Nutritional and osmoregulatory functions of betaine

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1 See discussions, stats, and author profiles for this publication at: Nutritional and osmoregulatory functions of betaine ARTICLE in WORLD'S POULTRY SCIENCE JOURNAL JUNE 1997 Impact Factor: 1.09 DOI: /WPS CITATIONS 77 READS AUTHORS, INCLUDING: Peter Ferket North Carolina State University 175 PUBLICATIONS 2,813 CITATIONS SEE PROFILE Available from: Peter Ferket Retrieved on: 08 April 2016

2 Nutritional and osmoregulatory functions of betaine M.T. KIDD", P.R. FERKET and J.D. GARLICH Department of Poultry Science, North Carolina State University, Raleigh, North Carolina , USA Betaine, a donor of labile methyl groups, can spare choline and methionine but cannot replace these compounds in poultry diets. Betaine is synthesized from choline by choline oxidase and it can donate methyl groups to homocysteine to form methionine. Physiologically, betaine is one of several compounds used by cells to regulate osmotic pressure. Among the potential benefits of its inclusion in poultry feeds are sparing choline, carcass fat reduction and aiding cell osmoregulation. Some feed ingredients are natural sources of betaine per se. This review considers the metabolism, functions and applications of betaine in poultry. Keywords: Betaine; choline; methionine; osmoregulation; poultry. Introduction The significance of betaine in chick nutrition was first shown in the early 1940s (du Vigneaud et al., 1939; Jukes and Welch, 1942; McGinnis et al., 1942; Almquist and Grau, 1943, 1944). In order to determine the role of betaine in chicks, these researchers fed semi-purified diets supplemented with various levels of betaine, methionine, or choline and evaluated growth and occurrence of perosis in chicks. Perosis, a symptom of choline deficiency in birds, is defined as haemorrhages and swelling in the hocks due to abnormal cartilage formation. This condition, which causes the metatarsal joint to twist and the Achilles tendon to slip from the condyle, is commonly called 'slipped tendon' (Scott et al., 1982). Betaine inclusion improved the growth of chicks fed a semi-purified diet (McGinnis et al., 1942). Almquist and Grau (1944) showed that, although betaine improved growth, the improvement was less than that obtained with choline. These authors also established that the inability of betaine to stimulate growth when added to a purified diet was due to a higher methionine content. Choline improved growth *Present address: Nutri-Quest Inc., 1400 Eldbridge Payne Road, Suite 110, Chesterfield, MO 63017, USA. 0 World's Poultry Science Association World's Poultry Science Journal, Vol. 53, June 1997

3 Nutritional and osrnoregulatory functions of betaine: M.T. Kidd et al. as well as preventing perosis when included in both semi-purified and purified diets. Early biochemical studies led to the conclusion that betaine is formed from choline and that growth responses obtained from betaine are due to its ability to provide methyl groups. Subsequent biochemical studies established that betaine donates methyl groups to homocysteine to form methionine. Thus, betaine can spare methionine, a methyl donor, by recycling homocysteine. Apart from methyl group donation, other roles of betaine are less well known. Betaine alters fat metabolism of rats (de Ridder and van Dam, 1975) and is an osmoprotectant in bacteria (Sutherland et al., 1986), plants, (Yancey ef al., 1982) and animals (Law and Burg, 1991). The applications of these roles in poultry have not been studied thoroughly. The objective of this paper is to review the metabolism and function of betaine and to evaluate its new potential uses in poultry. Betaine metabolism Betaine is a metabolite of choline that donates methyl groups to homocysteine to form methionine, and also to the folate pool. A discussion of betaine metabolism is incomplete without reference to both choline and methionine. Choline Choline functions in nerve impulse transmission as a part of acetylcholine. Poultry require adequate acetylcholine for functions such as heart beat, oviduct contraction, and crop emptying (Scott ef al., 1982). Choline is incorporated into lecithin (phosphatidyl choline) and sphingomyelin, which are essential components of cell membranes throughout the body. Both lecithin and sphingomyelin are incorporated into lipoproteins in the liver. Fatty liver syndrome, involving the accumulation of triglycerides in the livers of rats and male chicks, occurs when dietary choline is inadequate for lipoprotein formation, while lecithin is the major phospholipid constituent of the membranes that maintain cell integrity in all organs (Zeisel, 1981). From this it can be concluded that the growth promoting effects of supplemental choline reflect a need for membrane synthesis and consequent cellular and tissue integrity. Hegsted et al. (1941) showed that choline is essential for growth and the prevention of perosis in chicks fed a semi-purified diet. Choline, but not betaine or methionine, prevented perosis in experiments by Pesti et al. (1980). Biosynthesis of choline involves methylation of phosphatidylethanolamine and is catalysed by phosphatidyl-ethanolamine-n-methyltransferase (EC ) (Combs, 1992). The methyl donor involved is S-adenosyl methionine. The activity of this enzyme for the biosynthesis of choline is negligible in young male poultry and is limited in young females (Combs, 1992). However, the enzyme is active in sexually mature females. A choline deficiency in male chicks can be readily produced. Hence, young chicks and poults require a dietary source of choline. Molitoris and Baker (1976) demonstrated that neither methionine nor betaine could replace choline in the semi-purified diet of male broiler chicks. These authors also determined that the availability of choline in soybean meal is > 60%. Laying fowl are capable of synthesizing enough choline for maintenance and egg production (Lucas et al., 1946). Nesheim ef al. (1971) demonstrated that 126 World s Poultry Science Journal, Vol. 53, June 1997

4 Nutritional and osrnoregulatory functions of betaine: M.T. Kidd et al. choline supplementation of practical rearing or laying diets resulted in no beneficial responses in layers. Keshavarz and Austic (1985) replaced methionine with choline in layer diets and concluded that supplemental choline only improves egg production when there is a dietary deficiency of total sulphur amino acids. Parons and Leeper (1984) fed low protein diets to layers and demonstrated that they have specific requirements for methionine, and to a lesser extent choline, rather than choline and methyl groups per se. Domestic fowl are therefore capable of synthesizing enough choline to meet their needs for phospholipid deposition in egg yolk. A typical whole egg contains 246mg of choline (Ensminger et al., 1986). Methionine Choline is oxidized to betaine by the enzyme choline oxidase (Figure 1). Betaine can sometimes spare methionine because betaine provides methyl groups for methionine regeneration. Quantitatively, most of the methionine, whether synthesized or of dietary origin, is utilized as building blocks for protein. Methionine plus adenosine triphosphate forms S-adenosyl methionine which is a major donor of methyl groups. Methionine is formed from homocysteine and betaine in the liver by the enzyme betaine-homocysteine methyltransferase (EC ) with production of the co-product N,N-dimethylglycine. In the process, only one methyl group of betaine is directly available for homocysteine methylation; however, N,N-dimethylglycine and sarcosine can transfer their methyl groups to tetrahydrofolate to form N5-methyltetrahydrofolate. Thus, methionine can also be formed by the transfer of a methyl group from N5-methyltetrahydrofolate to homocysteine which is catalysed by methyltetrahydrofolate-homocysteine methyltransferase (EC ) and mediated by the coenzyme methylcobalamin. Methionine is the source of sulphur for cysteine biosynthesis via the reaction of serine with homocysteine to form cystathionine. This reaction is catalysed by cystathionine gamma-synthase (EC ). Cystathionine gamma-lyase subsequently produces free cysteine. This series of reactions to form cysteine from methionine is not reversible and consequently cysteine cannot give rise to methionine. However, the compounds cysteine, choline and betaine can spare dietary methionine by meeting requirements to which methionine could contribute (Scott et al., 1982). Pesti et al. (1979) evaluated the interrelationship between choline and methionine in broiler chicks. Broilers were fed a high fat, maize-soybean meal basal diet containing 1500 mg/kg choline. In their experiment 2 the chicks responded in body weight gain to methionine and betaine but not to cysteine or sulphate. In experiment 5 the chicks responded to both methionine and choline. These authors concluded that their basal diet had adequate sulphur amino acids for growth but inadequate methyl groups. In addition, Pesti ef al. (1980) hypothesized that broiler growth can be hindered by insufficient numbers of metabolically active labile methyl donors rather than by methionine per se. This hypothesis was verified by feeding broilers a high fat maize-soybean meal basal diet supplemented with either methionine, choline, betaine or homocysteine. Methionine, choline and betaine improved growth and feed efficiency whereas homocysteine did not. A comprehensive review of the interrelationships between choline, methionine and sulphate in poultry is given elsewhere (Baker, 1977; Ruiz et al., 1983). World's Poultry Science Journal, Vol. 53, June

5 -_ Acetylcholine 4-(CHJ3 N+ CH2-CH,0H Phosphocholine Choline Nutritional and osrnoregulatory functions of betaine: M.T. Kidd et al. FADH I CDP-Choline NAD 2 I- Cys eine alpha-ketobutyrate Acceptor Methylated acceptor e.g. Creatine Epinephrine Camitine Phosphatidylethanolamine Figure 1 Betaine and choline metabolism in poultry. Oxidation of choline by choline oxidase yields betaine aldehyde. Betaine is then formed by the action of betaine aldehyde dehydrogenase on betaine aldehyde. The remethylation of homocysteine to methionine requires betaine. Numbered enzymes include: (1) choline oxidase (EC 3.1 l.27); (2) betaine aldehyde dehydrogenase; (3) betainehomocysteine methyltransferase (EC ); (4) methyltetrahydrofolate-homocysteine methyltransferase (EC ); (5) methionine adenosyl transferase (EC ). 128 World's Poultry Science Journal, Vol. 53, June 1997

6 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et a1. Betaine, a labile methyl donor Betaine and methyl-tetrahydrofolate can both serve as donors for the methylation of homocysteine to methionine, but they do not directly participate in most biosynthetic methylations (Stryer, 1988; and see Figure 1). Both betaine and 5-methyltetrahydrofolate donate methyl groups to homocysteine to produce methionine and yield N,N-dimethylglycine and tetrahydrofolate by the action of betaine-homocysteine methyltransferase (EC ) and methyltetrahydrofolate-homocysteine methyltransferase (EC ), respectively (Combs, 1992). S-adenosyl methionine (the activated methyl donor) transfers a methyl group to an acceptor for the synthesis of compounds such as creatine, phosphatidylcholine, and epinephrine (Stryer, 1988). Furthermore, choline liberated from phosphatidylcholine may be oxidized to betaine (Figure 1). If dietary methionine is low, then betaine deficiency may result in an inability to regenerate methionine from homocysteine because the 5-methyltetrahydrofolate-homocysteine methyltransferase reaction is limited, as observed in mammals (Finkelstein ef al., 1982, 1983; Barak and Tuma, 1983). There is considerable variation in the published literature regarding the efficiency of choline, betaine and methionine for methylation reactions (Molitoris and Baker, 1976; Lowry et al., 1987). Stekol et al. (1953) reported that, in chicks, betaine methylates homocysteine to methionine approximately three times more efficiently than choline. This difference in efficiency may be due to inefficiencies in the metabolic conversion of choline to betaine. Choline must be transported from the cytosol into the mitochondria where it is oxidized to betaine, and then betaine is transported to the cytosol where it can function as a methyl donor (Mann ef al., 1938). The efficiency of converting choline to betaine is further reduced by polyether ionophores by interfering with mitochondria1 membrane transport (Tyler, 1977). These ionophores are commonly used to control coccidiosis in poultry. Conversely, Pesti et al. (1980, 1981) demonstrated that betaine, choline and methionine appear equal as sources of methyl groups. Using a chick growth model, Pesti et al. (1981) concluded that sparing of methionine by the methylation of homocysteine to methionine is increased by providing dietary choline or betaine. This conclusion was confirmed by Finkelstein ef az. (1983) who evaluated the effect of supplemental betaine and choline at dietary levels of 0.2% on hepatic betaine-homocysteine methyltransferase activity in rats. Hepatic betaine-homocysteine methyltransferase activity increased as dietary levels of betaine and choline increased. In addition, intraperitoneal injection of betaine to rats fed choline-free diets increased the activity of this enzyme. It is important to note that the rat has a high hepatic choline oxidase activity, thus favouring the use of choline as a methyl donor. Saunderson and MacKinlay (1990) evaluated growth and hepatic enzymes in male broiler chicks as influenced by dietary supplementation with combinations of methionine, betaine and choline. Dietary supplementation of methionine with either betaine or choline did not produce chick weight gains that were different from those produced by methionine or choline supplements alone. However, hepatic betaine-homocysteine activity was much higher than 5-methyltetrahydrofolate-homocysteine methyltransferase (24.2 versus 10.3 nmol/hour/mg protein in chick liver). Remethylation of homocysteine to methionine in male broiler chicks therefore seems to be achieved primarily with methyl groups from betaine. World s Poultry Science Journal, Vol. 53, June

7 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al. Applications for betaine Lipid metabolism The involvement of choline in the synthesis of phospholipids and cell membranes has been discussed in a previous section. Betaine indirectly contributes labile methyl groups for the synthesis of carnitine via S-adenosylmethionine. Carnitine is required for transporting long-chain fatty acids across inner mitochondria1 membranes for oxidation (Stryer, 1988). Inadequate carnitine, caused by a deficiency in methyl groups, impairs fatty acid oxidation (Stryer, 1988). Supplemental dietary choline decreases urinary carnitine excretion in humans and guinea pigs (Daily and Sachan, 1995). Thus, fatty acid transport into cells and mitochondria may increase during carnitine deficiency. Choline deficiency results in fatty liver in both rats and chicks (Lombardi et al., 1968) and laying fowl (Schexnailder and Griffith, 1973; Wolford and Polin, 1975). Oxidation of choline in rat liver mitochondria results in the formation of betaine (de Ridder and van Dam, 1973). These authors demonstrated that betaine is present in high concentrations in rat liver mitochondria (de Rideer and van Dam, 1975). Suboptimal secretion of low density lipoprotein by cultured hepatocytes isolated from choline-deficient rats is corrected by supplemental betaine (Yao and Vance, 1989). These studies support the hypothesis that betaine functions to spare choline for the formation of phospholipids. Many consumers of poultry meat place high value on lean products. Moreover, the abdominal fat pad of broilers represents a waste product and contributes in part to waste water fat pollution as a consequence of fat pad removal during processing. Bell (1995) reported a 12% decrease in backfat thickness in swine fed a diet supplemented with betaine. Saunderson and MacKinlay (1990) observed a reduction in the carcass fat of chicks fed a basal diet supplemented with betaine and methionine combined compared with a basal diet supplemented with choline and methionine combined (88.2 versus 71.5 g crude fat/kg body weight). However, the combined betaine and methionine supplementation did not result in a difference in carcass fat compared with chicks fed basal diets supplemented with methionine or choline alone. Thus, although betaine is involved in phospholipid metabolism, a reduction in carcass fat in poultry from dietary betaine supplementation is not clearly established. More research data on the effect of betaine on carcass fat deposition need to be published to help clarify the issue. Osmoregulation Birds maintain the intracellular concentration of water that is crucial for homeostasis by osmoregulation. Osmoregulation is the ability of a cell to maintain its structure and function by regulating movement of water in and out of the cell. In the bodies of most animals water is the major component of virtually all the compounds present (Dick, 1979) and is vital for survival. For example, chick heart fibroblast cells contain 87% (vol/vol) water as measured at isotonic osmotic pressure (Dick, 1958). Most cells (animal, plant, and prokaryotic) adapt to external osmotic pressure or stress by altering the intracellular concentration of low molecular weight organic solutes (Wunz and Wright, 1993) and inorganic ions. Inorganic ions are limited osmotic effectors within cells because their concentration levels can affect protein structure and enzyme function (Burg, 1994; Petronini et al., 1992,1994). The purpose of this section is to review the role of betaine as an organic osmotic effector (i.e. osmoprotectant or osmolyte) with possible application to commercial poultry production. 130 World s Poultry Science journal, Vol. 53, June 1997

8 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al. Organic osmo2ytes. Organic osmotic osmolytes are major modulators of intracellular osmolarity (Gilles, 1979). Osmolarity is expressed in milliosmoles/l or milliosmolar (mosm). For example, the osmolarity of 1mM of glucose is 1 mosm, the osmolarity of 1 mm sodium chloride is 2mOsM. The normal osmolarity of chicken plasma is 300 mosm. Aproximately 10-20% of the particles that regulate the osmotic pressure of their intracellular fluid are organic. Animals with high blood osmolarity (e.g. marine invertebrates) use organic compounds to regulate approximately 60-70% of their intracellular fluid osmolarity. This is because organic osmolytes (especially betaine) are highly compatible with enzyme function and altering their intracellular concentration does not upset metabolism (Yancy et al., 1982; Draglovich, 1994). Because osmoregulation of marine animals has been studied in some detail, it serves as a basis for the consideration of osmoregulation in non-marine vertebrates, particularly in poultry. Robertson (1965) demonstrated that osmolarity due to organic compounds was 57% in the mantle muscle of Sepia officinalis (a cephalopod mollusc). Moreover, betaine represented 14% of the osmolarity from organic compounds (or 8% of total osmolarity in the mantle muscle). Therefore, amino acids and amino acid-like compounds are important for maintaining the control of intracellular fluid osmolarity in molluscs (Gilles, 1979; Chambelaine and Strange, 1989). For example, the betaine content of gill tissue in a marine mussel (Myths califurnianus) may exceed 45 mmol/kg wet weight, which is equivalent to an intracellular concentration of 180 mm betaine (Wright et al., 1992). The importance of organic osmolarity has been demonstrated by transferring marine animals and crustacea from fresh water to sea water and vice versa. For example, 40% of the osmoregulation of Chinese crabs (Eriucheir sinensis) is maintained by amino acids, taurine, betaine, and trimethylamine oxide (Gilles, 1979). Transferring these crabs from fresh water to sea water results in a twofold increase in free amino acids, but not betaine. Sea water tolerance of young Atlantic salmon, however, is improved when betaine is added to their diet (Virtanen et al., 1989). Osmoregulation by betaine. The osmoprotective properties of betaine are well conserved in many forms of life, including bacteria (Chambers and Kunin, 1987), plants (Yancey et al., 1982) and animals (Law and Burg, 1991). Osmoprotective substances are used by bacterial cells to prevent dehydration when growing in concentrated solutions of glucose, sodium chloride or other salts. Betaine is the most important osmoprotective compound in bacteria, although glycerophosphocholine, proline and glutamine are also osmoprotectants (Imhoff and Rodriguez-Valera, 1984). These authors demonstrated that halophilic eubacteria placed in solutions with increased external osmotic pressure increase the synthesis of betaine. Le Rudulier ef al. (1984) has shown that supplementing Escherichia culi with betaine allows them to grow in an environment with double the osmotic strength of sea water. Betaine is known to serve as an osmoprotectant in bacteria by replacing intracellular K' and restoring the osmotic turgor without accumulation of K+ when the environmental salinity increases (Sutherland et al., 1986). These beneficial osmoprotective properties may be due to the dipolar zwitterion characteristics of betaine and its high solubility in water (Chambers and Kunin, 1985). The unique chemical properties of betaine play a key role in providing osmoprotective properties in microorganisms and these attributes have a parallel in more complex organisms (Bagnasco et al., 1986; Chambers and Kunin, World's Poultry Science Journal, Vol. 53, June

9 Nutritional and osrnoregulatory functions of betaine: M.T. Kidd et al. 1987). There is evidence that the addition of betaine to the feed improves the seawater tolerance of charr (Staurnes and Eliassen, 1986), rainbow trout and young Atlantic salmon (Virtanen et al., 1989). In mammalian cells the common inorganic intracellular osmolytes are potassium, magnesium and phosphate. Common organic osmolytes include methylated amines (e.g. betaine), certain amino acids (e.g. arginine, lysine, taurine, proline, glutamine), and sugar alcohols (e.g. sorbitol, myo-inositol) (Bagnasco et al., 1986; Wunz and Wright, 1993). These osmolytes protect cells from environments with high levels of sodium chloride (Yancey et al., 1982) or other osmotically active compounds. Most experiments evaluating organic intracellular osmolytes are conducted in the presence of the perturbing solutes sodium chloride, potassium, chloride, and urea. These solutes inhibit or disturb cellular enzymes, non-enzyme proteins and nucleic acids if allowed to reach high intracellular concentrations (von Hippel and Scheich, 1969; Nakanishi et al., 1988; Burg, 1994; Petronini et al., 1992, 1994). In contrast, betaine is highly compatible with cellular enzyme function and structural proteins and membranes. Betaine can raise cytoplasmic osmotic pressure in stressed cells by increasing the temperature and ionic tolerance of critical enzymes and cellular membranes (Hanson et al., 1994). Mammalian cell culture medium made hyperosmotic by the addition of sodium chloride causes Madin-Darby canine kidney (MDCK) cells to increase their concentration of betaine, whereas this response does not occur with urea plus sodium chloride or urea alone (Nakanishi et al., 1988). The medium used in this study contained 10% fetal bovine serum which provided 18mM betaine. In addition, sodium chloride also increased the concentrations of myoinositol and glycerophosphorylcholine (GPC) in MDCK cells. Moreover, MDCK cells, as well as others tested, did not accumulate betaine during hyperosmotic stress if betaine was not present in the medium (Nakanishi et al., 1990). Thus, an uptake of extracellular betaine occurs from the medium rather than synthesis of betaine by MDCK cells (Nakanishi et al., 1990). Importantly, these authors have demonstrated that extracellular uptake of betaine by MDCK cells is dependent on a sodium-dependent betaine transporter. Media made hypertonic with sodium chloride cause a 10-fold increase in the maximal velocity of the sodium-dependent betaine transport system without affecting the Michaelis constant (Km). This dramatic increase in maximal velocity is induced by hypertonic conditions rather than by sodium chloride alone, since isotonic conditions with mannitol gave similar results. Under osmotic stress, MDCK cells increase the maximal velocity of sodium-dependent betaine uptake. The maximal velocity decreases as betaine and other osmolytes accumulate in these cells. In general, betaine transport increases in mammalian cells as external osmolarity rises. Betaine and GPC are methylamines that protect renal cells from urea (Law and Burg, 1991). Antidiuresis in mammals increases betaine in the inner medulla (Yancey and Burg, 1990) and, to a lesser extent, in the outer medulla and cortex (Bagnasco et al., 1986). Yancey and Burg (1989) have shown that the concentration of intracellular betaine is correlated with sodium and, to a lesser extent, with urea in rabbit kidney cells. These authors also demonstrated that betaine and GPC are the main osmolyte stabilizing cells against urea in renal medulla. Osmoregulation by betaine in poulty. The kidney, large intestine, caeca and cloaca maintain whole body osmoregulation in poultry (Shoemaker, 1972). Body 132 Worlds Poultry Science Journal, Vol. 53, June 1997

10 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al LV I I 1 I I I I a2 85 a9 93 Age (days) Figure 2 Percentage litter moisture of female turkeys supplemented with betaine for 24 hours at 69 days of age. Betaine supplementation was at a rate of 2.5g per litre drinking water. water from drinking and eating and that formed by oxidative metabolism must equal water lost by evaporation and through urine, faeces and glandular secretions in order for poultry to maintain osmoregulatory balance. Electrolyte ingestion must also equal electrolyte excretion. When affected by diarrhoea, the osmotic balance in poultry is altered. Diarrhoea may result from infections of protozoa (e.g. species of Eirneuia), bacteria (e.g. Escherichia coli, Pasturella rnultocida and species of Salmonella), viruses (e.g. rotavirus) and fungi (e.g. Candida albicans). The condition may also result from excessive consumption of water, sodium, chloride, potassium, magnesium sulphate and indigestible carbohydrates. Diarrhoea in poultry is of practical concern because it increases litter moisture and consequently increases atmospheric ammonia and odour emission. High litter moisture increases the susceptibility of a flock to pathogens. Ferket (1995) evaluated the efficacy of betaine in female turkeys with diarrhoea by adding it to the drinking water (approximately 2.5 g anhydrous betaine per litre of drinking water) for a 24 hour period at 69 days of age. This flock had been selected for a betaine field study due to excessive diarrhoea and wet litter. Litter moisture was sampled at various times after the initial betaine administration (Figure 2). The moisture content of litter sampled six days after the initial betaine administration (75 days of age) had decreased from 46% to 27%. Ferket (1995) also treated 60 flocks of commercial male turkeys showing symptoms of diarrhoea with 2.5g anhydrous betaine per litre of drinking water for a 48 hour period. This betaine treatment was effective in stopping diarrhoea in 96% of all male flocks over the age of 70 days, but the treatment was less effective in younger flocks ( < 60%). The effective diarrhoea treatment dosage ranged from 0.15 to 1.5g in anhydrous betaine per kg body weight. The causative agent that induced diarrhoea in the female and male turkeys could not be determined. Although many factors contribute to the World s Poultry Science Journal, Vol. 53, June

11 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al. condition in commercial poultry, betaine appears to have a beneficial effect and should be the subject of future research. Diarrhoea1 diseases caused by bacteria (e.g. Clostridium, Escherichia, or Salmonella) and viruses (e.g. adenovirus, coronavirus, parvovirus, or rotavirus) stimulate adenylate cyclase and increase CAMP which increases crypt cell secretory activity and decreases villous absorption of sodium and chloride (Fondacara, 1986). The result is a hyperosmolar solution in the intestinal lumen (Cantey, 1993). One may hypothesize that the potential effectiveness of exogenous or supplemental betaine in ameliorating diarrhoea in poultry may be related to the upgrading of betaine transport in response to the hyperosmolarity, and the consequent accumulation of betaine in the intestinal epithelial cells where betaine exerts protective effects on the integrity and functions of the cells. Coccidiosis in birds, caused by protozoan parasites of the genus Eimeuia, results in diarrhoea which may lead to dehydration (Pellerdy, 1974). E. tenella causes caecal coccidiosis and is of great importance in commercial broilers because infection results in high morbidity and mortality (McDougald and Reid, 1991). Susceptibility to Eimeuia species is dependent upon environmental conditions, medication and immunity (Chapman, 1988). Active coccidiosis can result in decreased nutrient absorption, enteritis and eventually death in severe cases. Fluid loss as a result of caecal coccidiosis may lead to impaired osmotic balance. Experimental infection from oocysts of E. fenella caused osmotic changes in chickens due to loss of water (Chadwick et al., 1985). These authors evaluated plasma concentrations of prolactin, glucose, sodium and potassium after E. tenella infection. Plasma electrolytes were unchanged, haematocrit was reduced, and glucose and prolactin were elevated seven days after E. fenella infection. They concluded that E. fenella induced water loss in birds and that the impaired osmotic balance is directly related to increased prolactin concentrations. Gwyther and Britton (1989) showed that male broiler chicks orally given 1 X lo5 sporulated oocysts of E. necatrix had a decrease in both sodium/potassium and calcium/ magnesium ATPase activities of 53% and 6076, respectively, in midgut tissue. The ATPase enzyme system facilitates active membrane transport. For example, the sodium/potassium ATPase pumps potassium in and sodium out of the cell with the concomitant hydrolysis of ATP (Voet and Voet, 1995). The net result is the maintenance of the correct intracellular osmolarity and differential concentrations of electrolytes. Renal cells possess an active sodium-dependent betaine transport system that increases in activity under osmotic stress in vitro (Nakanishi et al., 1990). Handler and Kwon (1993) reviewed the regulation of renal cell osmolytes. Madin-Darby canine kidney cells accumulate betaine and other osmolytes when cultured in a hypertonic medium. Cellular betaine transport activity peaks at 24 hours after betaine administration to the medium. Shifts in cellular tonicity increase transcription of the genes for the sodium chloride-betaine co-transporters. It is not known, however, whether poultry possess a betaine transport mechanism in intestinal cells. As previously stated in this review, betaine is an effective organic osmoregulator in bacteria (Chambers and Kunin, 19871, plants (Yancey et al., 1982) and animals (Law and Burg, 1991). The ability of betaine to provide osmotic stability to intestinal cells of poultry during parasitic infection warrants investigation. The effects of betaine on osmoregulation in poultry under the influence of severe stressors such as heat, high sodium intake, low phosphorus intake, intestinal infection, mycotoxin intake and corticosterone should also be subject to experimentation. 134 World s Poultry Science Journal, Vol. 53, June 1997

12 ~ Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al. Table 1 Betaine content of common feed ingredients Feed ingredient Alfalfa, 17% dehydrated meal Soybean meal Fish meal Meat meal Canola meal Corn, ground yellow Milo, ground Rice Oats Barley Wheat, ground Wheat bran Wheat standard middlings Betaine content (mg/kg) Below detection limit* Below detection limit* Below detection limit Below detection limit) Below detection limit* Reference Westberg (1951) Vertanen (1993) Vertanen (1993) Vertanen (1993) Vertanen (1993) Westberg (1951) Westberg (1951) Vertanen (1993) Vertanen (1993) Vertanen (1993) Vertanen (1993) Westberg (1951) Westberg (1951) Average detection limit for betaine in feedstuffs is about 150mg/kg Dietary sources of betaine In general, this review demonstrates that poultry may have a need for betaine depending on the dietary concentration of methyl donors and/or intracellular osmotic stress. This need is dependent upon age and physiological condition. If the bird s total betaine need cannot be met by endogenous metabolism, a dietary source may be necessary. The betaine contents of some common poultry feed ingredients are shown in Table 1. Diets composed primarily of maize, milo, soyabean meal and meat or fish by-product meals, as are common in the USA and some other parts of the world, have very low betaine contents. In such diets supplementation may be necessary to satisfy the apparent needs of animals for betaine. These can be provided by the inclusion of condensed beet molasses solubles ( g betaine/kg), feed grade betaine hydrochloride (722 g betaine/ kg), feed grade anhydrous betaine ( > 970 g betaine/kg), or purified betaine liquid ( > 470 g betaine/kg). Diets containing forage (e.g. alfalfa (lucerne)) and/or small grains (i.e. wheat, barley, etc.) or their products may contain sufficient betaine to satisfy apparent requirements. However, betaine contents and bioavailabilities may vary considerably, depending on crop growing conditions. In general, the betaine content of plant tissues rises as the soil moisture level decreases or salinity increases. Conclusions After reviewing the literature, one may conclude that poultry do not have a specific requirement for betaine provided they consume or synthesize sufficient choline. However, supplemental betaine may be advantageous during certain physiologically challenging conditions, including the high metabolic demand of rapid growth, disease and osmotic stress in different cell types. Because betaine is a labile donor of methyl groups, it can be used to spare the metabolic need for other methyl donor compounds, namely methionine and choline. Depending upon the application, dietary formulation or economic scenario, betaine may be World s Poultry Science Journal, Vol. 53, June

13 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al. the preferred methyl donor compound. On a molecular weight basis betaine contains about 3.75 and 0.90 times the methyl groups contained in methionine and choline, respectively. The methylation efficiency of betaine and methionine are similar, but choline is a less efficient methyl donor. On a practical basis, 1 kg of anhydrous betaine (97% activity) will provide the equivalent amount of methyl groups as 1.25 kg of D,L-methionine (activity 99%) or 1.65 kg of choline chloride (70% activity). Moreover, the osmoregulatory effects of betaine may be as important in poultry as its methyl donor function. Although the effects of betaine on osmoregulation have been elucidated primarily in non-avian species, the fact that it is conserved by most forms of life lends credance to the view that betaine may be an effective intracellular osmolyte in poultry as well. Betaine supplementation of feed or drinking water may therefore be applicable in the control of dysfunctional osmoregulatory conditions such as diarrhoea, catharsis, diuresis and ascites. The role of betaine in osmoregulation of poultry warrants further research. References ALMQUIST, H.J. and GRAU, C.R. (1943) Growth-promoting activity of betaine in the chick. Journal of Biological Chemistry 149: ALMQUIST, H.J. and GRAU, C.R. (1944) Interrelationship of methionine, choline, betaine, and arsenocholine in the chick. Journal of Nutrition 27: BAGNASCO, S., BALABAN, R., FALES, H.M., YANG, Y. and BURG, M. (1986) Predominant osmotically active organic solutes in rat and rabbit renal medullas. Journal of Biological Chemistry 261: BAKER, D.H. (1977) Sulfur in nonruminant nutrition. National Feed Ingredient Association Review, pp. 123 BARAK, A.J. and TUMA, D.J. (1983) Betaine, metabolic by-product or vital methylating agent? Life Sciences 32: BELL, A. (1995) What s the word on betaine? Pork95, February, pp BURG, M.B. (1994) Molecular basis for osmoregulation of organic osmolytes in renal medullary cells. Journal of Experimental Zoology 268: CANTEY, J.R. (1993) Escherichia coli diarrhea. Gastroenterology Clinics of North America 22: CHADWICK, A., RAPSON, E.B., CARLOS, G.M. and LEE, D.L. (1985) Circulating prolactin concentrations in chickens infected with Eimeria tenella. British Poultry Science 26: CHAMBELAINE, M.E. and STRANGE, K. (1989) Anisosmotic cell volume regulation: a comparative review. American Journal of Physiology 257 (Cell Physiology 26): C159-Cl73 CHAMBERS, S.T. and KUNIN, C.M. (1985) The osmoprotective properties of urine for bacteria: The protective effect of betaine and human urine against low ph and high concentrations of electrolytes, sugars, and urea. Journal of Infectious Diseases 152: CHAMBERS, S.T. and KUNIN, C.M. (1987) Osmoprotective activity for Escherichia coli in mammalian renal inner medulla and urine. Iournal of Clinical Investigation 80: CHAPMAN, H.D. (1988) Strategies for the control of coccidiosis in chickens. World s Poultry Science Journal 44: COMBS, G.F. (1992) The vitamins: fundamental aspects in nutrition and health. In: Quasi-Vitamins, Academic Press, New York, pp DAILY, J.W. and SACHAN, D.S. (1995) Choline supplementation alters carnitine homeostasis in humans and guinea pigs. Journal of Nutrition 125: DICK, D.A.T. (1958) Osmotic equilibria in fibroblasts in tissue culture measured by immersion refractometry. Proceedings of Royal Society of London B 149: DICK, D.A.T. (1979) Structural and properties of water in the cell. In: Mechanisms of Osmoregulation in Animals (Ed. Gilles, R.), John Wiley and Sons, New York, pp DRAGOLOVICH, J. (1994) Dealing with salt stress in animal cells: the role and regulation of glycine betaine concentrations. Journal of Experimental Zoology 168: DE RIDDER, J.J.M. and VAN, DAM, K. (1973) The efflux of betaine from rat-liver mitochondria, a possible regulating step in choline oxidation. Biochimica et Biophysica Acta 291: DE RIDDER, J.J.M. and VAN DAN, K. (1975) Control of choline oxidation by rat-liver mitochondria. Biochimica et Biophysica Acta 408: World s Poultry Science Journal, Vol. 53, June 1997

14 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al. DU VIGNEAUD, V., CHANDLER, J.P., MOYER, A.W. and KEPPEL, D.M. (1939) The effect of choline on the ability of homocystine to replace methionine in the diet. loicrnal of Biological Chemistry 131: ENSINGMER, A.H., ENSMINGER, M.E., KONLANDE, J.E. and ROBSON, J.R.K. (1986) Choline. In: Food for Health: A Nutrition Encyclopedia, Pegus Press, Clovis, California, pp FERKET, P.R. (1995) Flushing syndrome in commercial turkeys during the grow-out stage. In: Proceedings, Smithkline Beeclzain Pacesetter Conference, National Turkey Federation Annual Meeting, 10 January, pp FINKELSTEIN, J.D., MARTIN, J.J., HARRIS, B.J. and KYLE, W.E. (1982) Regulation of hepatic betaine-homocysteine methyltransferase by dietary methionine. Biochemical and Biophysical Research Communications 108: FINKELSTEIN, J.D., MARTIN, J.J., HARRIS, B.J. and KYLE, W.E. (1983) Regulation of hepatichomocysteine methyltransferase by dietary betaine. Journal of Nutrition 113: FONDACARO, J.D. (1986) Intestinal ion transport and diarrheal disease. American Journal of Physiology 250 (Gasrointestinal Liver Physiology 13) Gl-GS GILLES, R. (1979) Intracellular organic osmotic effectors. In: Mechanisms of Osmoregulation in Animals (ed. Gilles, R.), John Wiley and Sons, New York, pp GWYTHER, M.J. and BRITTON, W.M. (1989) The influence of coccidial infections and ionophore treatment on tissue cations and anions in broiler chicks. In: Coccidia and Intestinal Coccidiomorphs, INRA Publication, Tours, France, pp HANDLER, J.S. and KWON, H.M. (1993) Regulation of renal cell organic osmolyte transport by tonicity. American journal of Physiology 265 (Cell Physiology) 34: C1449-Cl455 HANSON, A.D., RATHINASABAPATHI, B., RIVOAL, J., BURNET, M., DILLON, M.O., GAGE, D.A. (1994) Osmoprotective compounds in the Plumbaginacae: a natural experiment in metabolic engineering of stress tolerance. Proceedings of the National Academy of Sciences USA 91: HEGSTED, D.M., MILLES, R.C., ELVEHJEM, C.A. and HART, E.B. (1941) Choline in the nutrition of chicks. Journal of Biological Chemistry 138: HIPPEL, P.H., von and SCHLEICH, T. (1969) The effects of neutral salts on the structure and conformational stability of macromolecules. In: Structure and Stability of Biological Macromolecules (Eds Timsheff, S.N. and Fasman, G.D.), Dekker, New York, pp IMHOFF, J.F. and RODRIGUEZ-VALERA, F. (1984) Betaine is the main compatible solute of halophilic eubacteria. Journal of Bacteriology 160: JUKES, T.H. and WELCH, A.D. (1942) The effect of certain analogues of choline on perosis. Journal of Biological Chemistry 146: KESHAVARZ, K. and AUSTIC, R.E. (1985) An investigation concerning the possibility of replacing supplemental methionine with choline in practical laying rations. Poultry Science 64: LAW, R.O. and BURG, M.B. (1991) The role of organic osmolytes in the regulation of mammalian cell volume. In: Advances in Comparative and Environmental Physiology Vol. 9, Volume and Osmolality Control in Animal Cells (Eds Gilles, R., Hoffmann, E.K. and Bolis, L.), Springer-Verlag, New York, pp LE RUDULIER, D., STROM, A.R., DANDEKAR, A.M., SMITH, L.T. and VALENTINE, R.C. (1984) Molecular biology of osmoregulation. Science 224: LOMBARDI, B., PANI, P. and SCHLUNK, F.F. (1968) Choline-deficiency fatty liver: impaired release of hepatic triglycerides. Journal of Lipid Research 9: LOWRY, K.R., IZQUIERDO, Q.A. and BAKER, D.H. (1987) Efficacy of betaine relative to choline as a methyl donor. Poultry Science 55 (Supplement 1): 135 LUCAS, H.L., NORRIS, L.C. and HEUSER, G.F. (1946) Observations on the choline requirements of hens. Poultry Science 25: MANN, P.J.G., WOODWARD, H.E. and QUASTEL, J.H. (1938) Hepatic oxidation of choline and arsenocholine. Biochemistry Journal 32: McDOUGALD, L.R. and REID, W.M. (1991) Coccidiosis. In: Diseases of Poultry (Ed. Calnek, B.W.), 9th ed. Iowa State University Press, Ames, Iowa, pp McGINNIS, J., NORRIS, L.C. and HEUSER, G.F. (1942) Effect of ethanolamine and betaine on perosis in chicks. Experimental Biology and Medicine 51: MOLITORIS, B.A. and BAKER, D.H. (1976) The choline requirement of broiler chicks during the seventh week of life. Poultry Science NAKANISHI, T., BALABAN, R.S. and BURG, M.B. (1988) Survey of osmolytes in renal cell lines. American Journal of Physiology 255 (Cell Physiology 24): C18l-Cl91 NAKANISHI, T., TURNER, R.J. and BURG, M.B. (1990) Osmoregulation of betaine transport in mammalian renal medullary cells. American Journal of Physiology 258 (Renal Fluid Electrolyte Physiology 27): F1061-F1067 World s Poultry Science Journal, Vol. 53, June

15 Nutritional and osmoregulatory functions of betaine: M.T. Kidd et al. NESHEIM, M.C., NORVELL, M.J., CEBALLOS, E. and LEACH, R.M. Jr. (1971) The effect of choline supplementation of diets for growing pullets and laying hens. Poultry Science 50: PARSONS, C.M. and LEEPER, R.W. (1984) Choline and methionine supplementation of layer diets varying in protein content. Poultry Science 63: PELLERDY, L.P. (1974) Coccidia and Coccidiosis. 2nd ed. Akademiai Kiado, Budapest, Hungary and Paul Parey, Berlin, Germany. PESTI, G.M., HARPER, A.E. and SUNDE, M.L. (1979) Sulfur amino acid and methyl donor status of corn-soy diets fed to starting broiler chicks and poults. Poultry Science 58: PESTI, G.M., HARPER, A.E. and SUNDE, M.L. (1980) Choline/methionine nutrition of starting broiler chicks. Three models for estimating the choline requirement with economic considerations. Poultry Science 59: PESTI, G.M., BENEVENGA, N.J., HARPER, A.E. and SUNDE, M.L. (1981) Factors influencing the assessment of the availability of choline in feedstuffs. Poultry Science 60: PETRONINI, P.G., DEANGELIS, E.M., BORGHETTI, P., BORGHETTI, A.F. and WHEELER, K.P. (1992) Modulation by betaine of cellular responses to osmotic stress. Biochemistry Journal 282: PETRONINI, P.G., DEANGELIS, E.M., BORGHETTI, A.F. and WHEELER, K.P. (1994) Osmotically inducible uptake of betaine via amino acid transport system A in SV-3T3 cells. Biochemistry Journal 300: ROBERTSON, J.D. (1965) Studies on the chemical composition of muscle tissue The mantle muscle of cephalopod mollusks. Journal of Experimental Biology 42: RUE, N., MILES, R.D. and HARMS, R.H. (1983) Choline, methionine, and sulfate interrelationships in poultry nutrition: a review. World s Poultry Science Journal 39: SAUNDERSON, C.L. and MacKINLAY, J. (1990) Changes in body-weight, composition and hepatic enzyme activities in response to dietary methionine, betaine and choline levels in growing chicks. British Journal of Nutrition 63: SCHEXNAILDER, R. and GRIFFITH, M. (1973) Liver fat and egg production of laying hens influenced by choline and other nutrients. Poultry Science 52: SCOTT, M.L., NESHEIM, M.C. and YOUNG, R.J. (1982) The vitamins. In: Nutrition of the Chicken. M.L. Scott and Associates, Ithaca, New York, pp SHOEMAKER, V.H. (1972) Osmoregulation and excretion in birds. In: Avian Biology, Vol. II. (Eds Farner, D.S., King, J.R. and Parkes, K.C.), Academic Press, New York, pp STAURNES, M. and ELIASSEN, R. (1986) Smoltifisering og sjovannstoleranse 11. (Smoltification and seawater tolerance 11.) Norsk Fiskeoppdrett STEKOL, J.A., HSU, P.T., WEISS, S. and SMITH, P. (1953) Labile methyl group and its synthesis de novo in relation to growth in chicks. Journal of Biological Chemistry 203: STRYER, L. (1988) Biosynthesis of amino acids and heme. In: Biochemistry, 3rd ed, W.H. Freeman and Company, New York, pp SUTHERLAND, L., CAIRNEY, J., ELMORE, M., BOOTH, I. and HIGGINS, C. (1986) Osmotic regulation of transcription: induction of the prou betaine transport gene is dependent on accumulation of intracellular potassium. Journal of Bacteriology 168: TYLER, D.D. (1977) Transport and oxidation of choline by liver mitochondria. Biochemistry Journall66: VIRTANEN, E., JUNNILA, M. and SOIVIO, A. (1989) Effects of food containing betaine/amino acid additive on the osmotic adaptation of young atlantic salmon, Salmo salar L. Aquaculture 83: VERTANEN, E. (1993) Analyzed values for betaine in feedstuffs. Personal communication, Finnsugar Bioproducts, Helsinki, Finland. VOET, D. and VOET, J.G. (1995) Transport through membranes. In: Biochemistry, 2nd ed., John Wiley and Sons, New York, pp WESTBERG, J.K. (1951) Betaine in the nutrition of chickens and turkeys. International Minerals and Chemical Corporation, Chicago, Illinois, pp. 3 WOLFORD, J.H. and POLIN, D. (1975) Effect of inositol, lecithin, vitamins (812 with choline and E), and iodinated casein on induced fatty liver-hemorrhagic syndrome in laying chickens. Poultry Science 54: WRIGHT, S.H., WUNZ, T.M. and SILVA, A.L. (1992) Betaine transport in the gill of a marine mussel, Mytilus cafifornianus. American Journal of Physiology 263 (Regulatory Integrative Comparative Physiology 32): R226-R232 WUNZ, T.M. and WRIGHT, S.H. (1993) Betaine transport in rabbit and renal brush-border membrane vesicles. Biochimica et Biophysica Acta 1062: World s Poultry Science Journal, Vol. 53, June 1997

16 Nutritional and osmoregulatovy functions of betaine: M.T. Kidd et al. YANCEY, P.H. and BURG, M.B. (1989) Distribution of major organic osmolytes in rabbit kidneys in diuresis and antidiuresis. American Journal of Physiology 257 (Renal Fluid Electrolyte Physiology 26): F602-F607 YANCEY, P.H. and BURG, M.B. (1990) Counteracting effects of urea and betaine in mammalian cells in culture. American Journal of Physiology 258 (Regulatory Integrative Comparative Physiology 27): R198-R204 YANCEY, P.H. and SOMERO, G.H. (1979) Counteracting of urea destabilization of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes. Biochemical Journal 183: YANCEY, P.H., CLARK, M.E., HAND, S.C., BOWLUS, R.D. and SOMERO, G.N. (1982) Living with water stress: evolution of osmolyte systems. Science 217: YAO, Z. and VANCE, D.E. (1989) Head group specificity in the requirement of phosphatidylcholine biosynthesis for very low density lipoprotein secretion from cultured hepatocytes. Iournal of Biological Chemistry 264: ZEISEL, S.H. (1981) Dietary choline: biochemistry, physiology, and pharmacology. Annual Reviezu of Nutrition 1: Worlds Poultry Science Journal, Vol. 53, June