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Effects of Amide-Protected and Lipid-Encapsulated Conjugated Linoleic Acid (CLA) Supplements on Milk Fat Synthesis^*

¹ Department of Animal Science, Cornell University, Ithaca, NY 14853
² BASF Aktiengesellschaft, Nutrition Research Station, Offenbach, Germany

Corresponding author: D. E. Bauman; e-mail: deb6{at}cornell.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis; its ability to reduce milk fat output in a controlled manner as a feed supplement, has potential management applications in the dairy industry. The effectiveness of dietary supplements of trans-10, cis-12 CLA is related to the extent to which their metabolism by rumen bacteria is minimized. A number of processes have been used to manufacture "rumen-protected" feed supplements, and their efficacy can be described by the extent of protection from rumen bacteria as well as postruminal bioavailability. The objective of this study was to investigate the effects of 2 rumen-protected CLA supplements on milk fat synthesis. Using the same initial batch of CLA, supplements were manufactured by the formation of fatty acyl amide bonds or by lipid encapsulation. Three rumen fistulated Holstein cows were randomly assigned in a 3 x 3 Latin square experiment. Treatments were 1) no supplement (control), 2) amide-protected CLA supplement, and 3) lipid-encapsulated CLA supplement. Supplements were fed to provide 10 g/d of the trans-10, cis-12 CLA isomer. Over the 7-d treatment period, 21 and 22% reductions in milk fat yield were observed for the amide-protected and lipid-encapsulated supplements, respectively. Transfer of trans-10, cis-12 CLA into milk fat was also similar for the amide-protected (7.1%) and lipid-encapsulated (7.9%) supplements. Overall, the amide-protected and lipid-encapsulated CLA supplements were equally effective at reducing milk fat synthesis and had no effect on milk yield or dry matter intake.

Key Words: conjugated linoleic acid • milk fat depression • rumen-protected supplement • cow

Abbreviation key: AP-CLA = amide-protected CLA supplement, CLA = conjugated linoleic acid, LE-CLA = lipid-encapsulated CLA supplement

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Conjugated linoleic acid (CLA) is a collective term for different positional and geometric configurations of octadecadienoic acid containing a pair of double bonds in a conjugated configuration. Supplementation with various mixtures of CLA isomers has been shown to reduce milk fat content in lactating cows (Loor and Herbein, 1998; Chouinard et al., 1999a, 1999b; Mackle et al., 2003). Comparisons of relatively pure CLA isomers infused into the abomasum identified trans-10, cis-12 CLA to be a potent inhibitor of milk fat synthesis, while cis-9, trans-11 CLA (Baumgard et al., 2000, 2002; Loor and Herbein, 2003) as well as trans-8, cis-10 CLA and cis-11, trans-13 CLA (Perfield et al., 2004) had no effect on milk fat synthesis. Subsequent studies abomasally infused various doses of trans-10, cis-12 CLA and demonstrated a curvilinear relationship between the percent reduction in milk fat yield and both the dose of trans-10, cis-12 CLA and the milk fat content of trans-10, cis-12 CLA (Baumgard et al., 2001; Peterson et al., 2002).

A technology to reduce milk fat output in a controlled manner has potential application as a management tool (Bauman et al., 2001). However, the effectiveness of dietary supplements of trans-10, cis-12 CLA will be related to the extent that their metabolism by rumen bacteria is avoided. A number of processes have been used to produce "rumen-protected" supplements, and their efficacy is characterized by the extent of their protection from rumen bacteria and postruminal bio-availability (Wu and Papas, 1997). These include formation of calcium salts, fatty acyl amides, formaldehyde-protein matrix protection, and lipid encapsulation. To date, the majority of research conducted with rumen-protected CLA has used supplements consisting of calcium salts of FFA (Giesy et al., 2002; Perfield et al., 2002; Bernal-Santos et al., 2003). Data from these studies provide incentive to continue evaluating the use of controlled milk fat depression as a management tool. However, CLA supplements formulated with other methods of rumen protection have not been adequately examined. The objective of the present study was to investigate 2 supplements; an FFA form of CLA that was bound by amide bonds and a lipid-encapsulated supplement that contained methyl esters of CLA.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The Cornell University Institutional Animal Care and Use Committee approved all procedures involving animals. Three multiparous lactating Holstein cows fitted with rumen fistulas (78 ± 13 DIM; mean ± SD) were randomly assigned in a 3 x 3 Latin square experiment. Cows were housed in tie stalls at the Cornell University Large Animal Teaching and Research Unit and fed a TMR formulated to meet or exceed nutrient requirements (NRC, 2001) using the Cornell Net Carbohydrate and Protein System (Fox et al., 1992). Chopped alfalfa was the major forage component with cracked corn as the major concentrate (Table 1). Cows were fed ad libitum with equal portions of feed offered at 0700 and 1900 h daily. Water was available at all times.

The CLA used for the manufacture of the rumen-protected supplements originated from the same batch and was supplied by BASF AG (Ludwigshafen, Germany). Treatments were: 1) no supplement (control), 2) amide-protected CLA (AP-CLA; 54 g/d) (Akzo Nobel Chemicals, Inc., Dobbs Ferry, NY), and 3) lipid-encapsulated CLA (LE-CLA; 138 g/d) (Balchem Encapsulates, New Hampton, NY). The fatty acid composition of the supplements is provided in Table 2, and the daily amount was designed to provide 10 g of trans-10, cis-12 CLA. To ensure exact intakes, rumen-protected supplements were placed directly into the rumen through the fistula and mixed into the rumen contents once per day after the evening milking. The AP-CLA supplement, which was a solid at room temperature, was heated and a predetermined quantity placed on a small amount of feed, and then allowed to cool before being placed in the rumen. The LE-CLA supplement was a powder at room temperature and was administered as such. Treatment periods were 7 d in duration, and each treatment period was followed by a 7 d washout period.

Fatty acid methyl esters were quantified using a gas chromatograph (GCD system HP 6890+; Hewlett Packard, Avondale, PA) equipped with a CP-SIL 88 fused silica capillary column (100 m x 0.25 mm (i.d.) with 0.2-µm film thickness; Varian, Inc., Walnut Creek, CA). Gas chromatograph conditions were as described by Perfield et al. (2002). Fatty acid peaks were identified using pure methyl ester standards (Nu-Chek Prep, Elysian, MN). Additional standards for CLA isomers were obtained from Natural ASA (Hovdebygda, Norway). A butter oil reference standard (CRM 164; Commission of the European Community Bureau of References, Brussels, Belgium) was used to determine recoveries and correction factors for individual fatty acids.

Statistical Analysis
Data were statistically analyzed as a 3 x 3 Latin square design using the PROC MIXED procedure of SAS (2001). The model is: Y_ijk = µ + T_i + P_j + C_k + E_ijk, where Y_ijk is observation, µ is overall mean, T_i is treatment (i = 1, 2, and 3), P_j is period (j = 1, 2, and 3), C_k is cow (k = 1, 2, and 3), and E_ijk is residual error.

	RESULTS

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The AP-CLA and LE-CLA supplements are novel products, and analysis of total fat as well as fatty acid composition for each posed unique challenges. Determining total fat for the AP-CLA supplement required acidic conditions that were severe enough to break the amide bonds (Christie, 1989). We found that this required acid hydrolysis of the amide-protected CLA, and results indicated that the supplement had a lipid content of 87 ± 1.5% (mean ± SD; n = 3). The extracted lipid was in the FFA form, and this was considered in selecting the methylation method.

The LE-CLA supplement was manufactured by binding methyl esters of CLA to a silica matrix, and then coating this complex with hydrogenated soybean oil, which contained fatty acids in the triglyceride form. To extract total fat, an ether soxhlet extraction was used; the refluxing involved in this procedure was important for extracting the stearic acid which otherwise adhered to the silica once cooling began. Extraction was performed in triplicate, and the fat content averaged 72 ± 0% for the LE-CLA supplement. This supplement was manufactured from CLA methyl esters so that only the hydrogenated soybean oil component of the supplement required methylation and this was done using a base-catalyzed transmethylation as described previously.

Fatty acid analysis of the rumen-protected CLA supplements is presented in Table 2. Unique to the AP-CLA supplement was a sizable peak that eluted between the peaks for the cis-9, trans-11 CLA and trans-10, cis-12 CLA isomers. The lower proportion of trans-10, cis-12 CLA, suggests that this unique fatty acid is cis-11, trans-13 CLA produced by a thermal [1,5] sigmatropic rearrangement, which is common under the temperature conditions required for the manufacture of the AP-CLA supplement (Sæbø, 2003). Although an acid methylation was used, we are confident that the observed rearrangement was due to manufacturing conditions as this unique peak was not present when we conducted a test where the LE-CLA supplement and pure trans-10, cis-12 CLA were methylated under the same conditions (data not shown). Most likely, trans-8, cis-10 CLA was also formed through the same transformation from the cis-9, trans-11 CLA isomer, but was not identified due to coelution with the cis-9, trans-11 CLA isomer under the analytical conditions we used. The addition of hydrogenated soybean oil to the LE-CLA product resulted in a stearic acid content of this supplement that was about 10 times greater than that of the AP-CLA supplement.

The AP-CLA and LE-CLA supplements were supplied in quantities so that each provided 10.0 g/d of trans-10, cis-12 CLA. The temporal pattern for milk fat yield demonstrated a progressive reduction for cows receiving the CLA supplements that had reached nadir by d 4, while no change in milk fat yield was observed for the control (Figure 1). When supplements were terminated, the temporal pattern indicated a return to previous milk fat yield for the AP-CLA and LE-CLA treatment groups. Overall, the 2 CLA supplements caused similar reductions in milk fat content and milk fat yield (Table 3). In contrast to milk fat, CLA supplements had no effect on DMI or milk yield (Table 3). A trend for increased milk protein content (P < 0.07), as well as a small but statistically significant increase in protein yield was observed when cows received the CLA supplements.

View larger version (17K): [in this window] [in a new window]   Figure 1. Temporal pattern of milk fat yield during supplementation of rumen-protected CLA supplements [amide-protected CLA (AP-CLA); lipid-encapsulated CLA (LE-CLA)]. Animals received supplements for 7 d (dotted lines). Values represent means from 3 cows; SEM = 0.079 kg/d.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

In the present study, the amide-protected supplement provided CLA as free fatty acids, whereas the lipid-encapsulated supplement provided CLA as methyl esters. Recent work has shown that the FFA and methyl ester forms of trans-10, cis-12 CLA are equally effective in reducing milk fat synthesis when supplied by abomasal infusion (de Veth et al., 2004). We observed a gradual reduction in milk fat yield over the first few days of treatment and a return to control values when the supplement was terminated (Figure 1), and this is similar to changes observed when trans-10, cis-12 CLA was abomasally infused (Baumgard et al., 2000, 2001; Mackle et al., 2003) or provided intravenously (Viswanadha et al., 2003). The AP-CLA and LE-CLA supplements resulted in decreased secretion of milk fatty acids of all chain lengths, but the reduction was relatively greater for milk fatty acids containing 16 carbons (Table 4). These reductions are similar to those observed when milk fat depression is caused by abomasal infusion of trans-10, cis-12 CLA (Baumgard et al., 2001; Loor and Herbein, 2003; Mackle et al., 2003) or by feeding of calcium salts of CLA (Giesy et al., 2002; Perfield et al., 2002; Bernal-Santos et al., 2003). A lack of an effect on the ratio of fatty acid pairs reflecting ⁹-desaturase activity has also been observed when low doses of trans-10, cis-12 CLA were abomasally infused (Peterson et al., 2002) or when calcium salts of CLA were fed (Perfield et al., 2002; Bernal-Santos et al., 2003). In contrast, higher doses of trans-10, cis-12 CLA have been shown to inhibit ⁹-desaturase causing a decrease in the desaturase index (Baumgard et al., 2001; Loor and Herbein, 2003; Mackle et al., 2003).

The AP-CLA and LE-CLA supplements caused similar reductions in milk fat content and milk fat yield, with no effects on DMI or milk yield (Table 3). Specific effects of CLA on milk fat are consistent with results from a longer term study using rumen-protected CLA in the form of calcium salts (Perfield et al., 2002) as well as short-term studies involving abomasal infusions (Chouinard et al., 1999a, 1999b; Baumgard et al., 2000; 2001; Loor and Herbein, 2003; Mackle et al., 2003). In contrast, studies conducted with pasture-fed animals have reported significant increases in protein yield as well as milk yield when CLA was supplemented (Medeiros et al., 2000; Gulati et al., 2001); these are situations in which the energy content of the diet is limiting and protein is present in excess. Reducing energy demands through decreased milk fat synthesis in these situations may allow for a repartitioning of energy for the production of milk and milk protein. Similarly, CLA supplementation caused an increased milk yield in early lactation cows, but this was not accompanied by an increased protein yield consistent with the fact that the supply of amino acids would be limited in early lactation (Bernal-Santos et al., 2003). We utilized mid-lactation cows that were fed a dry diet in our metabolic unit, and performance was lower then when cows were housed at the University farm and fed a traditional TMR. Thus, in the present study an apparent repartitioning of energy also occurred when cows received the CLA supplements as indicated by the significant increase in milk protein yield (+7%) and the small nonsignificant increase in milk yield (+5%).

The efficacy of a rumen-protected trans-10, cis-12 CLA supplement is reflected by the extent to which it causes milk fat depression. The proportional transfer of trans-10, cis-12 CLA from the abomasum into milk fat has been shown to be relatively constant (de Veth et al., 2004) and increasing the trans-10, cis-12 CLA content of milk corresponds to a curvilinear reduction in milk fat synthesis (Peterson et al., 2002). The transfer of trans-10, cis-12 CLA from a rumen-protected supplement into milk fat is influenced by the ability of the supplement to provide protection from rumen metabolism as well as postruminal availability (Wu and Papas, 1997). Therefore, incorporation of trans-10, cis-12 CLA into milk fat is a good method for evaluating rumen-protected CLA products because protection from rumen metabolism as well as postruminal availability are assessed.

A unique feature of the present study is that it provided an identical daily dose of trans-10, cis-12 CLA originating from the same batch of CLA as was used in a separate study by de Veth et al. (2003) that compared calcium salt and formaldehyde-protected products. The reduction in milk fat yield, as well as the transfer efficiency of trans-10, cis-12 CLA into milk fat for each of the 4 rumen-protected supplements are presented in Table 5. The 2 studies were completed independently, so statistical comparison is not possible. However, it is interesting to note that all supplements caused similar degrees of reduction in milk fat secretion (Table 5). In general, the transfer efficiencies of trans-10, cis-12 CLA from rumen-protected supplements are much lower than the ~20% transfer efficiencies reported for abomasal infusion studies (Chouinard et al., 1999a, 1999b; Baumgard et al., 2000, 2001; Peterson et al., 2002). Thus, a large proportion of the CLA in the rumen-protected supplements must be metabolized in the rumen or unavailable for postruminal absorption. Transfer of trans-10, cis-12 CLA from the formaldehyde-protected CLA supplement was similar (7.0%) to the transfer observed for the 2 rumen-protected supplements used in the present study (Table 5). In contrast, the calcium salts of CLA had a transfer efficiency of only 3.2%, which is similar to the transfer efficiencies reported for longer-term studies using calcium salts of CLA (Perfield et al., 2002; Bernal-Santos et al., 2003). In these situations the milk fat content of trans-10, cis-12 CLA does not effectively explain the observed milk fat depression when plotted on the curve developed by Peterson et al. (2002). It has been hypothesized that the reason for this is that other unique fatty acids are formed in the rumen, which are also potent inhibitors of milk fat synthesis (Bauman and Griinari, 2003; Bauman et al., 2003). This could provide an explanation for why the calcium salt supplements of CLA have about one-half the transfer efficiency of trans-10, cis-12 CLA found for other rumen-protected products, yet the reduction in milk fat synthesis is similar (Table 5). Of course, the most dependable CLA supplement would transfer a specific quantity of trans-10, cis-12 CLA into milk fat per amount fed without variations introduced by particular rumen conditions or rumen fermentation.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Authors gratefully acknowledge the contributions of J. Mikko Griinari (Clanet Ltd, Espoo, Finland) in the development of this investigation and evaluation of the results. The authors also thank P. Lee and D. Putnam (Balchem Encapsulates) for the preparation of the lipid-encapsulated supplement and T. Jenkins (Clemson University, Clemson, SC) for consultation and assistance in obtaining the amide-protected supplement. The support of the following students and colleagues at Cornell University in implementing the study is also gratefully acknowledged and appreciated: S. Bean, J. McFadden, D. Dwyer, B. English, L. Furman, G. Birdsall, B. Jones, and D. Ceurter.

	FOOTNOTES

 
^* Supported in part by BASF AG (Ludwigshafen, Germany), Balchem Encapsulates (New Hampton, NY), and Cornell Agricultural Experiment Station.

Received for publication January 27, 2004. Accepted for publication March 9, 2004.

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