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© 2008 American Society for Nutrition J. Nutr. 138:710-717, April 2008

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Ruminal Infusions of Cobalt-EDTA Reduce Mammary {Delta}9-Desaturase Index and Alter Milk Fatty Acid Composition in Lactating Cows1–3,

Kevin J. Shingfield4,*, Anu Arölä4, Seppo Ahvenjärvi4, Aila Vanhatalo4,6, Vesa Toivonen4, J. Mikko Griinari5 and Pekka Huhtanen4,7

4 Animal Production Research, MTT Agrifood Research Finland, Jokioinen, FIN 31600, Finland; and 5 Department of Animal Science, University of Helsinki, FIN 00014, Finland

* To whom correspondence should be addressed. E-mail: kevin.shingfield{at}mtt.fi.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Ruminal administration of a triple indigestible marker system comprised of cobalt EDTA (CoEDTA), ytterbium acetate (YbAc), and chromium-mordanted straw (CrS) decreases product:substrate ratios for {Delta}9-desaturase in bovine milk fat. This experiment was designed to identify the marker(s) responsible and develop an alternative system for simultaneous determination of nutrient flow in the gastro-intestinal tract and milk fatty acid composition. Five lactating dairy cows were used in a 5 x 5 Latin square with 21-d periods to evaluate the effects of YbAc, CoEDTA, and CrS independently or as part of a triple marker system (TMS), and CrEDTA as an alternative to CoEDTA on milk fat composition. Markers were administered in the rumen over a 7-d interval and samples of milk were collected on d –1, 3, 7, and 11. Both TMS and CoEDTA alone reduced the concentrations of milk fatty acids containing a cis-9 double bond, whereas YbAc, CrS, and CrEDTA had no effect. Reductions in product:substrate ratios for {Delta}9-desaturase were time dependent and evident within 3 d of administration. Ruminal infusion of CoEDTA for 7 d induced mean decreases in milk cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 18:1/18:0, and cis-9, trans-11 conjugated linoleic acid/trans-11 18:1 concentration ratios of 47.7, 26.7, 40.3, and 42.6%, respectively. In conclusion, ruminal infusion of CoEDTA alters milk fatty acid composition and appears to inhibit {Delta}9-desaturase activity in the bovine mammary gland. Results indicate that a TMS based on CrEDTA, YbAc, and indigestible neutral detergent fiber can be used for estimating nutrient flow without altering milk fat composition in lactating cows.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Based on the potential benefits to human health there is considerable interest in altering the composition of ruminant milk fat with reductions in 12:0, 14:0, and 16:0 and increases in cis-9 18:1, cis-9, trans-11 conjugated linoleic acid (CLA)8, and 18:3(n-3) concentrations being targeted (1–5). Cis-9, trans-11 CLA secreted in ruminant milk is derived directly from the incomplete metabolism of 18:2(n-6) in the rumen or via conversion of trans-11 18:1 by the {Delta}9-desaturase enzyme in animal tissues (6). Several studies have provided clear evidence that endogenous synthesis is the major source contributing between 64 and 97% of cis-9, trans-11 CLA incorporated into milk fat (7–9). The appearance of cis-9 18:1 in milk originates from cis-9 18:1 in the diet that escapes metabolism in the rumen and via the action of {Delta}9-desaturase enzyme on 18:0 (10–12).

Nutrition is the major environmental factor regulating milk fat composition (1–3,5). To understand the mechanisms underlying milk fatty acid responses to changes in diet composition, an evaluation of the effects on both ruminal and mammary lipid metabolism is required. Measurements of fatty acids at the duodenum in lactating cows have been based on the collection of spot samples and estimating digesta flow using Cr2O3 (13), YbCl3, and polyethylene glycol (14) or YbCl3 and cobalt EDTA (CoEDTA) (15) as markers. Use of a single marker is known to introduce bias in estimates of dry matter flow (16,17) and also prevents correction of the composition of spot digesta for errors due to unrepresentative sampling (17). Lipid is not evenly distributed in digesta, with evidence that the majority of fatty acids are associated with particulate matter in the rumen (18). Fatty acid composition and content also differ between liquid- and solid-associated rumen bacteria (18–20). Sampling at the omasum rather than the duodenum reduces the requirements for surgical intervention, results in measurements of nutrient flow being less dependent on endogenous secretions, and minimizes the digestion of protein and microbial cells (21,22). However, accurate determination of digesta flow at the omasum requires the use of a triple marker system (TMS) due to the propensity of omasal digesta to separate during sampling (17). A system based on CoEDTA, ytterbium acetate (YbAc), and chromium-mordanted straw (CrS) has been used to assess the flow of fatty acids at the omasum (23,24), but this combination of markers decreases milk fat concentrations of fatty acids containing a cis-9 double bond, suggesting an inhibitory role on {Delta}9-desaturase (25).

The objectives of this study were to identify the marker or markers responsible for the effects on milk fat composition and provide relevant information for developing an alternative marker system for the simultaneous determination of fatty acid flow in the gastro-intestinal tract and milk fatty acid composition in lactating cows.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Cows and experimental design. All experimental procedures were approved by the Animal Experiment Committee of MTT Agrifood Research Finland in accordance with the 1985 Use of Vertebrates for Scientific Purposes Act. Five multiparous Finnish Ayrshire cows (85 ± 15.8 d postpartum and 595 ± 27.6 kg live weight; mean ± SD) fitted with a rumen cannula (i.d. 100 mm; Bar Diamond) were used in a 5 x 5 Latin square with 21-d experimental periods.

Treatments consisted of ruminal administration of the lithium salt of CoEDTA, YbAc, CrS, or simultaneous administration of all 3 markers as components of a TMS. In addition, an additional treatment comprised of ruminal infusion of CrEDTA was also examined to establish the possible role of EDTA on milk fat composition and evaluate the potential of CrEDTA to replace CoEDTA as a liquid phase marker in conjunction with the use of indigestible neutral detergent fiber (iNDF) as an alternative particulate marker to CrS. Administration of CrS, CoEDTA, and YbAc, separately or as TMS, were designed to provide 2.9 g/d Cr, 1.6 g/d Co, and 1.5 g/d Yb in accordance with previous studies (24,25). Ruminal infusions of CrEDTA were established to supply 2.2 g/d of Cr. Coarsely chopped barley straw was soaked in tap water overnight, washed in neutral detergent solution, rinsed with hot water, and labeled with Cr as outlined previously (26). CoEDTA (26) and CrEDTA (27) were prepared according to standard procedures and YbAc was obtained from a commercial source (Dasico A/S). To facilitate rapid equilibration of the marker concentration in the rumen, a priming dose of CrS (50 g/d) was given at 1800 on d 4. Thereafter, CrS (40 g/d) was administered as 2 equal doses at 12-h intervals at 1800 and 0600 for 7 d. Priming doses of CoEDTA (18 g/d), YbAc (6 g/d), and CrEDTA (18 g/d) were administered directly into the rumen at 1800 on d 5. Following the priming dose, CoEDTA (12 g/d), CrEDTA (12 g/d), and YbAc (4 g/d) were dissolved in 6 L of distilled water and infused via separate lines into the rumen at a constant rate for 144 h until 1800 on d 11. Ruminal infusions were made using polyamide tubing (i.d. 4 mm) that passed through the rumen fistula and a peristaltic pump (Watson-Marlow) calibrated to deliver 6 kg/d of infusate. Relatively long washout periods between marker administrations were applied (13 d) to minimize possible treatment carry-over effects.

    Sampling and analysis. Cows were housed in individual stalls in a dedicated metabolism unit and consumed water and a diet (Table 1) formulated to meet or exceed nutrient requirements (28). Individual cow intakes and milk yields were measured daily. Representative samples of fresh diets and feed refusals were collected daily and composited for each experimental period. Chemical composition and nutritive value of experimental feeds were determined using reference methods (29). Cows were milked twice daily at 0700 and 1645. Samples of milk for the determination of fat, crude protein, lactose, and fatty acid composition were collected over 2 consecutive milkings (1645 and 0700) on 4 occasions during each experimental period starting in the afternoon on d 3, 7, 11, and 15, corresponding to d –1, 3, 7, and 11 relative to the start of marker administration. Milk samples treated with Bronopol preservative (Valio) were analyzed for milk fat, crude protein, and lactose by near infrared analysis (29). Untreated samples of milk were composited according to yield on each day of sampling and stored at –20°C until submitted for fatty acid determinations.


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  		TABLE 1 Ingredient and chemical composition of the experimental diet

 
Total urine collection was made over a 96-h period starting at 1800 on d 8 of each experimental period. Urine was collected by means of a light harness attached around the vulva secured using an adhesive and voided urine was collected into a container via a flexible tube. Urinary pH was maintained below 3.0 using 10 mol/L H2SO4. Samples of urine voided over 24-h intervals were collected, composited for each sampling period, and stored at –20°C for measurements of Cr, Co, and Yb. Urine, CrS, and samples of administered marker solutions were prepared according to reference procedures (30) and analyzed for Co, Yb, and Cr using inductively coupled plasma spectroscopy (Iris Advantage, Thermo Jarrel Ash).

    Milk fatty acid analysis. Lipid in 1 mL of milk was extracted using diethylether/hexane and transesterified to FAME using freshly prepared methanolic sodium methoxide (24). Methyl esters were quantified by GLC using a gas chromatograph fitted with a 100-m fused silica capillary column (CP-SIL 88; Chrompack 7489) and hydrogen as the carrier gas. Total FAME profile in a 2-µL sample volume at a split ratio of 1:50 was determined using a temperature gradient program (24). Individual isomers of 18:1 and 18:2 were further resolved in a separate analysis under isothermal conditions at 170°C (24). Peaks were identified using authentic standards and GC-MS analysis of FAME (24). Methyl esters not contained in commercially available standards were formally identified by GC-MS analysis of 4,4-dimethyloxazoline fatty acid derivatives prepared from FAME of selected samples of milk (31,32). The distribution of CLA isomers in lipid supplements and milk was determined by HPLC using 4 silver-impregnated silica columns (ChromSpher 5 Lipids, 250 x 4.6 mm; 5-µm particle size, Varian Ltd) coupled in series using 0.1% (v:v) of acetonitrile in heptane (24,33) as the mobile phase.

    Statistical analysis. Dry matter intake, milk yield, and composition data recorded during the last 4 d of marker infusions were averaged and analyzed by ANOVA for a 5 x 5 Latin Square design with a statistical model that included the random effect of cow and fixed effect of period and treatment using the mixed procedure of SAS (version 8.2, SAS Institute). Measurements of milk fatty acid composition during marker administration were analyzed by repeated measures ANOVA using a model that included the fixed effect of period, treatment, time, and their interaction and random effects of cow assuming an auto regressive order 1 covariance structure fitted on the basis of Akaike Information and Schwarz Bayesian model-fit criteria. Mean fatty acid composition of milk collected before and at the end of marker administration was compared using a paired t test. Least square means ± SEM are reported and effects were considered significant at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
Marker treatments supplied the targeted amounts of rare earths into the rumen (Table 2). Dry matter intake, milk yield, milk fat, crude protein, lactose, and urea concentrations did not differ among treatments (Supplemental Table 1).


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  		TABLE 2 Amounts of rare earth markers administered into the rumen of lactating cows1

 
Concentrations of Yb in urine were below detection limits (<0.1 mg/kg) on all treatments. Urinary Cr excretion was not altered (P > 0.05) by administration of CrS into the rumen. Ruminal infusions of CoEDTA in isolation or as part of TMS increased Co excretion (mean recoveries of 2.56 ± 0.366 and 2.40 ± 0.388%, respectively) and 2.39 ± 0.346% of Cr administered as CrEDTA was recovered in urine.

Administration of CrEDTA, CrS, and YbAc for 7 d did not affect milk fatty acid composition, whereas both TMS and CoEDTA increased (P < 0.01) total SFA content and decreased (P < 0.01) total monounsaturated fatty acid and PUFA concentrations in milk (Table 3). Concentrations of cis-9 10:1, cis-9 12:1, cis-9 14:1, cis-9 16:1, cis-9 17:1, cis 18:1, cis-9 20:1, and cis-9 22:1 all decreased (P < 0.01) with ruminal administration of CoEDTA (39.4, 43.8, 44.7, 30.1, 18.4, 20.5, 17.3, and 34.0%, respectively). Administration of TMS resulted in corresponding reductions of 32.5, 37.2, 36.7, 24.7, 15.7, 15.8, 14.9, and 25.2%, respectively. Measurements of milk fat 18:1 isomer concentrations (Table 4) indicated that the alterations in cis 18:1 content to CoEDTA and TMS were due to decreases (P < 0.01) in cis-9 18:1 (21.4 and 16.3%, respectively), with evidence that CoEDTA also increased (P < 0.01) milk fat trans-11 (15.3%) and -13/14 concentrations (28.0%).


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  		TABLE 3 Effects of marker administration on milk fatty acid composition in lactating cows1

 

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  		TABLE 4 Effects of marker administration on milk 18:1 concentration in lactating cows1

 
With the exception of 18:2(n-6), concentrations of all 18:2 fatty acids in milk containing a cis-9 double bond in milk were reduced (P < 0.01) following administration of CoEDTA and TMS into the rumen (Table 5). CrS and YbAc had no effect on the distribution of 18:2 isomers in milk, whereas CrEDTA increased (P < 0.01) cis-9, trans-12 18:2, cis-9, trans-13 18:2, and trans-11, trans-13 CLA concentrations (Table 5).


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  		TABLE 5 Effects of marker administration on milk 18:2 concentration in lactating cows1

 
Due to the reductions in the concentration of cis-9 containing fatty acids, the ratios of cis-9 10:1/10:0, cis-9 12:1/12:0, cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 17:1/17:0, cis-9 18:1/18:0, trans-7, cis-9 CLA/trans-6–8 18:1, cis-9, trans-11 CLA/trans-11 18:1, cis-9 20:1/20:0, and cis-9 22:1/22:0 concentrations in milk decreased (P < 0.01) after 7 d of administration of CoEDTA (13.4–47.7%) or TMS (13.3–39.4%). Reductions in product/substrate concentrations for {Delta}9-desaturase to CoEDTA and TMS were evident within 3 d and continued to decrease until the end of CoEDTA or TMS administration (Fig. 1). Fatty acid ratios dependent on {Delta}9-desaturase increased after the withdrawl of CoEDTA and TMS treatments but remained lower (P < 0.01) than the ratios in milk collected before the start of marker administration (Fig. 1). Changes in cis-9 17:1/17:0, trans-7, cis-9 CLA/trans-6–8 18:1, cis-9 20:1/20:0, and cis-9 22:1/22:0 concentration ratios also varied in a similar manner in response to markers (data not presented).


Figure 1
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  		FIGURE 1  Temporal changes in milk cis-9 10:1/10:0 (A), cis-9 12:1/12:0 (B), cis-9 14:1/14:0 (C), cis-9 16:1/16:0 (D), cis-9 18:1/18:0 (E), and cis-9, trans-11 CLA/trans-11 18:1 (F) concentration ratios in response to ruminal administration of CrS, YbAc, CoEDTA, CrEDTA, or a TMS comprised of CrS, YbAc, and CoEDTA in lactating cows. Administration of markers (indicated by dotted lines) started from 1800 on d 4 and continued through to 1800 on d 11 during each 21-d experimental period. CrS was administered into the rumen 24 h before ruminal infusions of other markers. Pooled SEM for the concentration ratio of product:substrate for cis-9 10:1, cis-9 12:1, cis-9 14:1, cis-9 16:1, cis-9 18:1, and cis-9, trans-11 CLA = 0.008, 0.002, 0.009, 0.005, 0.089, and 0.016, respectively. Labeled means (n = 5) within a treatment without a common letter differ, P < 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Administration of a TMS comprised of CoEDTA, CrS, and YbAc altered milk fatty composition, increasing 4:0 concentrations and reducing the ratios of product:substrate for {Delta}9-desaturase in milk fat, confirming earlier observations (25). Relative reductions in the concentrations of milk fatty acids containing a cis-9 double bond are consistent with the previous observations (25) despite differences between individual cows and variations in the stage of lactation, milk production, and diet composition. Responses to constituent markers identified CoEDTA as responsible for the reduction in the {Delta}9-desaturase product:substrate ratios in milk fat. Ruminal infusions of CoEDTA altered milk fat composition within 3 d, with further changes in milk fatty acids evident after 7 d. No differences in fat composition have been found between milk samples collected 6 and 9 d after ruminal administration of CrS, YbAc, and CoEDTA (25), suggesting that the changes in milk fat composition reach a nadir after 6 d.

Measurements of urinary excretion confirmed earlier observations that absorption of Cr from mordanted straw (26) or Yb infused as YbAc (34) is negligible within the gastro-intestinal tract of ruminant animals, but ~3% of Co or Cr is recovered in urine when administered as a chelate with EDTA in the rumen (26). Because ruminal infusions of CoEDTA but not CrEDTA altered milk fat composition despite similar absorption, the possible contribution of EDTA can be excluded, with the implication that Co is the bioactive compound.

Ruminal infusions of CoEDTA alone or as a component of TMS reduced the product:substrate ratios for {Delta}9-desaturase for fatty acids of chain length from C10 to C22. De novo synthesis in the mammary gland is the major source of 10:0, 12:0, and 14:0 in milk fat triacylglycerides, fatty acids that serve as substrates for {Delta}9-desaturase yielding cis-9 10:1, cis-9 12:1, and cis-9 14:1, respectively (35). Decreases in milk fat {Delta}9-desaturase ratios for 10:0, 12:0, and 14:0 substrates strongly suggest that milk fat composition responses to Co are mediated via an effect on the mammary gland and ruminant tissues rather than by inducing changes in ruminal lipid metabolism and the supply of fatty acids available for incorporation into milk fat. Changes in milk fat composition to ruminal infusions of CoEDTA were also independent of alterations in the proportion of fatty acids derived from de novo synthesis (<16 carbon fatty acids; Table 3) or the peripheral circulation (>16 carbon fatty acids; Table 3). Such findings tend to suggest that alterations in milk fat composition occur in the absence of changes in pathways of de novo lipid synthesis or long-chain fatty acid uptake. Furthermore, cis-9 10:1/10:0, cis-9 12:1/12:0, cis-9 14:1/14:0, cis-9 16:1/16:0, and cis-9 18:1/18:0 concentration ratios are known to be highly correlated with mRNA abundance and activity of {Delta}9-desaturase in the mammary gland of mice (36) and goats (37,38). The ratios of all fatty acid pairs dependent on this enzyme were altered over time in response to CoEDTA and TMS consistent with Co directly or via other factors inhibiting {Delta}9-desaturase.

Palmitoyl- and stearoyl-CoA are the preferred substrates of {Delta}-9 desaturase (7) with conversion of 18:0 to cis-9 18:1 estimated as 40–52% (11,12,39), being quantitatively the most important in the bovine mammary. Previous studies have reported reductions in the cis-9 18:1/18:0 concentration ratio in bovine milk of 63.1–75.1% during abomasal infusions of sterculic oil (40–43), which contains 2 known inhibitors (7–2-octyl-1-cyclopropenyl heptanoic acid and 8–2-octyl-1-cyclopropenyl octanoic acid) of {Delta}9-desaturase. Postruminal infusions in lactating cows have also established that trans-10, cis-12 CLA (44,45), trans-9, trans-11 CLA (46), and trans-10, trans-12 CLA (47,48) also induce 17.5–42.4% decreases in cis-9 18:1/18:0 concentration ratios in milk fat. However, the mode of action on mammary metabolism may differ. Inhibitory effects of sterculic oil are mediated via a direct effect on enzyme activity rather than by reductions in {Delta}9-desaturase gene or protein expression (49). In contrast, trans-10, cis-12 CLA has been reported to decrease mRNA abundance for {Delta}9-desaturase in bovine mammary tissue (45,50) and bovine mammary epithelial cells (51,52), with evidence that the downregulation of genes encoding for {Delta}9-desaturase in vivo involves sterol response element binding protein-1 and thyroid hormone responsive spot 14 transcription factors as common regulatory elements (50). Further studies characterizing the bovine {Delta}9-desaturase gene promoter have identified a critical 36-bp fragment designated as the stearoyl-CoA desaturase transcriptional enhancer element region that contains binding sites for 3 transcriptional factors, including sterol response element binding protein-1 (52). It is not clear if the regulation of mammary {Delta}9-desaturase by trans-10, cis-12 CLA in the lactating cow involves a direct inhibitory effect on enzyme activity or if it arises from decreases in protein abundance as part of an overall downregulation of lipogenic enzymes. Based on in vitro studies, it has been postulated that the effects of trans-10, cis-12 CLA are a result of direct transcriptional downregulation due to a lack of SRBEP protein activation resulting in the loss of binding to the transcriptional enhancer element region and inhibition of the {Delta}9-desaturase promoter (52). Current data provide support for an inhibitory role of Co on mammary {Delta}9-desaturase, but the mode of action is not known. Previous studies have demonstrated that {Delta}9-desaturase activity in liver microsomes is reduced during incubation with Cu (53) or Cd (54) and administration of Cd decreases hepatic {Delta}9-desaturase activity in rats (55). The {Delta}9-desaturase system is known to be comprised of 3 components: NADH-cytochrome b5 reductase, cytochrome b5, and the terminal desaturase (56). In vitro studies have shown that the inhibitory effects of Cu (53) and Cd (54) on {Delta}9-desaturase are due to a reduction in terminal desaturase activity. During desaturation, electrons are transferred sequentially from NAD(P)H, via NADH-cytochrome b5 reductase and cytochrome b5, to the terminal desaturase, and finally to active oxygen, which is reduced to H2O (56). One possible working hypothesis to explain the current findings is that specific metal ions, including Co, interfere with the transfer of electrons from cytochrome b5 to the di-iron protein center (57) of the terminal desaturase central to the oxidation-reduction reactions required for the conversion of acyl-CoA substrates.

Ruminal administration of markers had no effect on milk fat synthesis. A lack of change in milk fat content to CoEDTA is consistent with milk fat synthesis being independent of acute decreases in {Delta}9-desaturase product/substrate concentration ratios during abomasal infusions of sterculia foetida oil (40–43), trans-9, trans-11 CLA (46), or trans-10, trans-12 CLA (47,48). Overall, the data from this experiment support the view that reductions in {Delta}9-desaturase activity are not a prerequisite or major component of diet-induced milk fat depression (46–48,58). Furthermore, the lack of significant treatment effects on concentrations of CLA isomers known to reduce {Delta}9-desaturase product:substrate ratios in bovine milk (44–48) indicate that the effects of Co on milk fat composition are not mediated by increases in ruminal outflow of these biohydrogenation intermediates.

Studies based on duodenal (59) or omasal (17,22) sampling techniques have shown that estimates of nutrient flow based on the use of single markers are subject to considerable variation. Provided that the assumption of homogenous digesta phases holds true, unrepresentative samples can be reconstituted to represent true digesta based on 2 or more distinct digesta phases (60). Current results indicate that CoEDTA is unsuitable as liquid phase marker in studies examining the effect of nutrition on ruminal lipid metabolism and milk fat composition. Data also indicated that CrEDTA could be used as an alternative to CoEDTA without altering milk fat composition, but this precludes the use of Cr-mordanted fiber as a large particle marker. Previous studies have established that the use of iNDF as a particulate marker results in smaller variations in nutrient flows at the duodenum compared with Cr-mordanted fiber as part of a double-marker system (61). Furthermore, iNDF is more evenly distributed between different particle size fractions in digesta compared with CrS (62), indicating that iNDF is a more ideal large particle marker. Rare earth markers that associate with all digesta phases or Cr2O3, which is not associated with any digesta phase, may provide reasonably accurate estimates of dry matter flow, but the composition of sampled digesta can be unrepresentative of true digesta (17,59). Lipid in digesta are mainly associated with particulate matter (18), which can introduce bias in estimates of fatty acid flow when the contribution of small and large particles is under- or overrepresented in digesta samples, even though dry matter flow may be accurately estimated. Therefore, a system based on a combination of markers that intimately associate with specific digesta phases represents the most reliable approach to assess fatty acid flows in the ruminant gastro-intestinal tract.

In conclusion, this study provided evidence that Co alters milk fat composition and reduces the mammary {Delta}9-desaturase index. Further research is required to establish the possible mode of action. Substituting CrEDTA for CoEDTA and using iNDF rather than Cr-mordanted fiber can be used as part of a TMS for the simultaneous determination of nutrient flow and milk fatty acid composition in the lactating cow.


   ACKNOWLEDGMENTS
 
We thank Minna Aalto for technical assistance during sample lipid analysis.


   FOOTNOTES
 
1 Supported by central funds received from MTT Agrifood Research Finland. Back

2 Author disclosures: K. J. Shingfield, A. Arölä, S. Ahvenjärvi, A. Vanhatalo, V. Toivonen, J. M. Griinari, and P. Huhtanen, no conflicts of interest. Back

3 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org. Back

6 Present address: Department of Animal Science, University of Helsinki, P.O. Box 28, FIN 00014, Finland. Back

7 Present address: Department of Animal Science, Cornell University, Ithaca, NY 14853. Back

8 Abbreviations used: CLA, conjugated linoleic acid; CoEDTA, cobalt EDTA; CrEDTA, chromium EDTA; CrS, chromium-mordanted straw; iNDF, indigestible neutral detergent fiber; NDF, neutral detergent fiber; TMS, triple marker system; YbAc, ytterbium acetate. Back

Manuscript received 5 December 2007. Initial review completed 30 December 2007. Revision accepted 23 January 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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