**1. Introduction**

Recent developments in nutrition have established that choline is an essential nutrient for mammals when a sufficient supply in methionine and folates are not available in the diet. Vitamin B12 is also involved in this process. The dynamic interactions between these components introduced the concept of choline as vitamin-like compound. Two types of choline functions are known: as choline *per se*, for which the choline moiety is required, and functions as a methyl donor. Choline per se plays a major role in lipid metabolism, particularly in lipid transport, as lipotropic agent. Choline is also an important source of labile methyl groups for the biosynthesis of other methylated compounds. Based on this second function choline and methionine are interchangeable, as sources of methyl groups. Accordingly choline occupies a key position between energy and protein metabolism in mammals.

Choline and methyl group metabolism in ruminants however are different. In adult ruminants, choline is extensively degraded in the rumen; for this reason dietary choline contributes insignificantly to the choline body pool and methyl group metabolism is generally conservative with a relatively low rate of methyl catabolism and an elevated rate of de novo synthesis of methyl groups via the tetrahydrofolate (THF) system. This can be exacerbated in lactating dairy ruminant, in which the dietary availability of choline is nearly non-existent, but the output of methylated compounds in milk is high, while methionine as well as other sources of methyl groups are likely to be in short supply, especially at the onset of lactation. In light of this, the hypothesis that choline can be a limiting nutrient for milk production has been formulated and tested in several studies. Accordingly this chapter will focus on the effects of rumen-protected choline (RPC) supplementation to transition and early lactating dairy ruminants (cows and goats) on milk production and on metabolic health. Experimental data will be discussed in a systematic analysis, in order to define possible recommendations for high yielding dairy ruminants.

© 2012 Pinotti, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Pinotti, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **2. What is choline?**

In 1849 Adolph Strecker, a German chemist, isolated a compound from pig bile, to which he subsequently (in 1862) applied the name choline (from the Greeke, *chole*, bile). In 1867, Bayer, determined the chemical structure of choline (McDowell, 1989).

Choline is a beta-hydroxyethyltrimethylammonium hydroxide (figure 1), which is widely distributed in nature as free choline, acetylcholine, and more complex phospholipids and their metabolic intermediates. Its role in the body is complex. It is needed for neurotransmitter synthesis (acetylcholine), cell-membrane signaling (phospholipids), lipid transport (lipoproteins), and methyl-group metabolism (homocysteine reduction) (Zeisel & Da Costa, 2009).

**Figure 1.** Structural formula for free choline, acetylcholine, and phosphatidylcholine (lecithin). R and R' refers to any fatty acids (adapted from McDowell, 1989).

#### **2.1. Vitamin or not?**

In mammals choline has been classified as one of the B-complex vitamins but it does not satisfy the standard definition of a vitamin (Pinotti et al., 2002): it is synthesised endogenously and there is no evidence that it is an enzyme co-factor; furthermore, unlike other water-soluble vitamins, it is difficult to identify a deficiency syndrome for choline in healthy mammals because of its interrelation with methionine, folic acid, and vitamin B12 (Scott, 1999; Zeisel, 1988). Finally, choline is a vital component of tissues and is required in the diet of non-ruminant species at much higher levels than the water-soluble vitamins (g vs. mg) (Whitehead & Portsmouth, 1989). However, the presence of an endogenous synthetic pathway does not render choline dispensable and deficiency results in several dysfunctions when other nutrients are limiting (Zeisel et al., 1991). For this reason it has been suggested that choline may be an essential nutrient (or vitamin like compound) for mammals when excess methionine and folic acid are not available in the diet (Zeisel & Da Costa, 2009). Consequently choline was officially recognized as an essential nutrient for humans by the Institute of Medicine in 1998 (Food and Nutrition Board, 1998).

## **2.2. How requirement for choline can be met**

66 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

Bayer, determined the chemical structure of choline (McDowell, 1989).

 CH3 ⏐

 ⏐ CH3

 O ║ CH2OC⎯R'

 O ║ CHOC⎯R

Choline

In 1849 Adolph Strecker, a German chemist, isolated a compound from pig bile, to which he subsequently (in 1862) applied the name choline (from the Greeke, *chole*, bile). In 1867,

Choline is a beta-hydroxyethyltrimethylammonium hydroxide (figure 1), which is widely distributed in nature as free choline, acetylcholine, and more complex phospholipids and their metabolic intermediates. Its role in the body is complex. It is needed for neurotransmitter synthesis (acetylcholine), cell-membrane signaling (phospholipids), lipid transport (lipoproteins), and methyl-group metabolism (homocysteine reduction) (Zeisel &

CH3 ⎯ N+⎯ CH2 ⎯ CH2 ⎯ OH

 O CH3 ║ ⏐

Acetylcholine

 O CH3 ║ ⏐

CH3⎯C⎯ O ⎯CH2⎯CH2⎯N+⎯ CH3

 ⏐ CH3

**Figure 1.** Structural formula for free choline, acetylcholine, and phosphatidylcholine (lecithin). R and R'

 ⏐ CH3

CH2O⎯P⎯ O ⎯CH2⎯CH2⎯N+⎯ CH3

Phosphatidylcholine

In mammals choline has been classified as one of the B-complex vitamins but it does not satisfy the standard definition of a vitamin (Pinotti et al., 2002): it is synthesised endogenously and there is no evidence that it is an enzyme co-factor; furthermore, unlike other water-soluble vitamins, it is difficult to identify a deficiency syndrome for choline in

refers to any fatty acids (adapted from McDowell, 1989).

**2.1. Vitamin or not?** 

**2. What is choline?** 

Da Costa, 2009).

The metabolic need for choline can be met in two ways: either by dietary choline and via de novo biosynthesis through the methylation of phosphatidylethanolamine to phosphatidylcholine. From the point of view of animal nutrition, relatively rich sources of choline are soybean, soybean meal, rapeseed meal, fish meal and dried yeast, even though its bioavailability is considered "moderate" (Pinotti et al., 2002; Whitehead & Portsmouth, 1989). Dietary choline from a variety of choline-containing feed and food is absorbed by the intestine and uptake is mediated by choline transporters nutrient (McDowell, 1989). *De novo* synthesis of choline occurs by the sequential methylation of phosphatidylethanolamine, the methyl groups being supplied by S-adenosyl-L-methionine (SAM) (Mato et al., 1994). However, *de novo* synthesis of choline alone is not sufficient or rapid enough to satisfy all the animal's need. Methyl groups needed in this pathway may be derived from exogenous sources such as methionine, and betaine but can arise de novo in the body from the tetrahydrofolate (THF) system (Zeisel, 1988; Zeisel, 1992); vitamin B12 is involved in this process (Kennedy et al., 1995). Thus, dietary factors such us methionine, betaine, myoinositol, folic acid, and vitamin B12 or combination of different levels of fat, carbohydrates, and protein in the diet, as well as the physiological state, all have influence on the "requirements" of choline (McDowell, 1989; Zeisel & Da Costa, 2009).

## **2.3. Functions of choline**

Choline is considered a vitamin-like compound with two main functions: as choline *per se*, for which the choline moiety is required, and as methyl donor, although the two roles overlap.

Choline *per se* is an essential constituent of all cell membranes, where it is required to make the phospholipids phosphatidylcholine, lysophosphatidylcholine, choline plasmalogen, and sphingomyelin. Phosphatidylcholine, one of the most abundant phospholipids in higher plants and animals, is the predominant phospholipid (>50%) in most mammalian cell membranes (Kuksis & Mookerjea, 1978; Ruiz et al., 1983; Zeisel, 1988; Zeisel, 1992). Choline *per se* plays a major role in lipid metabolism, particularly in lipid transport, as lipotropic agent because of its ability to prevent or correct excess fat deposition in the liver generally arising as a result of its dietary deficiency (Kuksis & Mookerjea, 1978; Zeisel, 1988). Impaired triglyceride secretion to very low density lipoproteins (VLDL) is considered a major cause of fatty liver in dietary choline deficiency (Zeisel, 1988). In this context, it is noteworthy that

bovine VLDL phospholipids are mainly phosphatidylcholine, with smaller proportions of sphingomyelin and phosphatidylethanolamine (Moore & Christie, 1981). Thus, when massive mobilisation of fatty acids – as at the onset of lactation in dairy ruminants- is associated with lipotropic factor deficiency (e.g. choline) triglycerides accumulate in the liver and may lead to the development of fatty liver (Gruffat et al., 1996). Choline *per se* is required to prevent hemorrhagic kidney lesions in rats (Kuksis & Mookerjea, 1978); and together with other nutrients particularly manganese salts, is required to prevent perosis, a bone disease of poultry (Ruiz et al., 1983). In the prevention of perosis, choline is needed as constituent of phospholipids required for normal maturation of the cartilage matrix of the bone, whereas in the prevention of hemorrhagic kidney lesions choline seems involved in the renal phospholipids turnover (Kuksis & Mookerjea, 1978).

Choline *per se* is also essential for the synthesis of the neurotransmitter acetylcholine (Kuksis & Mookerjea, 1978; Zeisel, 1988). It plays important roles in brain and memory development in the fetus and appears to decrease the risk of the development of neural tube defects (Zeisel & Da Costa, 2009).

As methyl donor, choline, like methionine, is an important source of labile methyl groups for biosynthesis. Actually, the two principal methyl donors in animal metabolism are betaine, a choline metabolite, and S-adenosyl-L-methionine (SAM) a metabolite of methionine (Pinotti et al., 2002 for references). At least 50 SAM-dependent reactions have been identified in mammals, and it is likely that the number is much higher. Such methylation reactions play major roles in biosynthesis of lipids, the regulation of several metabolic pathways, and detoxification in the body (Zeisel & Da Costa, 2009). Accordingly it has been suggested that choline, and methionine are closely interrelated metabolically (Mato et al., 1994); it is also clear that choline has only little capacity to reduce the requirement for methionine, even though choline seems important to spare methionine as a methyl donor.

Although the choline-methionine interrelationship has been studied extensively (see Pinotti et al, 2002 for reference), less emphasis has been placed on the link between choline and its oxidation product, betaine. Betaine serves as an osmoregulator and is a substrate in the betaine–homocysteine (HCy) methyltransferase reaction, which links choline and betaine to the folate-dependent one-carbon metabolism (Kempson & Montrose, 2004; Ueland, 2011). The availability of choline and its metabolite betaine appear to influence the conversion or recycling of the HCy moiety of methionine, through betaine-HCy methyltransferase activity (Stipanuk, 1986). However, betaine fails to prevent fatty livers and haemorrhagic kidney (McDowell, 1989), indicating that the requirement for choline per se must be met as choline, and that betaine can substitute only the methyl donor function of choline, probably because betaine cannot be reduced to choline (Zeisel, 1988). Thus, choline *per se* must provide at least 50% of the total choline requirement, while the remaining portion of the choline requirement can be replaced by betaine (Dilger et al., 2007).

## **3. Choline in ruminants**

Ruminants differ from most other mammals in regard to their choline and methyl group metabolism. In adult ruminants, choline is extensively degraded in the rumen. Quantitative studies in sheep showed that 76% of [14C]-choline injected into the rumen was expired as methane over 6h, whereas approximately 15% accumulated as trimethylamine. Under such conditions, less than 10% of choline escapes degradation by incorporation, as phosphatidylcholine, into the structural membranes of ciliate protozoa (Neill et al., 1979). Nevertheless, the concentration of phosphatidylcholine in ruminal digesta was higher than in abomasal digesta, suggesting that protozoa are selectively retained in the rumen (Neill et al., 1979). In sheep with a defaunated rumen, the concentration of phosphatidylcholine in the abomasal digesta was higher than in the rumen (Dawson et al., 1981), leading to the suggestion that some of this abomasal Phosphatidylcholine was derived from non-dietary sources (regurgitation of bile from the lower digestive tract) (Robinson et al., 1984). In fact, intravenous injection of labelled choline in sheep indicated that the small amount of phosphatidylcholine present in abomasal digesta is largely (69%) of non-dietary or ruminal origin (Dawson et al., 1981).

68 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

the renal phospholipids turnover (Kuksis & Mookerjea, 1978).

(Zeisel & Da Costa, 2009).

can be replaced by betaine (Dilger et al., 2007).

**3. Choline in ruminants** 

bovine VLDL phospholipids are mainly phosphatidylcholine, with smaller proportions of sphingomyelin and phosphatidylethanolamine (Moore & Christie, 1981). Thus, when massive mobilisation of fatty acids – as at the onset of lactation in dairy ruminants- is associated with lipotropic factor deficiency (e.g. choline) triglycerides accumulate in the liver and may lead to the development of fatty liver (Gruffat et al., 1996). Choline *per se* is required to prevent hemorrhagic kidney lesions in rats (Kuksis & Mookerjea, 1978); and together with other nutrients particularly manganese salts, is required to prevent perosis, a bone disease of poultry (Ruiz et al., 1983). In the prevention of perosis, choline is needed as constituent of phospholipids required for normal maturation of the cartilage matrix of the bone, whereas in the prevention of hemorrhagic kidney lesions choline seems involved in

Choline *per se* is also essential for the synthesis of the neurotransmitter acetylcholine (Kuksis & Mookerjea, 1978; Zeisel, 1988). It plays important roles in brain and memory development in the fetus and appears to decrease the risk of the development of neural tube defects

As methyl donor, choline, like methionine, is an important source of labile methyl groups for biosynthesis. Actually, the two principal methyl donors in animal metabolism are betaine, a choline metabolite, and S-adenosyl-L-methionine (SAM) a metabolite of methionine (Pinotti et al., 2002 for references). At least 50 SAM-dependent reactions have been identified in mammals, and it is likely that the number is much higher. Such methylation reactions play major roles in biosynthesis of lipids, the regulation of several metabolic pathways, and detoxification in the body (Zeisel & Da Costa, 2009). Accordingly it has been suggested that choline, and methionine are closely interrelated metabolically (Mato et al., 1994); it is also clear that choline has only little capacity to reduce the requirement for methionine, even though choline seems important to spare methionine as a methyl donor.

Although the choline-methionine interrelationship has been studied extensively (see Pinotti et al, 2002 for reference), less emphasis has been placed on the link between choline and its oxidation product, betaine. Betaine serves as an osmoregulator and is a substrate in the betaine–homocysteine (HCy) methyltransferase reaction, which links choline and betaine to the folate-dependent one-carbon metabolism (Kempson & Montrose, 2004; Ueland, 2011). The availability of choline and its metabolite betaine appear to influence the conversion or recycling of the HCy moiety of methionine, through betaine-HCy methyltransferase activity (Stipanuk, 1986). However, betaine fails to prevent fatty livers and haemorrhagic kidney (McDowell, 1989), indicating that the requirement for choline per se must be met as choline, and that betaine can substitute only the methyl donor function of choline, probably because betaine cannot be reduced to choline (Zeisel, 1988). Thus, choline *per se* must provide at least 50% of the total choline requirement, while the remaining portion of the choline requirement

Ruminants differ from most other mammals in regard to their choline and methyl group metabolism. In adult ruminants, choline is extensively degraded in the rumen. Quantitative The physiological state however, is important (Table 1). In the pre-ruminant lamb the activities of liver Cho-oxidase and betaine-HCy methyltransferase increase markedly after birth, but subsequently decrease as the animals reach the ruminant state (Xue & Snoswell, 1986a). The increasing activities of liver choline oxidase and betaine-HCy methyltransferase in pre-ruminant lambs are probably related to the abundance of choline-containing compounds in the milk (Xue & Snoswell, 1986a- Figure 2). By contrast, in adult ruminants only small quantities of methyl group nutrients are available from the diet and methionine synthase assumes a much more important role (Dawson et al., 1981; Neill at al., 1978; Neill et al., 1979; Kennedy et al., 1995). Methionine synthase is responsible for the de novo synthesis of methionine methyl groups from one-carbon units furnished by tetrahydrofolate (THF) (figure 1). The enzyme utilises methyl-THF as methyl donor and methylcobalamin as tightly-bound coenzyme. De novo synthesis of methyl groups via this system appears to be minimal when labile methyl group intake is sufficient or excessive (Stipanuk, 1986).

Dietary choline therefore contributes insignificantly to the choline body pool in adult ruminants (Table 1; Figure 3). Methyl group metabolism is generally conservative with a relatively low rate of methyl catabolism and an elevated rate of de novo synthesis of methyl groups via the tetrahydrofolate (THF) system (Girard et al., 2010; Henderson et al., 1983; Robinson et al., 1984; Snoswell & Xue, 1987; Xue & Snoswell, 1986a; Xue & Snoswell, 1986b). In dairy ruminants the situation can be even worse (Table 1): the dietary availability of choline is still low while the output of methylated compounds to milk is particularly high, and methionine, as well as other methyl group sources - one-carbon units for methylneogenesis via the THF system-, is likely to be in short supply, especially at the onset of lactation (Pinotti et al., 2002). In dairy ruminants producing large quantities of milk in fact, methionine is also the first limiting amino acid. This means that the elevated requirement for methionine for transmethylation reactions and milk protein synthesis may lead to altered methyl group metabolism (Girard et al., 2010; LaCount et al., 1995; Lobley et al., 1996). The effects of lactation on methyl group metabolism have been examined in sheep (Xue & Snoswell, 1985); it was found that the activities of hepatic phospholipid methyltransferase (for phosphatidylcholine synthesis) and methionine synthase were significantly higher (+ 33% and +34%, respectively) in lactating than non-lactating ewes (Xue & Snoswell, 1985).

**Figure 3.** Choline and methyl groups metabolism in adult ruminants in positive energy balance (adapted from Pinotti et al., 2002). SAM, S-adenosyl-L-methionine. HCys, Homocysteine. TM, Transamethylation pathway. THF Tetrahydrofolate system. Choline oxidase (EC 1.1.3.17). Betainehomocysteine methyltransferase (EC 2.1.1.5). Methionine synthase (EC 2.1.1.13). (adapted from Pinotti et al., 2002).


**Table 1.** Evolution of metabolism in methyl groups in ruminants in three physiological stages[\*at the onset of lactation, the substrates of this path often become limiting](adapted from Pinotti et al., 2002).
