased milk production, improved tissue-energy balance and nitrogen retention in the first 15 wk of lactation; these effects were more pronounced then those observed by Bernal-Santos et al. (2003) using calcium salts of CLA mixtures in early lactation. Abbreviations: CLA, conjugated linoleic acid; RP-CLA, rumen protected conjugated linoleic acid; RP-C/SB, rumen protected canola/soybean oilseeds

2 64 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 1. Composition and rumen protection of fat supplements Fatty acid (%;wt/wt) cis-9, trans-10, trans-11 and cis-12 and Fat % in vitro Supplement C16:0 C18:0 C18:1c C18:2 C18:3 trans-8 cis-10 cis-11 trans-13 (%) protection z RP-CLA ± 0.21 RP-C/SB ND y ND ± 0.46 z In vitro protection values are based on the content of CLA; C18:2 and C18:3 before and after incubation (n = 6). Dry matter content of supplement was 93%. RP-CLA rumen-protected triacylglycerol of conjugated linoleic acid/casein (1/1; wt/wt). RP-CSB rumen-protected canola/soybean (70/30; wt/wt). y ND, not detected. Table 2. Intake of rumen protected (RP) supplements Fatty acid composition (% of total) Fatty acid intake (g d 1 ) Fat Fat cis-9, trans-10, cis-9, trans-10, Fat content intake trans-11 and cis-12 and trans-11 and cis-12 and Supplement (%) (g d 1 ) trans-8, cis-10 cis-11, trans-13 trans-8, cis-10 cis-11, trans-13 Trial 1 RP-CLA C18:1cis C18:2 C18:3 C18:1cis C18:2 C18:3 RP-C/SB Trial 2 RP-CLA C18:1cis C18:2 C18:3 C18:1cis C18:2 C18:3 RP-C/SB RP-C/SB RP-CLA rumen-protected triacylglycerol of conjugated linoleic acid/casein (1/1; wt/wt). RP-CSB rumen-protected canola/soybean (70/30; wt/wt). RP-CLA fed at 150 g per day. RP-C/SB fed at 200 g per day; RP-C/SB1 fed at 1 kg per day; RP-C/SB2 fed at 2 kg per day. Baumgard et al. (2000) identified the trans-10, cis-12 isomer as the principal inhibitor of milk fat synthesis. In cows fed a total mixed ration, infusion of the trans-10, cis-12 isomer into the abomasum has been shown to result in a curvilinear reduction in milk fat yield (Baumgard et al. 2001; Peterson et al. 2002). In recent experiments with pasture-fed dairy cows, abomasal infusions of CLA equivalent to 3.7, 7.4 and 14.8 g d 1 of trans-10, cis-12 isomer reduced milk fat yield by about 32, 36 and 60%, respectively, (Mackle et al. 2003). Furthermore, in the study of Mackle et al. (2003), infusion of CLA decreased the milk fat content of C 4:0 (butyric) to C 16:0 (palmitic) and C 18:1 (oleic) fatty acids and increased the proportion of C 18:0 (stearic), C 18:2 n-6 (linoleic) and C 18:3 n-3 (linolenic) fatty acids; these results are similar to those of previous studies (Chouinard et al. 1999b; Baumgard et al. 2001). These changes in milk fatty acid composition indicate that the effect of the trans-10, cis-12 isomer involves an inhibition of fatty acid synthesis and uptake of circulating fatty acids within the mammary gland (Peterson et al. 2002) and expression of genes that encode for the enzymes involved (Baumgard et al. 2002). The aim of the present investigation was to determine the magnitude of change in milk fat content and composition by feeding a rumen protected triacylglycerol enriched in CLAs CLA and secondly to investigate whether the CLA-induced milk fat depression could be overcome by increasing the supply of circulating fatty acids to the mammary gland using protected dietary fat supplements. MATERIALS AND METHODS Rumen Protected Fat Supplements RP-CLA was prepared as follows: A 15% casein solution (wt/vol) was prepared and a commercial source of CLA-TG (triacylglycerol) (Gruenau Illertissen, GmbH, Germany) was added on a 1:1; (wt/wt; oil: protein basis). The mixture was homogenized using a colloid mill (Fryma Model MZ80/A) and the emulsified product was transferred to a mixing vessel. Formaldehyde (37%, wt/vol) was added to form a rubber like gel, which was flash-dried (Scott and Ashes 1992). Using these procedures the fat content of the RP-CLA was in the range of 40 43%. The RP-canola/soybean oilseed supplement (RP- C/SB; 70/30, wt/wt) was prepared using the procedures outlined by Gulati et al. (2000b). The fatty acid composition and degree of rumen protection of the fat supplements was measured using the procedures described by (Gulati et al. 2000a). The degree of rumen protection, fat content and fatty acid composition of the supplements for both trial 1 and trial 2 are given in Table 1.

3 GULATI ET AL. REVERSING CLA-INDUCED MILK FAT SUPPRESSION 65 Table 3. Effect of feeding cows RP-CLA and RP-C/SB fat supplements on milk parameters (trial 1) Milk yield (L d 1 ) Fat (%) Fat yield (g d 1 ) Protein (%) Fat supplement Day 0 Day 10 Day 18 Day 0 Day 10 Day 18 Day 0 Day 10 Day 18 Day 0 Day 10 Day 18 Pasture RP-CLA P* < RP-C/SB Z P** Protein yield (g d 1 ) Lactose (%) SNF (%) Fat Supplement Day 0 Day 10 Day 18 Day 0 Day 10 Day 18 Day 0 Day 10 Day 18 Pasture RP-CLA P* < RP-C/SB z P** z RP-C/SB supplement provided an equivalent of fat without CLA (see Table 1). * P value for test of equality of RP-CLA and Pasture means. ** P value for test of equality of RP-C/SB and Pasture means. Feeding Trials Animals and Diets Trial 1 In trial 1, 27 Holstein cows, approximately 100 d in milk were randomly allocated into three groups of nine cows. The cows grazed a predominantly kikuyu based pasture in December and were fed 2 kg d 1 of a protein pellet (42% protein), (Millmaster Feeds Pty Ltd., Sydney, Australia) in the milking parlour during the 18-d trial. For the first 10 d, group 1 were fed a mixture of lucerne chaff and molasses (1.5 kg; 1:1; wt/wt); group 2 were fed 150 g d 1 of RP-CLA mixed with lucerne chaff and molasses (1.5 kg; 1:1; wt/wt); group 3 were fed 200 g d 1 of RP-C/SB mixed with lucerne chaff and molasses (1.5 kg; 1:1; wt/wt) to provide an equivalent amount of fat intake from the supplements (see Table 2). Trial 2 In trial 2, five Holstein cows approximately 100 d in milk, grazing predominantly kikuyu based pasture in February were fed 2 kg d 1 of a protein pellet (42% protein), (Millmaster Feeds Pty Ltd., Sydney, Australia) and 4 kg of concentrate in the milking parlour due to the poor quality of pasture available. Cows were then supplemented with fat supplements mixed with lucerne chaff and molasses (1.5 kg, 1:1; wt/wt) for 4 d in individual pens after milking at 0500 in the following sequence: days 1 4 RP-CLA alone, days 5 8 RP-CLA + RP-C/SB1 (1 kg supplying 328 g fat), days 9 12 RP-CLA + RP-C/SB2 (2 kg supplying 656 g fat) (see Table 2); days no fat supplementation. Collection and Storage of Milk Samples Cows were machine milked twice daily, at 0500 before feeding and again at Milk volumes were recorded on Fig. 1. The effect of feeding pasture alone ( ), pasture + RP CLA (, 150 g equivalent to 61.5g fat) and pasture + RP C/SB (, 200 g equivalent to 65.5g fat) on the magnitude of milk fat depression in dairy cows; fat supplements were fed for the initial 10-d period. See Table 2 for details. Mean ± SEM is shown. Mean fat yield for Pasture + RP CLA is significantly lower (P < 0.05) than that for Pasture alone and/or Pasture + RP C/SB (non-significant). pre-calibrated flow meters and 100 ml milk samples were collected at every milking and stored at 4 C for analysis. Analysis of Milk Fatty acids Milk fat was extracted by the Rose-Gottlieb procedure [Association of Official Analytical Chemists (AOAC) 1997] and fatty acid methyl esters (FAMEs) were prepared by

4 66 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 4. Effect of feeding RP-CLA alone and in combination with RP-C/SB fat supplements on milk composition (trial 2) Milk yield (L d 1 ) Fat (%) Fat (g d 1 ) Fat supplement Fat intake (g d 1 ) Mean SEM P Mean SEM P Mean SEM P A Unsupplemented B RP-CLA < < C RP-CLA+RP-C/SB < D RP-CLA+RP-C/SB < < E Unsupplemented For details of fat supplements see legend to Table 1. Values are the means of milk samples collected from five cows on the PM and AM milking after 4 d of fat supplementation. P values refer to treatment mean comparisons A vs. B; B vs. C; B vs. D; and A vs. E. the procedures described by Gulati et al. (1999). The FAMEs were separated by gas chromatography (Perkin Elmer, auto sampler XL), on a BPX70 column (50 m, bonded fused silica phase, SGE Melbourne, Australia) with a split ratio of 20:1, using helium as a carrier gas. The column was temperature programmed from 150 C to 190 C at 2 C per minute. Each peak was identified and quantified using pure methyl ester standards (Nu Check Prep, Inc, Elysian, MN). The CLA isomers identified and quantified in the protected feed supplements were very similar to the manufacturers CLA isomer specifications. Using these procedures it is likely that some of the CLA isomers may co-elute, including cis-9, trans-11; trans-8, cis-10; trans-10, cis-12; cis-11, trans-13 (Destaillats and Angers 2002). Milk protein, fat, lactose and solids non-fat were determined by MIR (Mid infra red reflectance; milko-scan 130 series, Foss Electric, Denmark). All analysis was undertaken on milk samples collected in the afternoon and morning after 4 d of feeding supplements. The values presented were the means of all cows for both milking. Proximate Analysis Dry matter content of RP fat supplements was determined by the procedure of the AOAC (1997). Total fat was extracted with chloroform/methanol (2/1; vol/vol) and fatty acid composition analysed by procedures of Gulati et al. (1999). In Vitro Rumen Incubations The degree of rumen protection for the fat supplements was measured by the procedures of Gulati et al. (1999). Protection was calculated using the formula: Protection (%) = (% C 18:2 n-6 or CLA isomer after incubation)/(% C 18:2 n-6 or CLA isomer before incubation) 100. Statistical Analysis Data were analyzed by fitting linear mixed models, with fixed effects for treatment, day and treatment day interaction (where appropriate), with cow being considered a random effect. Additional correlation due to the repeat measures nature of the data was also taken into account. This was conducted using the REML procedure of GenStat V8.2 (Lawes Agricultural Trust 2005). Formal comparison of pairs of treatment was conducted using Wald z-tests. Animal Ethics All experimental protocols were approved by the CSIRO Animal Ethics Committee according to the recommendations of the Australian National Health and Medical Research Council. RESULTS Milk Fat Content and Yield In trial 1 feeding RP-CLA, equivalent to 16.8 g d 1 of the trans-10, cis-2 and cis-11, trans-13 isomer, resulted in a decrease in milk fat content (pasture 3.6%, RP-CLA 2.4%) and yield (pasture 835 g d 1, RP-CLA 514 g d 1 ) throughout the 10 day period of supplementation (Table 3, Fig. 1). During the first 4 d of CLA supplementation there was a 27% reduction in fat yield that declined further to 29 and 38% after 7 and 10 d of feeding, respectively (Fig. 1, Table 3), whereas feeding an equivalent amount of protected fat supplement (RP-C/SB; 65.6 g d 1 ) containing no CLA did not change the fat content or yield. Other components such as milk yield, protein, lactose and solids non-fat were unchanged (Table 3). In trial 2, feeding 16.8 g d 1 of the trans-10, cis-12 and cis-11, trans-13 isomer for 4 d reduced milk fat yield (pasture 826 g d 1, RP-CLA 594 g d 1 ) and content (pasture 3.4%, RP-CLA 2.4%) by 28 and 30%, respectively (Table 4). This magnitude of fat reduction was similar to the values obtained at day 4 in the 10-d feeding study in trial 1 (see Fig. 1 and Table 3). In trial 2, feeding RP-CLA plus 1 kg of RP- C/SB1 increased the fat content and yield by 32 and 29% respectively and the inclusion of 2 kg of RP-C/SB2 restored the fat parameters to control levels (Table 4). Feeding 1 kg of RP-C/SB1 supplement provided an additional 328 g d 1 of fat and the difference in yield of fat secreted between the RP-CLA alone and RP-CLA plus 1 kg of RP-C/SB1 was 172 g d 1 (Table 4). The major increases occurred in the C 18 unsaturated fatty acids (see Table 6) and this indicates that approximately 52% of the supplemental fat was transferred into milk fat; likewise, when 2 kg of the RP-C/SB2 supplement was fed there was an extra 264 g of fat secreted per day and this was equivalent to 40% transfer. Fatty Acid Composition of Milk Fat Feeding RP-CLA supplement reduced the yield (g d 1 ) of C 10:0 -C 18:0, C 18:1, C 18:2 and C 18:3 (Tables 5 and 6). In trial 1 feeding RP-CLA resulted in a gradual decline in the yield of C 10 -C 14, C 16 and C 18 fatty acids during the 10-d period and the values tended to revert towards the pasture control fol-

5 GULATI ET AL. REVERSING CLA-INDUCED MILK FAT SUPPRESSION 67 Table 5. Fatty acid %, (wt/wt) in milk of cows grazing pasture alone or supplemented with CLA (trial 1) Day 0 Day 4 Day 7 Day 10 Day 14 Day 18 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Fatty Acid pasture pasture P pasture CLA P pasture CLA P pasture CLA P pasture pasture P pasture pasture P C 10: < C 10: C 12: < C 14: < < C 14: C 16: < < < C 16: C 18: < C 18: < < < C18: C 18: CLA cis-9, trans < < < and trans-8, cis trans-10, cis ND < and cis-11, trans n = 9 cows per group; see methods for description of diet and composition. Pasture control vs. CLA supplementation on days 0, 4, 7, 10 and pasture control on days 14 and 18. Means are shown with their corresponding standard errors below. ND, not detected.

6 68 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 6. Effect of feeding RP-CLA alone and in combination with RP-C/SB fat supplements on milk fatty acid composition and yield in milk (trial 2) A Pasture B RP-CLA C RP-CLA + RP C/SB1 D RP-CLA + RP C/SB2 E Pasture Fatty acid Mean SEM Mean SEM P Mean SEM P Mean SEM P Mean SEM P C 8: < C 10: < < < C 12: < < < C 14: < < < C 16: < < C 18: < C 18: < < < < C18: < < < C 18: < < CLA cis-9, trans < and trans-8, cis-10 trans-10, cis < < and cis-11, trans-13 For details of fat supplements see legend to Table 1. Values are the means of milk samples collected from five cows on the PM and AM milking after 4 d of fat supplementation. P values refer to treatment mean comparisons A vs. B; B vs. C; B vs. D; and A vs. E. lowing cessation of CLA supplementation (Fig. 2). In trial 1, the concentrations of C 10 -C 16 fatty acids in the CLA-supplemented group compared with the pasture control decreased throughout the 10-d period, whereas the proportions of C 18:1 and to a lesser extent C 18:0 increased (Table 5b); similar trends were observed in trial 2 (Table 6). In trials 1 and 2, the proportion of trans-10, cis-12 plus cis-11, trans-13 increased from 0.08 to 0.25% and from 0.08 to 0.27%, respectively, and the cis-9, trans-11 plus cis-8, trans-10 increased from 1.1 to 1.7 and from 1.4 to 1.9%, respectively (Tables 5 and 6). In both trials feeding RP-CLA did not produce a consistent increase in the ratio of the saturated to monounsaturated fatty acids; for example in trial 1 the ratio of C 18:0 /C 18:1 was 0.45 after 10 d of CLA supplementation compared with 0.54 in the pasture-fed cows (see Table 5). Feeding RP-C/SB in combination with RP-CLA significantly increased the proportions of C 18:1 cis, C 18:2 n-6 and C 18:3 n-3 fatty acids and reduced the proportions of saturated fatty acids (Table 6). These changes are reflected in the major reductions that occur in the ratio of saturated/unsaturated fatty acids in milk fat; in pasture-fed cows this ratio was about 2 and this was reduced to approximately 1 in cows receiving the RP-C/SB supplement (Table 6). The proportions of C 18 unsaturated fatty acids in the final pasture period were similar to the initial period, but the proportions of individual saturates were lower, indicating a residual effect of the RP-CLA and RP-C/SB on those fatty acids synthesised in the mammary gland (Table 6). DISCUSSIONS Feeding rumen protected CLAs prepared by encapsulating them in a matrix of formaldehyde-treated protein significantly reduced milk fat content and yield (Tables 3 and 4). In trial 1, the protected CLAs were fed for 10 d, and during this period the milk fat yield continued to decline, although about 70% of the reduction was achieved in the first 4 d of supplementation. Perfield et al. (2002) observed that the decrease in milk fat yield had occurred at the end of the first week of feeding a calcium salt of CLAs and this reduction continued throughout the 140 d of supplementation. In short-term abomasal infusion studies i.e., 3 4 d with either pasture-fed or lot-fed dairy cows, CLA mixtures caused a rapid decline in milk fat content and yield, and their results suggest that a nadir in milk fat depression may not have been reached (Mackle et al. 2003; Chouinard et al. 1999b). The magnitude of the decrease in fat content and yield in the present study was similar to that reported by Baumgard et al. (2001) and Mackle et al. (2003) when trans-10, cis-12 isomer alone or a mixture of CLAs was infused into the abomasum at a rate of 3.5 to 7.4 g d 1. The intake of the trans- 10, cis-12 in the present study was 10.3 g d 1 (Table 2) and when adjusted for the degree of rumen protection in vivo (Ashes et al. 1979), this would be equivalent to about 6 g d 1 entering the abomasum. The 38% reduction in milk fat yield in the present study is similar to the 44% reduction observed by de Veth et al. (2005) who intra-ruminally infused a CLA methyl ester supplement (to deliver 10 g of trans-10, cis-12) prepared using procedures identical to that used in the present experiments. This demonstrates that different batches of RP-CLAs, either as methyl esters or triacylglycerols, are equally effective in inhibiting milk fat content and yield in lactating dairy cows. In the present study the CLA reduced the daily yield (g d 1 ) of C 10 to C 18 fatty acid into milk fat and this effect is in agreement with previous studies (Mackle et al. 2003; Chouinard et al. 1999b; Tables 5 and 6 and Fig. 2). This decrease in C 10 -C 16 fatty acids was also observed when RP- CLA together with RP-C/SB supplement was fed, although the total fat content and yield were restored due to the substantial increase in the C 18 unsaturated fatty acids (Table 6). Although there may be some residual carry over effects from feeding each supplement for 4-d periods; however, this does not alter the overall effect of feeding rumen-protected fat, which negates the CLA-induced milk fat suppression. This restoration of milk fat content and yield by feeding RP-

7 GULATI ET AL. REVERSING CLA-INDUCED MILK FAT SUPPRESSION 69 Fig. 2. The effect of feeding pasture alone or pasture + RP CLA (equivalent to 61.5 g fat; 10.3 g d 1 of trans-10, cis-12) on the mmoles secreted d 1 of the C 10 -C 14, C 16 and the C 18 fatty acids into milk fat on day 0, 4, 7, 10, 14 and 18. C/SB oilseeds tends to suggest that the mechanism of CLA inhibition may be directed more towards decreasing the expression of genes encoding for the enzymes involved in the synthesis of fatty acids within the mammary gland rather than the lipoprotein lipase enzyme, which regulates the uptake of circulating fatty acids. In previous studies, the infusion of CLAs into the abomasum resulted in an inhibition of the 9 desaturase enzyme, these changes are variable and seemed more pronounced at higher doses (i.e., 7 14 g d 1 of the trans-10, cis-12 isomer; Baumgard 2001, Peterson et al. 2002). For example, in the study of Mackle et al. (2003), CLAs inhibited this enzyme as indicated by an increase in the ratio of C 10:0 /C 10:1, C 14:0 / C14:1 and C 18:0 /C 18:1, but the ratio of C 16:0 /C 16:1 was unchanged, irrespective of the dose of the trans-10, cis-12 isomer, whereas in the study of Chouinard et al. (1999b) only the ratio of C 18:0 /C 18:1 was significantly increased. In the present study, no consistent increases were observed, For example, 18:1/18:0 was 2.1 for the control in trial 1 and trial 2, and 2.14 and 2.07 for CLA in trial 1 and 2, respectively (data calculated from Tables 5 and 6). These ratios suggested that the level of trans-10, cis-12 was below that required to induce a change in the activity of the 9 desaturase enzyme. This result is similar to that found by Peterson et al. (2002) who infused g of trans-10, cis- 12 isomer into the abomasum, and Baumgard et al. (2001) for their lower infusion level of 3.5 g d 1 of trans-10, cis-12. The present study demonstrates that if CLAs are protected from ruminal biohydrogenation by encapsulation in a matrix of inert protein they can exert a preferential action by significantly reducing milk fat synthesis and inhibiting mammary gland lipogenesis; these effects were reversed by feeding additional rumen-protected long chain fatty acids. This form of CLA fat supplement has the potential to significantly improve energy efficiency and tissue nitrogen retention during early to mid lactation (Shingfield et al. 2004). ACKNOWLEDGEMENTS We thank Mr. Jim Marsh and Mr. Kim McKean at May Farm (Sydney University) for assistance at the dairy and Gruenau Illertissen, GmbH, Germany, for providing the CLA-TG used in these studies. The Australian Centre for International Agricultural Research (ACIAR) for their support in this collaborative study. Association of Official Analytical Chemists Official methods of analysis of AOAC. 16th ed. P. Cunniff, ed. AOAC, Washington, DC. Ashes, J. R., Gulati, S. K., Cook, L. J., Scott, T. W. and Donnelly, J. D Assessing the biological effectiveness of protected lipid supplements for ruminants. J. Am. Oil. Chem. Soc. 56: Bassaganya-Riera, J. and Hontecillas Beitz, D. C Colonic ant-inflammatory mechanisms of conjugated linoleic acids. Clin Nutr. 2: Baumgard, L. H., Corl, B. A., Dwyer, D. A., Saebo, A. and Bauman, D. E Identification of the conjugated linoleic acid isomers that inhibit milk fat synthesis. Am. J. Phys. 278: R179-R184. Baumgard, L. H., Sangster, J. K. and Bauman D. E Milk fat synthesis in dairy cows is progressively reduced by increasing supplemental amounts of trans-10, cis-12 conjugated linoleic acid. J. Nutr. 131: Baumgard, L. H., Matitashvili, E., Corl, B. A., Dwyer, D. A. and Bauman, D. E trans 10, cis 12 Conjugated linoleic acid decreases lipogenic rates and expression of genes involved in milk lipid synthesis in dairy cows. J. Dairy. Sci. 83: Belury, M Inhibition of carcinogenesis by conjugated linoleic acid: potential mechanisms of action. J. Nutr. 132: Bernal-Santos, G., Perfield II., J. W., Barbano, D. M., Bauman, D. M. and Overton, T. R Production responses of dairy cows to dietary supplementation with conjugated linoleic acid during the transition period and early lactation. J. Dairy Sci. 83: Brown, J. M. and McIntosh, M. K Conjugated linoleic acid in humans: Regulation of adiposity and insulin sensitivity. J. Nutr. 133:

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