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Endogenous Synthesis of cis-9, trans-11 Conjugated Linoleic Acid in Dairy Cows Fed Fresh Pasture

J. K. Kay¹, T. R. Mackle^1,*, M. J. Auldist^1,, N. A. Thomson¹ and D. E. Bauman²

¹ Dexcel Ltd., Private Bag 3221, Hamilton, New Zealand
² Department of Animal Science, Cornell University, Ithaca, NY 14853

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
New Zealand Holstein-Friesian cows (n = 4) were used to quantify the importance of endogenous synthesis of cis-9, trans-11 conjugated linoleic acid (CLA) via ⁹-desaturase in cows fed a fresh pasture diet. The experiment was a 4 x 4 Latin square design with treatments arranged in a 2 x 2 factorial. Treatments lasted 4 d and were pasture only, pasture plus sterculic oil, pasture plus sunflower oil, and pasture plus sunflower oil plus sterculic oil. Abomasal infusion of sterculic oil inhibited ⁹-desaturase and decreased the concentration of cis-9, trans-11 CLA in milk fat by 70%. Using the changes in cis-9 10:1, cis-9 12:1 and cis-9 14:1 to correct for incomplete inhibition of ⁹-desaturase, a minimum estimate of 91% of cis-9, trans-11 CLA in milk fat was produced endogenously in cows fed fresh pasture. Dietary supplementation of a pasture diet with sunflower oil increased the proportion of long chain fatty acids in milk fat; however, the increase in vaccenic acid concentration was small (18%) and there was no increase in cis-9, trans-11 CLA concentration. Overall, results show that endogenous synthesis is responsible for more than 91% of the cis-9, trans-11 CLA secreted in milk fat of cows fed fresh pasture. However, the failure of plant oil supplements to increase the concentration of cis-9, trans-11 CLA in milk fat from pasture-fed cows requires further investigation.

Key Words: conjugated linoleic acid • ⁹-desaturase • milk fat • pasture

Abbreviation key: CLA = conjugated linoleic acid, DOM = digestible organic matter, ME = metabolizable energy, PAS = pasture treatment, PAS+SO = pasture plus sterculic oil treatment, PAS+SFO = pasture plus sunflower oil treatment, PAS+SFO+SO = pasture plus sunflower oil plus sterculic oil treatment, VA = vaccenic acid

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Conjugated linoleic acid (CLA) is a collective term for geometrical and positional conjugated dienoic isomers of linoleic acid. The primary CLA isomer in dairy products, cis-9, trans-11 CLA, is a potent anticarcinogen in animal models (Ip et al., 1999), and this has created a world-wide interest in the biology of CLA in dairy cows.

In ruminants, dietary polyunsaturated fatty acids undergo biohydrogenation in the rumen. cis-9, trans-11 CLA is an intermediate in rumen biohydrogenation of linoleic acid, and it was originally assumed that this was the source of cis-9, trans-11 CLA in milk fat (Harfoot and Hazlewood, 1988; Griinari and Bauman, 1999). Recently (Griinari et al. (2000) demonstrated endogenous synthesis of cis-9, trans-11 CLA also occurred, and this involved the enzyme ⁹-desaturase, with the precursor being trans-11 18:1 (vaccenic acid; VA), another intermediate in the biohydrogenation of linoleic acid. Consistent with this, diets supplemented with plant oils high in linoleic acid result in increases in milk fat concentration of CLA (Bauman et al., 2001; Stanton et al., 2003). Recent investigations have evaluated the importance of endogenous synthesis for TMR diets based on preserved forages, concentrates, and plant oil supplements; results uniformly indicate that endogenous synthesis was the major source of cis-9, trans-11 CLA in milk fat (Griinari et al., 2000; Corl et al., 2001; Lock and Garnsworthy, 2002; Piperova et al., 2002).

Pasture diets have several differences that relate to milk fat CLA when compared with TMR diets. Concentrations of cis-9, trans-11 CLA in milk fat from cows fed pasture diets are typically higher (Bauman et al., 2001; Stanton et al., 2003). In addition, the lipids in pasture are high in linolenic acid, and rumen biohydrogenation of linolenic acid does not produce cis-9, trans-11 CLA as an intermediate (Harfoot and Hazlewood, 1988; Griinari and Bauman, 1999). Thus, the importance of endogenous synthesis of CLA in milk fat of pasture-fed cows may differ.

The present study was undertaken to evaluate the importance of endogenous synthesis of cis-9, trans-11 CLA in milk fat of cows fed fresh pasture. A direct approach was used to examine this by inhibiting the activity of the ⁹-desaturase with sterculic oil. A secondary objective was to supplement the pasture diet with sunflower oil, a rich source of linoleic acid, and examine effects on the concentration of cis-9, trans-11 CLA in milk fat in the presence and absence of sterculic oil.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Animals, Experimental Design, and Treatments
The experiment was conducted at Dexcel, Hamilton, New Zealand, and lasted 48 d during October through November, 2000. All procedures were approved by the Ruakura Animal Ethics Committee (Hamilton, New Zealand).

Four multiparous, rumen-fistulated Holstein Friesian cows (541 ± 44 kg BW; mean ± SD) in early lactation (51 ± 17 DIM; mean ± SD) were randomly allocated to four treatments in a 4 x 4 Latin square design. Treatments were arranged as a 2 x 2 factorial. Treatments were pasture only (PAS), pasture plus sterculic oil (PAS+SO), pasture plus sunflower oil (PAS+SFO), and pasture plus sunflower oil plus sterculic oil (PAS+SFO+SO). Each experimental period consisted of a 2-d acclimation period, a 4-d treatment period, and an 8-d "washout" period.

Feeding and Milking Management
Cows were fed a pasture diet for the entire experiment. On a DM basis, the pasture sward consisted of approximately 65% perennial ryegrass (Lolium perenne L.), 30% other grasses (Dactylus glomerata, Holcus lanatus, and some Poa species), 2.5% white clover (Trifolium repens L.), and 2.5% weeds. Herbage mass was approximately 3000 kg of DM/ha when the pasture was harvested. During the acclimation and treatment periods, fresh pasture was cut twice daily at 0600 and 1500 h and offered for ad libitum intake to cows housed in individual metabolism stalls. The pasture was cut to approximately 40 mm above ground, and collected using a loader wagon. This cut and carry method has been used in previous trials to represent grazed pasture (Carruthers et al., 1996; Prosser and McLaren, 1997; Mackle et al., 2003; Roche et al., 2003). During the washout period, cows were allowed to graze pasture as a single herd and had unrestricted access to water. The pasture grazed during the washout period was similar in nutrient composition to the fresh pasture offered in the metabolism stalls (Table 1).

Sterculic Oil Infusions
Sterculic oil was derived from the seeds of the Sterculia foetida tree. The seeds were harvested from trees in India and imported to New Zealand. At the Dexcel laboratories (Hamilton, New Zealand), seeds were dehulled and crushed, and meats were refluxed in diethyl ether to extract the oil (AOAC, 1998). During the 4-d treatment period, cows in the PAS+SO and PAS+SFO+SO treatments received 9 g/d of sterculic oil. The daily dose of sterculic oil was chosen based on previous investigations with lactating ruminants (Bickerstaffe and Johnson, 1972; Porter, 1984; Griinari et al., 2000; Corl et al., 2001, 2002).

The sterculic oil emulsion was prepared based on the method described by Mackle et al. (2003). Briefly, skim milk powder (New Zealand Milk Products, Wellington, New Zealand) was rehydrated in water at 45°C for 20 min. The sterculic oil mixture was briefly heated to 50°C and was then incorporated into the skim milk using a high shear mixer (Ultra Turrax T50: Janke and Kunkel, IKA Labortechnik, Germany). The mixture was then heated to 74°C for 15 s to pasteurize, and passed through a homogenizer (APV Rannie, Copenhagen, Denmark) at 74°C with a first stage pressure of 13.0 MPa (2000 psi) and a second stage pressure of 3.45 MPa (500 psi). The final emulsion (2.25% sterculic oil, weight basis) was cooled to <7°C in an ice bath, and samples were taken for particle size testing (average size = 0.9 µm) and determination of TS. An identical mixture of skim milk was prepared for use in infusions of treatment groups not receiving sterculic oil (PAS and PAS+SFO treatments). In addition, for consistency the same batch of skim milk powder was used throughout the study.

The sterculic oil emulsions were infused into the abomasum to avoid alterations by rumen bacteria. To access the abomasum, a polyvinyl chloride tube (Nalgene 180 PVC; 0.47 cm i.d. x 0.78 cm o.d.; Nalgene Co., Rochester, NY) was passed through the rumen fistula and sulcus omasi into the abomasum (Spires et al., 1975) where it was secured by a 10-cm rubber flange. Infusions were via peristalic pumps (STA-131900; Desage, Heidelberg, Germany) calibrated to provide a constant delivery rate over the 24-h period (4 L/d). Sanitized containers (5 L) were placed on ice and served as reservoirs for the infusates. These were checked regularly to monitor flow rates and new containers of infusate were provided every 24 h. In addition, infusion lines and flanges were checked twice daily to ensure they remained secured in the abomasum.

Sunflower Oil Supplementation
Sunflower oil (James Gilmour and Co. Ltd., Hamilton, New Zealand) was used in this experiment. In New Zealand pasture-based dairy systems, cows are generally a considerable distance from facilities, and a common practice is to administer supplements, either once or twice daily, during milking (Roche et al., 2003). In the present study, 200 and 250 g of sunflower oil were administered daily into the rumen through the rumen fistula at approximately 0700 and 1600 h, respectively. Sunflower oil was squirted from a 300-mL syringe in 50-mL aliquots. Each aliquot was mixed with rumen contents by hand for approximately 60 s. This method was used in an attempt to combine the sunflower oil with the rumen contents, coat the surface of the feed particles, and subsequently allow the sunflower oil to undergo biohydrogenation. The daily dose of 450 g of sunflower oil equates to approximately 2.8% of DMI and is similar to dose rates used by other investigators (e.g. Kelly et al., 1998; Dhiman et al., 2000).

Sampling Procedures and Routine Analyses
During the treatment period, pasture intake for each cow was measured daily, and DMI was calculated as outlined by Roche et al. (2003). During the washout period, animals grazed the same paddock from which the pasture offered in the metabolism stalls was harvested. Representative samples of pasture offered and refused during the treatment period were dried daily at 95°C to constant weight to determine DM content. A representative daily sample of pasture was frozen, then bulked within each treatment period and freeze-dried (Cuddon instrument model 0610, Blenheim, New Zealand) at 40°C. The bulked sample was ground to pass through a 0.5-mm sieve (Christy Lab Mill, UK) and stored until extracted for analysis of fatty acid composition.

A second representative sample of pasture was collected daily, bulked within treatment period, and dried at 95°C for 12 h. The dried sample was ground to pass through a 0.5-mm sieve and analyzed for digestible organic matter (DOM), N, NDF, ADF, and ash. Digestible OM was determined by the method of Tilley and Terry (1963), and metabolizable energy (ME) was calculated from DOM (ME = DOM x 0.16/4.186; MAFF, 1975). Neutral detergent fiber and ADF were determined by reflux methods as outlined in AOAC (1990) and Van Soest et al. (1991), respectively. The N content was determined by a Kjeldahl method using an automated Tecator instrument, (Foss Denmark; AOAC, 1990) and CP was calculated from N (CP = N x 6.25). Ash was determined according to the method of AOAC (1990). The nutritive characteristics of pasture offered are presented in Table 1. Individual milk samples were taken at each milking, and daily composites were formed based on weight proportionality according to milk yields. One composite aliquot was analyzed for fat, lactose, CN, CP, and TS with an infrared milk analyzer (FT120; Foss Electric, Hillerød, Denmark). In addition, daily composite samples from the final day of the acclimation period and the 4-d treatment period were analyzed for fatty acid composition.

Fatty Acid Analysis
Milk fat was extracted from the fresh milk samples using the Röese-Gottlieb fat extraction procedure (IDF, 1987) and stored at -20°C until analyzed for fatty acid composition. Fatty acid methyl esters were quantified by gas chromatography after methylation using sodium methoxide as described by MacGibbon (1988). Gas chromatographic analyses of fatty acid methyl esters were performed on a GC-17A equipped with a flame ionization detector, autosampler, and autoinjector (Shimadzu Corporation, Kyoto, Japan). A 120-m BPX-70 column (120 m x 0.25 mm i.d. and 0.25 µm film thickness; SGE, Australia) was used and 0.2-µL solvent solution was injected using an on-column injection technique combined with programmed temperature volatilization. The initial temperature was set at 80°C for 0.1 min and then ramped to 230°C at a rate of 250°C/min. The initial oven temperature was 80°C, then ramped to 190°C at a rate of 2°C/min and held for 25 min. Injector and detector temperatures were set at 250°C.

Standards for CLA and other fatty acids were obtained from Matreya Inc. (Pleasant Gap, PA), and CLA isomer mixes were obtained from Sigma Chemical (St. Louis, MO) and NuCheck Prep (Elysian, MN). In addition, a butter reference standard (CRM 164; Commission of the European Communities, Community Bureau of Reference, Brussels, Belgium) was used as a qualitative reference for individual fatty acids and GLC 87 and 74X (NuCheck Prep) were used as quantitative methyl ester references.

Pasture lipids were extracted and methylated according to the one-step method of Garces and Mancha (1993). We added 6.6 mL of methanol:toluene:2,2-dimethoxypropane:sulphuric acid (39:20:5:2 vol/vol/vol) followed by 3.4 mL of n-heptane to 0.2 g of freeze-dried pasture. The mixture was heated to 80°C in dry heating blocks, shaken vigorously to form a single phase, and returned to the heating block for a further 2 h. The mixture was removed and allowed to cool to room temperature, then shaken vigorously to allow phases to separate. The upper phase was subsampled (1 mL) for subsequent GC analysis. Analyses of fatty acid compositions were performed on a Hewlett Packard 5890 Series II Gas Chromatograph equipped with a 30-m RTX-2330 column (30 m x 0.32 mm i.d. and 0.2 µm film thickness; Restek Corp). The initial oven temperature was 100°C for 1 min then ramped to 230°C at 10°C/min and held for 14 min. Injector and detector temperatures were maintained at 300°C, and the helium carrier gas flow rate was 2.5 mL/min.

For fatty analysis of sterculic oil, lipids were extracted using the method of Hara and Radin (1978) as described by Corl et al. (2001). Fatty acid methyl esters were prepared by transesterification with sodium methoxide (Christie, 1982) as adapted by Chouinard et al. (1999). Fatty acids were quantified by GC (Hewlett Packard GCD system HP G1800 A; Avondale, PA) equipped with a CP-Sil 88 column (100 m x 0.25 mm i.d. and 0.20 µm film thickness; Chrompack, The Netherlands). The oven temperature was initially 50°C for 1 min then ramped to 160°C at 5°C/min and held for 42 min. The temperature was then ramped again at 5°C/min to 190°C and held for 22 min. Injector and detector temperatures were maintained at 250°C and the helium carrier gas flow rate was 1 mL/min.

The fatty acids in sunflower oil were methylated by the same procedure as used for milk fat. Gas chromatographic analyses of sunflower oil fatty acids were performed on a Shimadzu GC17A (Shimadzu Corporation) equipped with a 15-m EC-1000 column (15 m x 0.53 mm i.d. and 1.2 µm film thickness; Alltech Associates, Inc., Deerfield, IL) with hydrogen as the carrier gas (10 mL/min). The flame ionization detector was set at 250°C. The column was operated at an initial temperature of 50°C for 1.5 min, then temperature programmed at 15°C/min to 150°C followed by 6°C/min to 220°C where it was held for 3 min. Cold on-column injection (1.0 µL) was used with an initial temperature of 35°C before ramping to 150°C at 250°C/min and then 220°C at 150°C/min.

Calculations
Sterculic oil was abomasally infused to inhibit ⁹-desaturase activity; inhibition may not be complete at the dose used, however. The milk fatty acids 10:0, 12:0, and 14:0 are synthesized almost exclusively in the mammary gland and can be acted upon by ⁹-desaturase to form cis-9 10:1, cis-9 12:1, and cis-9 14:1, respectively (Bauman and Davis, 1974). Consequently, the synthesis of cis-9 10:1, cis-9 12:1, and cis-9 14:1 is almost solely dependent on ⁹-desaturase, and the magnitude of reduction in these 3 fatty acids in milk fat following sterculic oil treatment can be used to estimate the extent of ⁹-desaturase inhibition. This provides a correction factor that can be applied to the sterculic oil-induced reduction of cis-9, trans-11 CLA, to estimate endogenous synthesis as detailed elsewhere (Griinari et al., 2000; Corl et al., 2001, 2002).

Statistical Analysis
All data were analyzed by REML for a factorial design using the statistical procedures of Genstat VI (Genstat VI, 2002) with cows as the experimental unit. Mean values for each treatment were determined for milk yield, milk composition, and DMI for d 4 of the treatment period using the last day of the acclimation period as a covariant. Where no significant interaction between sterculic oil infusion and sunflower oil administration (SO x SFO) was found, main treatment effects (sterculic oil; SO and sunflower oil; SFO) are discussed. The full model is:

where Y_ijk = observation, µ = overall mean, T_i = treatment (q = 1, 2, 3, and 4), P_j = period, (r = 1, 2, 3, and 4), C_k = cow (s = 1, 2, 3, and 4), and E_ijk = residual error; period and treatment factors are the fixed effects with cows as the random effect.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The fatty acid composition of pasture, sterculic oil, and sunflower oil is presented in Table 2 and the fatty acid composition and total fatty acid content of each treatment diet are presented in Table 3. The predominant fatty acid in pasture was linolenic acid, making up approximately 48% of total fatty acids in the PAS and PAS+SO treatments. The predominant fatty acid in the treatments involving sunflower oil supplementation (PAS+SFO and PAS+SFO+SO) was linoleic acid, making up approximately 33% of total fatty acids. Sterculic oil contains 2 cyclopropenoic fatty acids, sterculic acid (19:1) and malvalic acid (18:1), at 55.9 and 6.3% of total fatty acids, respectively.

Both sterculic oil infusion and sunflower oil supplementation resulted in distinct changes in the fatty acid composition of the milk and these effects were maximized by d 4 of treatment (temporal pattern not presented). Mean values from d 4 of the treatment period are presented in Table 5. Sterculic oil infusion substantially reduced the concentration of all milk fatty acids containing a cis-9 double bond, and in particular reduced (P < 0.01) cis-9, trans-11 CLA by 70%. As a consequence, the ratios of fatty acids pairs that represent product/substrate for ⁹-desaturase were decreased by 2- to 5-fold following sterculic oil infusion (Figure 1).

View larger version (24K): [in this window] [in a new window]   Figure 1. Ratios of milk fatty acid pairs that represent product/substrate for 9-desaturase. Values represent d 4 of the treatment period. Treatments were pasture only (PAS), pasture + abomasal infusion of 9 g/d sterculic oil (PAS+SO), pasture + 450 g/d sunflower oil (PAS+SFO), pasture + 450 g/d sunflower oil + abomasal infusion of 9 g/d sterculic oil (PAS+SFO+SO). Vertical brackets represent standard errors of the difference.

There was a significant interaction (P < 0.05) between treatments (SO x SFO) in the concentration of VA in milk fat. Sunflower oil administration (PAS+SFO) and sterculic oil infusion (PAS+SO) caused an 18 and 12% increase (P < 0.05) in VA concentration, respectively, when compared to the VA concentration in milk fat from the PAS treatment. However, sterculic oil infusion did not increase milk fat VA concentration in the PAS+SFO+SO treatment when compared with the PAS+SFO treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The present study is the first to examine the importance of endogenous synthesis of cis-9, trans-11 CLA in milk fat of pasture fed cows. Endogenous synthesis involves the enzyme ⁹-desaturase with VA produced in rumen biohydrogenation as the substrate. In lactating dairy cows, mammary epithelial cells have a high activity of ⁹-desaturase (Bickerstaffe and Annison, 1970; Kinsella, 1972; McDonald and Kinsella, 1973; Ward et al., 1998), which is consistent with the mammary gland being a major site for endogenous synthesis of cis-9, trans-11 CLA. However, ⁹-desaturase also occurs in the small intestine and adipose tissue of ruminants (Bickerstaffe and Annison, 1969; St. John et al., 1991; Ward et al., 1998), suggesting some endogenous synthesis may occur in these tissues as well.

We examined endogenous synthesis using a direct approach where sterculic oil was infused as a means to inhibit ⁹-desaturase. Sterculic oil contains sterculic acid and malvalic acid, 2 cyclopropenoid fatty acids that are very specific inhibitors of ⁹-desaturase (Phelps et al., 1965; Jeffcoat and Pollard, 1977). Abomasal infusion of sterculic oil resulted in reciprocal changes in the milk fat content of cis-9, trans-11 CLA and VA. However, on the basis of milk fatty acid yield the reduction in cis-9, trans-11 CLA was greater than the increase in VA. The explanation for this is not entirely clear, but several contributing factors can be identified. One is the endogenous synthesis of cis-9, trans-11 CLA in other tissues as discussed above. Sterculic oil infusion would also inhibit ⁹-desaturase in these tissues, thereby reducing their contribution to the CLA supplied to the mammary gland. A second reason is the presence of trans-7, cis-9 CLA. It is the second most abundant CLA isomer in milk fat and is derived almost exclusively from endogenous synthesis by ⁹-desaturase using rumen-derived trans-7 18:1 as the substrate (Corl et al., 2002; Piperova et al., 2002). In most GC analysis including that used in the present study, the trans-7, cis-9 CLA isomer co-elutes with cis-9, trans-11 CLA.

The inhibition by sterculic oil was reflected in the decreased milk fatty acids containing a cis-9 double bond and a 2- to 5-fold decrease in ratios for milk fatty acid pairs that represent product/substrate for ⁹-desaturase. The inhibition of ⁹-desaturase was not complete, but a correction factor for the extent of the inhibition can be estimated by changes in milk fatty acids dependent on ⁹-desaturase as detailed by Griinari et al. (2000) and Corl et al. (2001, 2002). In the present study, cis-9 10:1, cis-9 12:1, and cis-9 14:1 were used to calculate correction factors and treatment with sterulic oil decreased their content in milk fat by 77, 77, and 61%, respectively. Of these, the value of 77% represents a correction factor that gives a minimum estimate of endogenous synthesis. Applying this, it is estimated that over 91% of the cis-9, trans-11 CLA secreted in milk was derived from endogenous synthesis in cows fed fresh pasture.

Previous studies using a similar direct approach to estimate the importance of endogenous synthesis as a source of cis-9, trans-11 CLA in milk fat have involved TMR diets based on preserved forages, concentrates and plant oil supplements (Griinari et al., 2000; Corl et al., 2001). Pasture diets have a number of differences that are important considerations in the biology of CLA in ruminants. First, fresh pasture results in a milk fat content of cis-9, trans-11 CLA that is greater than observed with typical TMR diets. In the present study, milk fat cis-9, trans-11 CLA averaged approximately 1.2 g/100 g of fatty acid (Table 5) compared with 0.4 to 0.6 g/100 g of fatty acid in the studies by Griinari et al. (2000) and Corl et al. (2001). Second, the fatty acids present in concentrate feeds and most plant oils used in dairy cow diets are high in linoleic acid, and rumen biohydrogenation of linoleic acid produces both cis-9, trans-11 CLA and VA as intermediates. In contrast, pasture is high in linolenic acid (approximately 49% of total fatty acids in the present study; Table 2), and its biohydrogenation produces VA, but not cis-9, trans-11 CLA, as an intermediate (Harfoot and Hazlewood, 1988; Griinari and Bauman, 1999). These differences suggest that endogenous synthesis may be a relatively more important source of CLA from cows consuming pasture. Indeed, the estimate of >91% of milk fat cis-9, trans-11 CLA from endogenous synthesis in the present study compares with 64 and 78% reported for similar studies with TMR diets (Griinari et al., 2000; Corl et al., 2001).

The importance of endogenous synthesis of cis-9, trans-11 CLA has also been examined by an indirect approach where rumen outflow of cis-9, trans-11 CLA in TMR-fed cows was estimated and compared with the secretion of cis-9, trans-11 CLA in milk fat. These studies also demonstrated that endogenous synthesis was the major source of cis-9, trans-11 CLA in the milk fat of lactating dairy cows, with estimates of >80% by Lock and Garnsworthy (2002) and >93% by Piperova et al. (2002). This indirect approach has limitations and assumptions that differ from those associated with the direct approach discussed above (Bauman et al., 2003). Nevertheless, results from both approaches are consistent and when combined with the present study, results clearly demonstrate that endogenous synthesis is the major source of cis-9, trans-11 CLA in milk fat across a wide range of diets.

Our second objective sought to increase cis-9, trans-11 CLA content of milk fat by supplementing the pasture diet with sunflower oil. Plant oils high in linoleic acid have been shown to increase milk fat CLA by several-fold when supplemented to cows consuming a typical forage/concentrate based TMR (Chouinard et al., 1998; Kelly et al., 1998; Dhiman et al., 2000). However, in the present experiment, sunflower oil at 2.8% DMI had no affect on milk fat cis-9, trans-11 CLA concentration. Previous attempts to manipulate milk fat cis-9, trans-11 CLA concentration in pasture-fed cows have met with mixed success. Lawless et al. (1998) supplemented grazing cows with full fat soybeans (high in linoleic acid) and full fat rapeseeds (high in oleic acid), and observed increases in milk fat CLA concentration with both treatments. Conversely, Dhiman et al. (2002) found no change in the milk fat content of cis-9, trans-11 CLA when extruded full fat soybeans (high in linoleic acid) were added to a pasture diet and Stanton et al. (1997) reported mixed results when grazing cows were supplemented with rapeseed; a high rate of rapeseed supplement (1650 g/d) resulted in an increase in milk fat CLA, but a lower rate (825 g/d) produced no effect.

In addition to serving as the substrate for endogenous synthesis of cis-9, trans-11 CLA, VA is also incorporated into milk fat, and there is a strong correlation between the milk fat content of VA and cis-9, trans-11 CLA (Bauman et al., 2003). In the present study, sunflower oil supplementation increased VA concentrations in milk fat by only 18%, whereas the concentrations of linoleic, stearic, and oleic acids increased by 51, 35, and 29%, respectively. Both linoleic and oleic acids are high in sunflower oil and their increase in milk fat suggests that a portion of these fatty acids escaped biohydrogenation in the rumen. It may be that the 4-d treatment period was insufficient time for adaptation of the rumen microbial population involved in biohydrogenation. However Dhiman et al. (2002) observed only minimal effects on cis-9, trans-11 CLA concentration in milk fat following a 6-wk supplementation of a pasture diet with full-fat extruded soybeans. Rumen passage rate is more rapid in pasture-fed cows than those fed a TMR, and this may affect the rumen microbial population involved in biohydrogenation (Kolver, 1997). However, the 35% increase in the stearic acid concentration in milk fat suggests that some of the linoleic acid in the sunflower oil underwent complete biohydrogenation. Thus, additional studies will be needed to identify the basis for the lack of an effect of sunflower oil on milk fat content of VA and cis-9, trans-11 CLA in pasture-fed cows.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The present experiment is the first to quantify endogenous synthesis of cis-9, trans-11 CLA in lactating dairy cows fed pasture only where the predominant dietary fatty acid is linolenic acid. cis-9, trans-11 CLA in milk fat averaged 1.2 g/100 g of fatty acid for cows fed fresh pasture and endogenous synthesis accounted for more than 91% of the cis-9, trans-11 CLA secreted in milk fat. Thus, keys to increase milk fat content of cis-9, trans-11 CLA include increasing rumen production of VA and increasing the mammary activity of ⁹-desaturase. However, in the present short term study (4 d) supplementing pasture with sunflower oil at 2.8% DMI did not increase cis-9, trans-11 CLA concentration in milk fat and the basis for this lack of response requires further investigation.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Thanks to Amita Chand and Joelle Fleet for fatty acid analyses. Special thanks to John Roche, Erna Jansen, David Phipps, Pat Laboyrie, Dexcel No. 5 Dairy staff, Margaret Bryant, and Ross McKee for their skilled assistance, and to Barbara Dow for statistical analyses and advice on interpretation.

	FOOTNOTES

 
^* Present address: Fonterra, Private Bag 92032, Auckland, New Zealand.

Present address: Agriculture Victoria, Ellinbank, RMB 2460, Ellinbank VIC 3821, Australia.

Received for publication May 28, 2003. Accepted for publication October 2, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 


Association of Official Analytical Chemists. 1990. Official Methods of Analysis. 15th ed. AOAC, Washington, DC.

Association of Official Analytical Chemists. 1998. Official Methods of Analysis. 16th ed. AOAC, Gaithersburg, MD.

Bauman, D. E., B. A. Corl, L. H. Baumgard, and J. M. Griinari. 2001. Conjugated linoleic acid (CLA) and the dairy cow. Pages 221–250 in Recent Advances in Animal Nutrition. P. C. Garnsworthy and J. Wiserman, ed. Nottingham University Press, Nottingham, UK.

Bauman, D. E., B. A. Corl, and D. G. Peterson. 2003. The biology of conjugated linoleic acids in ruminants. Pages 146–173 in Advances in Conjugated Linoleic Acid Research. Vol. 2. J. L. Sebedio, W. W. Christie, and R. O. Adlof, ed. AOCS Press, Champaign, IL.

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