THE ROLE OF TRANS FATTY ACIDS IN THE REGULATION OF MILK FAT SYNTHESIS

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1 THE ROLE OF TRANS FATTY ACIDS IN THE REGULATION OF MILK FAT SYNTHESIS INTRODUCTION Dale E. Bauman,Adam L. Lock,and James W. Perfield II Department of Animal Science Cornell University Ithaca, NY Fat is the major energy component in milk and it accounts for many of the physical properties, manufacturing characteristics and organoleptic qualities of milk and milk products. It is estimated that the milk fat of dairy cows contains over 400 different fatty acids (Jensen, 2002). While most are present in minor amounts, it is generally recognized that the major fatty acids in milk fat include saturated fatty acids from C4:O to C18:O plus palmitoleic, oleic, linoleic and trans-18: 1 fatty acids. Milk fatty acids arise from two sources- de novo synthesis within the mammary gland and the uptake of preformed long chain fatty acids from circulation (Bauman and Davis, 1974). Fatty acids of chain length C4 to C14 and a portion of the C16 are derived from de novo synthesis with acetate and, to a lesser as the carbon sources. The remainder of the C16 and all of the longer chain fatty acids are taken up from circulating fatty acids that are absorbed from the digestive tract or originate from the mobilization of body fat reserves. Fat is the most variable component in milk and is affected markedly by physiological and environmental factors (see reviews by Palmquist et al., 1993; Doreau et al., 1999; Lock and Shingfield, 2004). However, nutrition is the predominant environmental factor affecting milk fat and represents a practical tool for altering its yield and composition. One of the most striking examples of nutritional effects on milk fat is the low fat milk syndrome, typically referred to as milk fat depression (MFD). Diet-induced MFD is a challenging problem that involves the interrelationship between digestive processes in the rumen and the synthesis of milk fat by the mammary gland. Recent developments have provided new insight in to the basis for diet-induced MFD, and these will be reviewed in the following sections. While not complete, our improved understanding of the cause of MFD is of value as an aid in troubleshooting situations in which diet-induced MFD is of concern. In addition, available technology provides the opportunity to manufacture feed supplements that can reduce milk fat production in a controlled manner. Such a supplement has potential for use as a management tool and research exploring this aspect will also be discussed.

2 BACKGROUND The earliest recognition of MFD was by Boussingault in Boussingault is often credited as being the world's first agricultural scientist, and he observed a reduction in milk fat yield when dairy cows were fed a diet of beets (Van Soest, 1994). Through the first half of the twentieth century, the feeding of dairy cows began to follow "scientific principles" and MFD was observed for a range of feeding situations, including diets supplemented with fish oils or plant oils and diets high in concentrates and low in fiber (HCILF) (see review by Bauman and Griinari, 2001). The fat content of milk is also affected by the physical characteristics of the roughage (e.g., grinding or pelleting); this was convincingly demonstrated during the 1939 New York World's Fair when the Borden Company's "Dairy World of Tomorrow" exhibit encountered MFD when cows were fed a pelleted diet (Erdman, 1996). Specific mechanisms involved in MFD have perplexed producers and scientists for over 150 years, but during the last 50 years there has been substantial research addressing MFD, and practical recommendations have been developed to minimize its occurrence (see reviews by Erdman, 1988; Palmquist et al., 1993). Several general characteristics have also been identified and these provide insight into the biology of MFD (Bauman and Griinari, 2003). First, the changes that occur with diet-induced MFD are specific for milk fat; fat yield can be reduced up to 50% with little or no change in milk yield or the yield of lactose or protein. Second, the yield of all fatty acids is reduced, but the decline is greatest for de novo synthesized fatty acids. As a result, milk fat composition shifts toward lower proportions of short and medium chain fatty acids and greater concentrations of long chain fatty acids. Third, changes in microbial processes are an essential component for the development of MFD. These changes in the rumen environment are often associated with a decrease in rumen ph and a shift in the acetate:propionate ratio. Fourth, for MFD to occur the diet must contain unsaturated fatty acids and the pathways of their biohydrogenation in the rumen must be altered. Thus, the induction of MFD is centered on three key components; an altered rumen environment, the presence of unsaturated fat in the rumen, and specific fatty acid intermediates produced by rumen bacteria due to the altered environment. Over the last century numerous theories have been postulated to explain the cause of MFD. These can be broadly summarized into theories that considered the reduction to be a consequence of a shortage in the supply of lipid precursors for mammary gland synthesis of milk fat and theories that attributed the reduction to a direct inhibition of one or more steps in the synthesis of milk fat in the mammary gland (see reviews by Bauman and Griinari, 2001; 2003). Theories in the former category have included acetate deficiency, and the glucogenic-insulin theory, and research has established that they are inadequate to explain the basis for diet-induced MFD. Theories that attribute MFD to a direct inhibition of milk fat synthesis include the effects of trans fatty acids and the biohydrogenation theory, and these are discussed in the next section.

3 TRANS FATTY ACIDS AND THE BIOHYDROGENATION THEORY Davis and Brown (1970) were among the first to recognize that increases in the milk fat content of trans fatty acids (TFA) was associated with MFD caused by feeding HCLF diets. In a subsequent study Pennington and Davis (1975) also reported an increase in milk fat TFA when MFD was induced by fish oil supplements. Dietary lipids are mainly composed of esterified fatty acids and when they enter the rumen bacterial lipases hydrolyze the ester linkages thereby producing free fatty acids. The major unsaturated fatty acids in dairy feeds, linoleic acid (C18:2) from plant seeds and oils and linolenic acid (C18:3) from forages, are toxic to the rumen bacteria, so the free unsaturated fatty acids are rapidly biohydrogenated to form saturated fatty acids. In this process trans fatty acids are formed as intermediates with trans :l (vaccenic acid) being the predominant isomer as illustrated for the biohydrogenation of linoleic acid (Figure 1). As the database grew, it became evident that MFD was often related to an increase in the TFAcontent of milk fat across a wide range of diets (Griinari et al., 1998; Erdman, 1999). However, there were also many situations where increases in milk fat content of TFA did not correspond to changes in milk fat production and thus, the basis for MFD had to be more complex than a simple relationship to the ruminal production of TFA. linoleic acid (cis-9, cis-12 18:2) conjugated linoleic acid (cis-9, trans-11 CLA) trans-11 18:l stearic acid (18:O) conjugated linoleic acid (trans-10, cis-12 CLA) trans-10 18:l stearic acid (18:O) Figure 1. Generalized scheme of ruminal biohydrogenation of linoleic acid under normal conditions and during diet-induced milk fat depression [dotted line]. Adapted from Griinari and Bauman 11999). A key development in understanding diet-induced MFD occurred when we utilized improved analytical techniques and discovered that it was the pattern of trans 18: 1 isomers rather than total TFA that was correlated to MFD. Specifically, our initial studies demonstrated that MFD which occurred with HCILF diets was associated with a marked increase in the milk fat content of trans-10 18: 1 (Griinari et al., 1998).

4 Subsequent work verified these results and demonstrated that a specific increase in trans-10 18: 1 also occurred with other types of diets that cause MFD (see review by Bauman and Griinari, 2003). Thus, under certain dietary situations a portion of the linoleic acid undergoes biohydrogenation via a pathway that produces trans-10 18: 1 (Figure 1). Note that trans-10, cis-12 CLA is also an intermediate in this pathway and we found that the milk fat content of this unique CLA isomer also increased. Of particular importance, there was a curvilinear relationship between the reduction in milk fat yield and the increase in milk fat content of trans-10, cis-12 CLA in cows fed a HC/LF diet or diets supplemented with plant oils (Bauman and Griinari, 2001). Over the same interval we were also conducting studies with pure CLA isomers and discovered that trans-10, cis-12 CLA was a potent inhibitor of milk fat synthesis (Baumgard et al., 2000). We established that the dose response relationship was also curvilinear and found that as little as 2.5 g/d of trans-10, cis-12 CLA delivered post-ruminally was sufficient to cause a 25% reduction in milk fat (Figure 2). Effects of trans-10, cis-12 CLA are specific for milk fat and its mechanism and that for dietinduced MFD involves coordinated reductions in mammary enzymes involved in milk fat synthesis (Piperova et al., 2000; Ahnadi et al., 2002; Baumgard et al., 2002; Peterson et al., 2003; 2004). Figure 2. Model of the relationship between the change in milk fat yield and the dose of trans-10, cis-12 CLA abomasally infused in lactating cows.adapted from deveth et al ) based on a computation of results from seven published studies.

5 Based on these results and the characteristics of the biology of MFD discussed earlier, we proposed the "biohydrogenation theory" (Bauman and Griinari, 2001; 2003). We hypothesized that "under certain dietary conditions the pathways of rumen biohydrogenation are altered to produce unique fatty acid intermediates which are potent inhibitors of milk fat synthesis." Clearly, trans- 10, cis- 12 CLA represents one example, and recent studies suggest the existence of additional, as yet unidentified, fatty acid intermediates that inhibit milk fat synthesis (Perfield et al., 2002; Bauman and Griinari, 2003; Peterson et al., 2003; Piperova et al., 2004). The application of improved analytical techniques also demonstrated that the pathways of rumen biohydrogenation are considerably more complex than first thought. Trans 18: 1 fatty acids and CLA isomers are generally not present in the dietary components fed to dairy cows, so their presence in milk fat is a consequence of their formation as intermediates in rumen biohydrogenation. As shown in Table 1, trans : 1 and cis-9, trans- 11 CLA are the predominant TFA and CLA respectively, consistent with the biohydrogenation pathway presented in Figure 1. These are the TFA and CLA isomers in milk fat that have been shown to be anticarcinogenic ( However, milk fat also contains minor amounts of many additional trans- 18: 1 and CLA isomers and thus rumen biohydrogenation must, to a limited extent, involve many different rumen bacteria and pathways of biohydrogenation (Table 1). Note, the two isomers that are correlated with the occurrence of MFD, trans : 1 and trans- 10, cis- 12 CLA, are just minor components of the total CLA and TFA found in milk fat. Considering this, it is important to be mindful that when troubleshooting MFD on commercial dairy farms, problems in milk fat test do not necessarily relate to total TFA or total CLA; rather it is the presence of specific fatty acid isomers that is important and small changes in the pattern of biohydrogenation intermediates leaving the rumen can have significant effects on milk fat synthesis. Table 1. Range of positional and geometric isomers of trans 18:l and conjugated linoleic acids [CLA] in milk and dairy products [adapted from Lock and Bauman, Trans 18.1 Conjugated Linoleic Acids Isomer % of total trans 18.1 isomers Isomer % of total CLA isomers trans trans-7, cis trans-5 < trans-7, trans-9 <O.l -2.4 trans trans-8, cis-10 < trans trans-8, trans trans cis-9. trans trans trans-9, trans trans trans-10, cis-12 < trans <O.l trans- 10, trans trans cis- 11, trans trans trans-1 1. cis trans-1 1, trans cis-12, trans-14 < trans-12, trans cis-cis isomers

6 Identification of additional fatty acid intermediates that may play a role in regulating milk fat synthesis is made possible by continuing advances in analytical techniques for the identification and quantification of fatty acids. However, these fatty acids will likely be present in very low concentrations complicating their identification. Even once identified, our ability to directly test their effects on milk fat synthesis is limited by the availability of pure fatty acids. So far, we have examined several additional CLA isomers via abomasal infusions to lactating cows and these include trans-8, cis-10 CLA, cis-9, trans-11 CLA, trans-10, trans-12 CLA and cis-11, trans-13 CLA (Perfield et al., 2004a; 2004b). Despite being taken up by the mammary gland and incorporated into milk fat, none of these CLA isomers affected milk fat synthesis or any other variables related to milk production. To date, the only trans- 18: 1 isomers that have been directly examined for effects on milk fat synthesis in dairy cows are trans-9, trans-11, and trans-12, and none of these affected milk fat yields at the dose levels tested (Rindsig and Schultz, 1974; Griinari et al., 2000). Trans : 1 is of obvious interest, but it is not available as a pure isomer in quantities needed to investigate its effect on milk fat synthesis. Partially hydrogenated vegetable oils (PHVO) generally contain about 40-50% TFA so these have been abomasally infused or fed as rumen-protected formulations to investigate MFD. Selner and Schultz (1980) were among the first to do so and they concluded "it appears that the trans acids caused the fat depression". Subsequent studies found similar affects and attributed the reduction in milk fat to the trans 18.1 fatty acids in the PHVO (Gaynor et al., 1994; Romo et al., 1996; Piperova et al., 2004; Selberg et al., 2004). However, PHVO contain a number of conjugated, partially conjugated, and other unique fatty acids formed during the chemical hydrogenation process, and effects of these cannot be excluded as the cause of observed MFD (see discussion in Bauman and Griinari, 2003). Overall, the biohydrogenation theory represents a unifying theory that may explain all types of diet-induced MFD. However, verification and additional approaches are needed for this to be firmly established. To date, trans-10, cis-12 CLA is the only biohydrogenation intermediate that has been unequivocally shown to inhibit milk fat synthesis, although as stated earlier several lines of evidence suggest there must be additional unique fatty acids that affect rates of milk fat synthesis. Identification of these fatty acids is essential to be able to adequately troubleshoot milk fat problems on commercial dairy farms. MANAGEMENT APPLICATIONS Identification of rumen-derived inhibitors of milk fat synthesis and the conditions which result in their formation will enable us to more effectively troubleshoot problems in low fat test on commercial farms. This will also help increase our understanding of the biology of diet-induced MFD and the specific mechanism(s) by which these compounds act. While working to further our knowledge in this area,

7 the benefits of using trans-10, cis-12 CLA to induce a controlled milk fat depression are also being investigated. Potential management benefits include applications to increase milk production without penalty in a system regulated by a milk fat quota or to reduce the energy demands on cows during nutritional situations where energy intake is inadequate to meet requirements. These nutritional situations include the onset and early lactation period, as well as adverse environmental conditions such as heat stress or weather-related feed shortages (see review by Griinari and Bauman, 2003). Initial studies with CLA-induced milk fat depression have demonstrated a consistent reduction in milk fat during treatment periods lasting 3 to 20 weeks, involving primiparious and multiparious cows under different dietary and management practices (Giesy et al., 2002; Perfield et al., 2002; Bernal-Santos et al., 2003; Moore et al., 2004; Selberg et al., 2004; Piperova et al., 2004). Perfield et al. (2002) reported the first long term study where a CLA supplement was fed for the last 20 weeks of lactation; we observed an average 23% reduction in milk fat synthesis over the treatment period while yields of milk and other milk components, maintenance of pregnancy and cow well-being were unaffected. The same supplement and CLA dose fed during the first 20 weeks of lactation also caused a reduction in milk fat yield; however the initial decrease in milk fat percent did not occur until after the first two weeks of lactation (Bernal-Santos et al., 2003). Subsequently Moore et al. (2004) demonstrated that higher doses of CLA are required to elicit a reduction in milk fat synthesis during the first two weeks postpartum. Research involving CLA supplements has been limited, but several studies have reported that when energy intake was inadequate to meet requirements, a shift in nutrient partitioning occurred whereby the energy spared by the CLA-induced reduction in milk fat was utilized to support an increase in milk yield (early lactation) or an increase in both milk and milk protein production (pasture-fed cows) (see review by Griinari and Bauman, 2003). However, some of these results have been presented only as abstracts and an increase in milk yield has not always been observed. Obviously, this will continue to be an active area of research. There is also some indication that CLA supplements may impact reproductive performance, and this is also an area requiring further investigation. Despite the small treatment group sizes, both Bernal-Santos et al. (2003) and Castaneda-Gutierrez et al. (2004) observed improved conception rates in cows receiving CLA supplements. Effects of CLA on reproduction parameters may be related to both energy and non-energy properties. Supplementation of CLA not only impacts bioenergetics due to the reduction in energy use for milk fat synthesis, but CLA isomers may also have effects on prostaglandin production and ovarian steroidogenesis. Dietary lipids are metabolized in the rumen, and as a consequence, an effective commercial supplement of trans-10, cis-12 CLA must have two characteristics; first, it must offer protection from alterations by rumen bacteria and second, the CLA must subsequently become available for absorption in the small intestine. Most rumen

8 protection methods are ph-dependent and take advantage of the transition in ph occurring between the rumen (ph -5.8 to 6.7) and the abomasum (ph -2 to 4). To date, most of the research with rumen-protected CLA has used supplements consisting of calcium salts of free fatty acids (Giesy et al., 2002; Perfield et al., 2002; Bernal- Santos et al., 2003; Moore et al., 2004; Selberg et al., 2004; Piperova et al., 2004). Rumen-protected supplements of CLA have also been manufactured using formaldehyde treatment, amide bond formation, and lipid-encapsulation (de Veth et al., 2003; Perfield et al., 2004~). Reductions in milk fat were observed for supplements utilizing all of these protection methods as well as the calcium salt supplements, although none of the methods completely protected the trans-10, cis-12 CLA from metabolism in the rumen. The form of fatty acid required for the production of these supplements varies (free fatty acid vs. methyl ester), which becomes an important consideration due to the variation in manufacturing processes and costs associated with the production of different lipid forms (Szeb~, 2003). A lipid-encapsulated form of CLA methyl esters is currently being marketed in Europe, while approval in Canada and the US is still pending. Use of CLA supplements to reduce milk fat has increased our understanding of the biology of MFD. Experience gained from this research can be used to help us better understand diet-induced MFD and will eventually enable us to more effectively troubleshoot low fat tests on commercial farms. We are seeing many more problems with MFD in the last few years as compared to the recent past. The magnitude of milk fat decrease (i.e., 10-25%) of economic and producer concern can be caused by as little as 1 to 2 g of trans-10, cis-12 CLA passing to the small intestine. The increased occurrence of MFD in recent years is likely due to a number of reasons; for example, changes in rumen biohydrogenation pathways may have been caused by poor silage making conditions the past several growing seasons, increased occurrence of sorting of TMR due to the attempt to increase effective dietary fiber, and increased use of unsaturated fat sources in diets. In addition, higher DM1 will increase passage rates from the rumen potentially increasing washout of biohydrogenation intermediates, including those that could cause MFD (Overton and Bauman, 2003). Further research is needed to fully evaluate these interrelationships and to develop nutritional strategies designed to avoid dietary-induced MFD problems in today's high producing dairy cows. SUMMARY The problem of diet-induced MFD has challenged producers and scientists for over a century. Over the last few years the biohydrogenation theory has been advanced to explain MFD, and while investigations to date offer strong scientific support, it still needs to undergo rigorous evaluation to determine if it provides a unifying concept applicable to all situations of diet-induced MFD. Ultimately our ability to predict and troubleshoot commercial problems related to milk fat is dependent on a complete understanding of the dynamic interactions in the fermentation

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