betaine as an osmoregulant

Osmoregulation is the ability of a cell to maintain its structure and function by regulating movement of water in and out of the cell (Kidd et al., Reference KIDD, FERKET and GARLICH1997). This is an important issue because cell activity is affected by changes in cell water volume. For instance, a slight increase in the water volume of liver cells directs the cells into a more anabolic state, whereas a lower cell water volume will favour a more catabolic state (Häussinger, Reference HÄUSSINGER1998). Hence, water homeostasis is relevant for cells exposed to different osmotic pressures. Inorganic ions are limited osmotic effectors within cells because their concentration levels may affect protein structure and enzyme functions (Petronini et al., Reference PETRONINI, DE ANGELIS, BORGHETTI and BORGHETTI1992; Reference PETRONINI, DE ANGELIS, BORGHETTI and WHEELER1994). Therefore, compatible osmolytes are particularly important in situations of cellular dehydration since these compounds assist to minimize water loss against a prevailing osmotic gradient (Kettunen et al., Reference KETTUNEN, PEURANEN and TIIHONEN2001b; Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002).

Betaine is a widespread compatible osmolyte for various prokaryotes, animals, algae and salt-tolerant plants (e.g., Da Costa et al., Reference DA COSTA, GARNER, CHANG and ZEISEL1995). Its osmotic activities have been attributed to the dipolar zwitterion characteristics and high solubility in water (Chambers and Kunin, Reference CHAMBERS and KUNIN1985). Betaine exerts an osmoprotective effect by accumulating in cell organelles and cells exposed to osmotic and ionic stress, thereby replacing inorganic ions as well as protecting enzymes and cell membranes from inactivation by inorganic ions (Petronini et al., Reference PETRONINI, DE ANGELIS, BORGHETTI and BORGHETTI1992). Overall, betaine transport is mediated by the Na⁺-dependent amino acid transport system ‘A’ and by the Na⁺ and Cl^- dependent betaine-γ-aminobutyric acid (GABA) transporter (Kempson, Reference KEMPSON1998; De Angelis et al., Reference DE ANGELIS, PETRONINI, BORGHETTI, BORGHETTI and WHEELER1999). These transporters are found in a variety of tissues of mammals including, among others, those of skeletal muscles, kidneys, leukocytes and intestine (e.g., Bonadonna et al., Reference BONADONNA, COBELLI, SACCOMANI and DE FRONZO1993; Kempson, Reference KEMPSON1998; Sutinen et al., Reference SUTINEN, JYRKKIO, GRONROOS, HAAPARANTA, P. and NAGREN2001). There is also evidence that betaine is involved in the osmoregulation of the small intestine of poultry, since the Na⁺-dependent active transport system of betaine occurs in the duodenum and jejunum of broiler chicks (Kettunen et al., Reference KETTUNEN, PEURANEN, TIIHONEN and SAARINEN2001a). Dietary inclusion of betaine increases the number of Na⁺-dependent active transporters and the total quantity of betaine taken up by the duodenum (Kettunen et al., Reference KETTUNEN, PEURANEN and TIIHONEN2001b), although these effects have not been observed in ileal tissue. Differences in the absorption capacity of betaine in the duodenum and ileum appear to be related to the higher osmotic pressure normally present in the duodenum of chickens. The osmoprotectant function of betaine may be especially pronounced in animals exposed to osmotic disturbances originating from intestinal infections such as diarrhoea, but also from coccidiosis and high environmental temperatures (e.g., Virtanen and Rosi, Reference VIRTANEN and ROSI1995; Cronjé, Reference CRONJÉ2005).

betaine and coccidiosis

Coccidiosis in avian species is caused by intracellular protozoan parasites of the genus Eimeria, and is an important enteric disease in chickens. The infection is associated with metabolic and structural changes in the intestinal mucosa as well as osmotic and ionic disorders (Ruff and Allen, Reference RUFF and ALLEN1990). In poultry, various pathogenic Eimeria species colonise different segments of the digestive tract. For example, E. acervulina, E. maxima and E. tenella parasitize the duodenum, midgut and caeca, respectively. One of the major pathologic effects of coccidiosis, associated with a considerable reduction in growth performance, is intestinal malabsorption of amino acids, carbohydrates and micro nutrients such as carotenoids and vitamin E, with the main effect in the specific part of the intestine where the infection occurs (Allen and Fetterer, Reference ALLEN and FETTERER2000). Histological evidence shows that dietary supplementation of betaine can counteract morphological changes in the small intestine generally occurring during coccidial challenge thereby maintaining villus integrity and structure of the gut mucosa (Kettunen et al., Reference KETTUNEN, K., PEURANEN, SAARINEN and REMUS2001c; Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002). In this respect, Klasing et al. (Reference KLASING, ADLER, REMUS and CALVERT2002) reported that the shortening of duodenal villi due to E. acervulina was less severe in chicks fed 0.1% betaine than in those fed an unsupplemented diet. Similarly, 0.1% betaine improved the jejunal crypt:villus ratio of broilers challenged with E. maxima (Kettunen et al., Reference KETTUNEN, K., PEURANEN, SAARINEN and REMUS2001c). Furthermore, supplementation of diets with betaine has shown to improve nutrient absorption and growth performance during coccidiosis. For example, malabsorption of methionine was significantly reduced in E. acervulina-infected chicks following dietary supplementation with betaine (Augustine et al., Reference AUGUSTINE, MCNAUGHTON, VIRTANEN and ROSI1997). Overall, the efficiency of betaine to improve nutrient absorption and growth performance may be related, at least in part, to the individual Eimeria species. In this context, Fetterer et al. (Reference FETTERER, AUGUSTINE, ALLEN and BARFIELD2003) reported that betaine had no effect on weight gain of chicks infected with E. acervulina and E. tenella, but significantly improved growth performance in birds infected with E. maxima on the sixth day of infection. Augustine et al. (Reference AUGUSTINE, MCNAUGHTON, VIRTANEN and ROSI1997), in turn, observed an increase in body weight gain in chicks infected with a mixed culture of Eimeria species (E. tenella, E. maxima, E. acervulina) following dietary supplementation with 0.15% betaine and 66 ppm salinomycin on the seventh day after challenge, respectively. In addition, the combination of both products gave an additional significant improvement over either alone. A second major effect of coccidiosis in poultry is dehydration and the occurrence of hyperosmotic conditions in the intestine. Due to its function as an osmolyte, betaine may, at least partly, counteract the osmotic stress produced by diarrhoea and dehydration (Kettunen et al., Reference KETTUNEN, PEURANEN and TIIHONEN2001b; Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002) by minimising water loss despite a prevailing intestinal osmotic gradient (Kidd et al., Reference KIDD, FERKET and GARLICH1997).

Augustine et al. (Reference AUGUSTINE, MCNAUGHTON, VIRTANEN and ROSI1997) suggest that betaine may improve performance of Eimeria-infected chickens directly, by partial inhibition of coccidial invasion and development, and indirectly, by support of intestinal structure and function in the presence of coccidial infection. In fact, with respect to the invasion of the different Eimeria species, betaine or salinomycin alone or in combination reduced the invasion of E. tenella and E. acervulina sporozoites. Only the development of E. acervulina was adversely affected by feeding the combination of betaine and salinomycin compared with feeding salinomycin alone (Augustine et al., Reference AUGUSTINE, MCNAUGHTON, VIRTANEN and ROSI1997). Similarly, Remus et al. (Reference REMUS, VIRTANEN, ROSI and MCNAUGHTON1995) reported that the beneficial effects of salinomycin, lasolocid and halofuginone hydrobromide on lesion score, mortality, feed conversion and nutrient digestibility could be increased by betaine. Thus, betaine seems to potentiate the inhibitory effect against coccidial infection of certain types of coccidiostat used. This was clearly demonstrated in the study of Remus et al. (Reference REMUS, VIRTANEN, ROSI and MCNAUGHTON1996) feeding broiler chickens infected with a mixed culture of Eimeria (E. acervulina, E. maxima and E. tenella) with two levels of betaine (0 and 0.15%) and different kinds of coccidiostats such as salinomycin (66 ppm), monensin (110 ppm), narasin (79 ppm), nicarbazin (199 ppm), diclazuril (1 ppm), robenidine (33 ppm), zoalene (125 ppm), maduramycin (5 ppm). These authors observed increased body weight gain at three weeks of age in birds fed the combination of betaine plus salinomycin, narasin or zoalene. They also reported improved feed conversion and reduced lesion score in broilers fed the combination of betaine plus salinomycin, monensin, narasin, diclazuril, zoalene or maduramycin compared with addition of the coccidiostats alone. However, nicarbazin and robenidine did not respond when fed in combination with betaine. Overall, the improved efficacy of coccidiostats when fed to broiler chickens in combination with betaine (Remus et al., Reference REMUS, VIRTANEN, ROSI and MCNAUGHTON1995, Reference REMUS, VIRTANEN, ROSI and MCNAUGHTON1996; Augustine et al., Reference AUGUSTINE, MCNAUGHTON, VIRTANEN and ROSI1997) may be linked to the function of betaine as osmolyte. In fact, coccidiostats appear to disturb the osmotic balance of the gut which may be attenuated by dietary betaine. In a more recent study, Remus and McNaughton (Reference REMUS and MCNAUGHTON2006) confirmed that supplementation of a diet with 0.15% betaine and 60 ppm salinomycin is likely to have the greatest impact in reducing oocyst output and lesion score than feeding these supplements alone. In contrast, results of Matthews et al. (Reference MATTHEWS, WARD and SOUTHERN1997) do not support synergistic effects of monensin and betaine to alleviate the infection with E. acervulina in chicks. These authors pointed out that betaine alone may have a positive effect on production traits of E. acervulina-infected chicks, but this effect was limited to a dietary betaine level of 0.1%.

In addition, during E. acervulina-infection, supplementation of 0.1% betaine was shown to modulate the immune response by increasing intraepithelial and lamina propria leukocytes in the duodenum and functional properties of phagocytes, i.e. increased chemotaxis of monocytes and release of nitric oxide by macrophages (Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002). Thus, chicks fed dietary betaine had more leukocytes in the epithelium and in the lamina propria during an E. acervulina infection than those fed diets without betaine. Klasing et al. (Reference KLASING, ADLER, REMUS and CALVERT2002) assumed that this observation may be the result of more effective clearance of sporozoites, and may account for the decreased numbers in the epithelium as previously observed by Augustine et al. (Reference AUGUSTINE, MCNAUGHTON, VIRTANEN and ROSI1997). Thus, increased numbers of leukocytes in response to E. acervulina suggest that betaine either increased the chemotactic signals responsible for recruitment of leukocytes to the villi or had a direct effect on the leukocyte's responsiveness to the signals (Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002). The shortening of duodenal villi due to E. acervulina infection was less severe in chicks fed 0.1% betaine than in those fed the non-supplemented diet which could be attributed to increased functional properties of phagocytes and preservation of water balance of the enterocytes supporting normal intracellular environment for the metabolic activities of the cells (Augustine and Danforth, Reference AUGUSTINE and DANFORTH1999; Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002).

As coccidiosis is considered to be strongly related to osmotic disturbance, it could be rather expected that, in the absence of severe pathology, the intestinal cells of infected chicks would accumulate more betaine than those of uninfected chicks in an attempt to maintain a normal water balance. However, betaine levels in intestinal tissue and plasma decreased during coccidiosis compared with non-infected chicks (Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002; Fetterer et al., Reference FETTERER, AUGUSTINE, ALLEN and BARFIELD2003). For example, in chicks infected with E. acervulina or E. maxima and fed a betaine supplemented diet, levels in the duodenum and midgut were lower than levels in uninfected chicks, but they remained higher than the levels in either uninfected or infected chicks fed the control diet (Fetterer et al., Reference FETTERER, AUGUSTINE, ALLEN and BARFIELD2003). The lower betaine level may be related to malabsorption of betaine due to intestinal lesions resulting in lower plasma concentrations of betaine in infected chicks. In addition, coccidia-challenged chicks had higher levels of S-adenosylmethionine (SAM) in the liver (Tiihonen et al., Reference TIIHONEN, KETTUNEN, REMUS, SAARINEN and VIRTANEN1997). Because betaine is used as a methyl group donor in the conversion of homocysteine to methionine, lower betaine levels in plasma and intestinal tissue may indicate that more methyl groups are required for transmethylation in the liver of challenged birds compared with healthy control chicks (Tiihonen et al., Reference TIIHONEN, KETTUNEN, REMUS, SAARINEN and VIRTANEN1997; Klasing et al., Reference KLASING, ADLER, REMUS and CALVERT2002).

betaine and nutrient digestibility

There is growing evidence that betaine supports intestinal growth and function, thereby improving nutrient digestibility (Eklund et al., Reference EKLUND, BAUER, WAMATU and MOSENTHIN2005). This becomes particularly apparent in chicks challenged with coccidial infection. Given the fact that betaine counteracts hyperosmolarity, the osmotic effects of betaine appear to change cell proliferation patterns in an osmotically disturbed intestinal epithelium, e.g. during coccidial infection (Kettunen et al., Reference KETTUNEN, K., PEURANEN, SAARINEN and REMUS2001c). Additionally, it stabilises the mucosal structure in healthy chicks by decreasing the crypt:villus ratio (Kettunen et al., Reference KETTUNEN, K., PEURANEN, SAARINEN and REMUS2001c). In general, it is assumed that damaged rather than healthy tissue requires more methyl groups (Chiang et al., Reference CHIANG, CORDON, TAL, ZENG, DOCTOR, PARDHASARADHI and MCCANN1996). Indeed, the digestibilities of various nutrients including fat, protein, amino acids (methionine, lysine) and carotinoids were improved in coccidia-challenged broilers supplemented with betaine compared with challenged birds fed the control diet (Remus and Virtanen, Reference REMUS and VIRTANEN1996; Augustine and Danforth, Reference AUGUSTINE and DANFORTH1999). Enhanced feather growth in broilers suggested a higher availability of sulphur amino acids following dietary betaine supplementation (Garcia Neto et al., Reference GARCIA NETO, PESTI and BAKALLI2000). However, Attia et al. (Reference ATTIA, HASSAN, SHEHATTA and ABD EL-HADY2005) and Hassan et al. (Reference HASSAN, ATTIA and EL-GANZORY2005) did not observe any effects of betaine on the digestibility coefficients of dry matter, protein, fat and fibre in slow-growing El-Salam broiler chicks.

Transepithelial electrophysiological studies suggest that betaine changes the transport of ions in the intestinal epithelium of other monogastrics (Skadhauge cited by Kettunen et al., Reference KETTUNEN, PEURANEN and TIIHONEN2001b). Betaine may also alter the fluidity of artificial lipid membranes. Alterations in the lipid environment of the cells may modify the activity of enzymes responsible for the active transport of monovalent ions (Gibbs and Somero, Reference GIBBS and SOMERO1990; Madsen et al., Reference MADSEN, MEDDINGS and FEDORAK1992). Moreover, the presence of betaine in the intestinal tissue may reduce the energy requirements for ion- and osmoregulation, hence lowering the energy requirement for maintenance (Campbell et al., Reference CAMPBELL, MORLEY and ZABARAS-KRICK1997; Schrama et al., Reference SCHRAMA, HEETKAMP, SIMMINS and GERRITS2003). In fact, in vitro incubation of erythrocyte cell membranes with betaine resulted in a decrease of 64% in the activity of the Na⁺/K⁺ pump and a decrease of 73% in Ca²⁺ pump activity (Moeckel et al., Reference MOECKEL, SHADMAN, FOGEL and SADRZADEH2002). Inhibition of gut ion-pump activity of this magnitude would save a considerable amount of energy. A 64% decrease in Na⁺/K⁺ activity alone would decrease the total energy consumption of the intestine by 32%. Results showing that dietary betaine supplementation reduced the maintenance requirements of growing pigs by 5.5% (Schrama et al., Reference SCHRAMA, HEETKAMP, SIMMINS and GERRITS2003) illustrate the practical impact of the effect of betaine may have in poultry on the energy costs associated with ion pumping.

Improved ileal and faecal digestibility of crude fibre (ADF, NDF) in mammalian species suggests that betaine enhances the bacterial activity along the gastrointestinal tract as well (Eklund et al., Reference EKLUND, MOSENTHIN, TAFAJ and WAMATU2006a,b). In fact, it has been shown in vitro that betaine functions as an osmoprotectant in a range of bacterial groups, such as enterobacteria and lactobacilli, in preventing dehydration in a hyperosmotic environment (Perroud and Le Rudulier, Reference PERROUD and LE RUDULIER1985; Hutkins et al., Reference HUTKINS, ELLEFSON and KASHKET1987).

betaine and heat stress

An important issue challenging the poultry industry is the high ambient temperature in many countries which can exert deleterious effects on productive performance in poultry, including turkeys (e.g., McKee and Sams, Reference MCKEE and SAMS1997), broiler chickens (e.g., Cooper and Washburn, Reference COOPER and WASHBURN1998) and laying hens (e.g., Mashaly et al., Reference MASHALY, HENDRICKS, III, KALAMA, GEHAD, ABBAS and PATTERSON2004). Episodes of acute heat stress may be experienced by broiler chickens under normal production conditions and during procedures such as transportation and pre-slaughter holding (Kettlewell et al., Reference KETTLEWELL, MITCHELL and MEEHAN1993; Warriss et al., 1993). For instance, feed intake, egg production and egg weight due to impaired ovarian and immune functions, such as total white blood cell counts and antibody production, were significantly reduced in laying hens exposed to heat stress (e.g. Mashaly et al., Reference MASHALY, HENDRICKS, III, KALAMA, GEHAD, ABBAS and PATTERSON2004; Rozenboim et al., Reference ROZENBOIM, TAKO, GAL-GARBER, PROUDMAN and UNI2007). Similarly, in broiler chickens heat stress reduces feed consumption and weight gain (Deeb and Cahaner, Reference DEEB and CAHANER2001; Farooqi et al., Reference FAROOQI, KHAN, KHAN, RABBANI, PERVEZ and KHAN2005; Star et al., Reference STAR, KEMP, VAN DEN ANKER and PARMENTIERI2008). Pre-slaughter exposure of broiler chickens to acute heat stress causes disturbances in blood acid-base status and has detrimental effects on skeletal muscle membrane integrity. Both negatively affect breast muscle quality, though not necessarily causally (Sandercock et al., Reference SANDERCOCK, HUNTER, NUTE, MITCHELL and HOCKING P.M.2001). Several factors influence the bird's responses to heat stress including age, body size, previous exposure to heat stress (acute or chronic) and breed (Teeter and Belay, Reference TEETER and BELAY1996). However, when the environmental temperature surpasses a critical level, poultry attempt to compensate for their reduced ability to dissipate heat during heat stress. This is achieved by increasing the physiological processes responsible for heat dissipation and lowering heat production. At the critical temperature level, both a sharp increase in panting frequency of birds from 25 to 250 breaths per minute and reduced feed intake has been observed (Teeter and Belay, Reference TEETER and BELAY1996). However, the increased alveolar ventilation rate, necessary for evaporative cooling, results in a reduction in blood partial pressure of CO₂, bicarbonate levels and an increase in blood pH, causing acid-base perturbations, also described as respiratory alkalosis (Bottje and Harrison, Reference BOTTJE and HARRISON1985; Teeter et al., Reference TEETER, SMITH, OWENS, ARP, SANGIAH and BREAZILE1985). The birds attempt to adjust blood pH to a physiological level by excreting bicarbonates via the urine. Bicarbonates are negatively charged ions that must be coupled with cations such as potassium to be excreted in urine. However, as potassium is important in maintaining intracellular water balance, a loss of potassium ions via urine reduces the ability of the bird to maintain its water balance. As a result, heat stressed broilers double their total urine excretion (as uric acid) over a period of 12 hours (Belay et al., Reference BELAY, WIERNUSZ and TEETER1992). Moreover, higher urinary and faecal excretion of other minerals, such as phosphorus, sulphur, sodium, magnesium, calcium, manganese and copper, in broiler chickens housed at higher ambient temperature further depletes the body of electrolytes (Belay and Teeter, Reference BELAY and TEETER1996). Consequently, while birds do compensate for water losses associated with panting by consuming more water, its retention in body cells is limited because of the simultaneous loss of electrolytes in the urine (Belay et al., Reference BELAY, WIERNUSZ and TEETER1992). In laying hens, reduced egg production during heat stress can be linked to respiratory alkalosis because it affects the formation of calcium carbonate required for eggshell formation. Indeed, respiratory alkalosis reduces the amount of ionized calcium in the blood due to the higher blood pH and lowered levels of blood bicarbonate (Odom et al., Reference ODOM, HARRISON and BOTTJE1986).

Several management procedures have been developed to minimize the deleterious effects of heat stress. The addition of minerals to the diet or water beneficially affects the acid-base equilibrium of broiler chickens (Borges et al., Reference BORGES, FISCHER DA SILVA, MAJORKA, HOOGE and CUMMINGS2004). Supplementation of betaine as an osmolyte to feed or drinking water may help to minimize the effects of heat stress when the reduction in bird's performance due to heat stress is associated with damage to the intestinal integrity (Cronjé, Reference CRONJÉ2005). Thus, in addition to the involvement of betaine in reducing parasite-induced damage to gut cells (Kettunen et al., Reference KETTUNEN, PEURANEN and TIIHONEN2001b), betaine may reduce the osmotic damage to gut tissue during heat stress. Due to its osmotic properties, betaine may support the prevention of dehydration during exposure to hot environments at several situations (Cronjé, Reference CRONJÉ2005). In fact, betaine is involved in the reduction of increasing vascular permeability and consequent loss of blood plasma water in cases of hyperthermia (Matthew et al., Reference MATTHEW, DUBOSE, SILS and TARTARTINI2000). In addition, epithermal dehydration in sweating animals may be reduced, and the decrease in kidney function, that is often observed in cases of hyperthermia, may be prevented (Terui et al., Reference TERUI, ISHINO, MATSUDA, SHOJI, AMBO and TSUDA1980; De Galan and Hoekstra, Reference DE GALAN and HOEKSTRA1995). As a result, the efficiency of electrolyte and water retention is improved, thereby preventing dehydration (Cronjé, Reference CRONJÉ2005). Moreover, osmotic properties of betaine may help maintain normal cell volume because metabolic processes in the cell are profoundly influenced by small changes in cell volume including protein turnover, metabolism of amino acids, ammonia, carbohydrates and fatty acids, membrane transport and endocytosis (Häussinger, Reference HÄUSSINGER1996). In this context, ion pumps consume the major part of intracellular energy. In the heat-stressed animal, a reduction of gut energy expenditure for ion pump activity (Moeckel et al., Reference MOECKEL, SHADMAN, FOGEL and SADRZADEH2002) could delay or even abrogate the cascade of events that is initiated by depletion of intracellular energy reserves. As intracellular ion pumps are damaged during hyperthermia, the osmotic properties of betaine may also assist to prevent cell swelling caused by the increased entry of Ca²⁺. Increased intracellular concentrations of Ca²⁺ activate proteases that hydrolyse proteins. Since proteins are essential to the functional integrity of the cell membrane, the membrane permeability is progressively increased and the cell wall will be destroyed. As shown, for example, under in vitro conditions in Escherichia coli cells, betaine can protect protein from denaturation (Diamant et al., Reference DIAMANT, ELIAHU, ROSENTHAL and GOLOUBINOFF2001) and thus protect the cell membrane integrity. However, if intestinal membrane permeability progresses due to protein denaturation, junctions between the cells are damaged, allowing components of the digesta to enter the blood supply. For example, when the barrier function of the gut lining is compromised, endotoxins from the gut lumen can be translocated into the blood stream; thereby causing immune responses (cytokines), and finally precipitating multiple organ failure. The role of the gut in the etiology of heat stress has significant implications for livestock production because concentrated diets, which are frequently used in intensive production systems, result in damage to the gut lining of pigs (ulcers), poultry (gizzard erosion) and ruminants (parakeratosis). It is thus possible that such diets increase the susceptibility of livestock to the adverse effects of heat stress (Cronjé, Reference CRONJÉ2005).

Cells normally respond to thermal stress by synthesising heat shock proteins (HSP) which restore the functionality of denatured proteins by refolding them and preventing them from aggregating. In vitro studies with Madin-Darby canine kidney cells showed that, in addition to the expression of genes for HSP to prevent denaturation of cell proteins, heat stress resulted in the expression of genes for betaine transporters as well. Provided that betaine is available for the cells before the heat stress occurred, betaine accumulates in the cells and prevents the gene expression of HSP-70, presumably during heat stress due to its own stabilising effect on proteins (Sheikh-Hamad et al., Reference SHEIKH-HAMAD, GARCIA-PEREZ, FERRARIS, PETERS and BURG1994). However, betaine should be made available to the cells before heat stress occurs, because the Na⁺-transporter for betaine uptake is induced by extracellular hyperosmolarity, but not by heat itself (Sheikh-Hamad et al., Reference SHEIKH-HAMAD, GARCIA-PEREZ, FERRARIS, PETERS and BURG1994). Therefore, in order to be effective in preventing heat stress in poultry, betaine should be fed prior to thermal events when the feed intake and osmotic stress on the cells lining the lumen of the gut are at their highest levels, and not only during heat waves in situations of reduced feed intake.

Under heat stress conditions, supplementation of broiler diets with 0.1% betaine has shown to improve weight gain compared with control birds (Farooqi et al., Reference FAROOQI, KHAN, KHAN, RABBANI, PERVEZ and KHAN2005), and to increase breast muscle yield in broilers reared under tropical conditions (Pirompud et al., Reference PIROMPUD, ATTAMANGKUNE, BUNCHASAK and PORMBBON2005). Similarly, dietary betaine (0.2%) supplementation improved egg production and egg shell quality in hens kept under heat stress (Ryu et al., Reference RYU, CHO, SHIN and RYU2002). By measuring the antibody production after vaccination against Newcastle disease and mortality rate in broiler chickens, Zulkifli et al. (Reference ZULKIFLI, MYSAHRA and JIN2004) found that dietary supplementation of betaine is less efficient than its addition to the drinking water. Overall, to increase bird's tolerance towards higher temperatures, betaine may be useful as a part of an overall strategy to minimize the negative effects of heat stress in order to sustain performance, feed efficiency and survival of birds (Cronjé, Reference CRONJÉ2005).

methyl group transfer

The principles of methyl group transfer in the liver of poultry are illustrated in Figure 1. Since vertebrates are unable to synthesise methyl groups, they are an essential component of their diet (Kidd et al., Reference KIDD, FERKET and GARLICH1997). Potential dietary sources of methyl groups are betaine, choline, methionine and folic acid. However, their methyl groups are not equally available for the use in methylation reactions. Most of dietary methionine is needed for protein synthesis, while choline is predominantly used in cell membranes and neurotransmitter (Stryer, Reference STRYER1988). Folic acid is both a methyl group donor and a methyl group acceptor that can use the second and third methyl groups of betaine via the one-carbon pool (Finkelstein, Reference FINKELSTEIN1990).

Figure 1

Metabolism of sulphur amino acids, choline, and betaine. Numerals indicate the following enzymes: 1) methionine adenosyltransferase, 2) various enzymes, 3) S-adenosylhomocysteine hydrolase, 4) cystathionine β-synthase, 5) cystathionine γ-lyase, 6) N5,N10-methylenetetrahydrofolate reductase, 7) methionine synthase, 8) choline dehydrogenase, 9) betaine aldehyde dehydrogenase, and 10) betaine-homocysteine methyltransferase (adapted from Pillai et al., Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a).

The liver is an important site for metabolism of methionine, which is the first-limiting amino acid in commercial poultry diets. In its role as a methyl donor (transmethylation), methionine is converted to homocysteine, which lies at the crossroads of sulphur amino acid (SAA) metabolism (Figure 1). Thereby, SAM, synthesized from methionine and adenosine triphosphate, is the principal methyl group donor in vertebrate tissue (Finkelstein, Reference FINKELSTEIN1990). When SAM donates a methyl group, it forms S-adenosylhomocysteine, which is converted to homocysteine. Formation of cysteine occurs if homocysteine proceeds through the irreversible transsulphuration pathway; alternatively, homocysteine can be converted back to methionine after addition of a methyl group from tetrahydrofolate, or betaine by folate-vitamin B₁₂-dependent methionine synthase (MS; EC 2.1.1.13) or betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5) (Finkelstein and Martin, Reference FINKELSTEIN and MARTIN1984). Enzymes involved in transsulphuration are found at significant activity levels in liver, pancreas, and kidney tissues in rats (Mudd et al., Reference MUDD, FINKELSTEIN, IRREVERRE and LASTER1965); similarly, BHMT activity is found predominantly in the liver. Some activity is also present in kidney tissue (Finkelstein et al., Reference FINKELSTEIN, KYLE and HARRIS1971; Finkelstein and Martin, Reference FINKELSTEIN and MARTIN1984; Cao et al., Reference CAO, CAI and COON1995). In contrast, MS is widely expressed in avian and mammalian tissues (Stipanuk, Reference STIPANUK2004). BHMT specifically catalyses the transport of the preformed labile methyl group from the betaine molecule to homocysteine, while MS catalyses the labile methyl group transport from 5-methyltetrahydrofolate to homocysteine. Betaine used in transmethylation can come from dietary sources or by oxidation of free (non lipid-bound) choline in the cell mitochondria. Choline is oxidized to betaine in a two-step enzymatic reaction, taking place exclusively in the mitochondria of liver and kidney cells. However, the conversion efficiency of choline to betaine is not 100% (Simon, Reference SIMON1999). According to Stekol et al. (Reference STEKOL, HSU, WEISS and SMITH1953), dietary betaine methylates homocysteine to methionine three times more efficiently than dietary choline in the chicken. More recent work of Saarinen et al. (Reference SAARINEN, KETTUNEN, PULLIAINEN, PEURANEN, TIIHONEN and REMUS2001) shows that dietary betaine is nearly twice as efficient as the same mole equivalent level of choline for increasing liver betaine levels in broiler chickens. The methyl group transfer results in the formation of betaine to N,N-dimethylglycine which still contains two methyl groups. These methyl groups can be split off through oxidation in the form of one-carbon fragments. During this reaction N,N-dimethylglycine is degraded to sarcosine, and finally to glycine. The one-carbon fragments from N,N-dimethylglycine as well as from other sources (e.g. carboxyl groups of organic acids) are infused into the one-carbon pool and used for remethylation reactions again via the tetrahydrofolate pathway. The one-carbon fragments bind to the tetrahydrofolate molecule and are transferred via MS to homocysteine.

regulation of the transmethylation cycle

In metabolic reactions, protein synthesis and the formation of SAM compete for available methionine. Consequently, homocysteine has to be distributed between two competing pathways (Finkelstein and Martin, Reference FINKELSTEIN and MARTIN1984). Regulation of the transmethylation cycle is necessary to maintain physiological levels of homocysteine and methionine in the cell (Finkelstein, Reference FINKELSTEIN1998), because accumulation of these substances in the face of a deficiency of either methyl donors or acceptors is toxic to the cell. Thus, methionine-conserving and catabolising enzymes help to regulate the content of methionine and homocysteine at a physiological level. On the other hand, transfer of free methyl groups from choline or betaine to other important components through the methionine metabolism can recycle methionine, thereby reducing the dietary methionine requirement (Kidd et al., Reference KIDD, FERKET and GARLICH1997).

In this context, two mechanisms for the distribution of homocysteine are discussed (Eklund et al., Reference EKLUND, BAUER, WAMATU and MOSENTHIN2005). Firstly, low homocysteine levels induce the transmethylation pathway since the enzymes involved in the transmethylation process show high affinity to homocysteine. If the capacity of these enzymes is reached, the transsulphuration pathway is favoured in which SAM acts as a switch. Secondly, high SAM levels as a consequence of high dietary methionine levels activate the transsulphuration pathway and limit the remethylating enzymes. Low homocysteine levels, in turn, promote the conservation of homocysteine. Moreover, glycine may have a fundamental role in maintaining the balance in the transmethylation cycle (Rowling et al., Reference ROWLING, MCMULLEN, CHIPMAN and SCHALINSKE2002). Particularly, in cases of short supply of methyl acceptors, the methyl groups of SAM are transferred to glycine. The involved enzyme shows high affinity, the products are non-toxic and the methyl groups can be regenerated if they are required for methylation processes afterwards. Hepatic serine levels may also be relevant. In a study on hepatic homocysteine remethylation, Cuskelly et al. (Reference CUSKELLY, STACPOOLE, J., BAUMGARTNER, GREGORY and III2001) noted that methyl groups from 5,10-methylenetetrahydrofolate may be used either for the synthesis of serine (from glycine) or for the remethylation of homocysteine. When the availability of labile methyl groups is limited, methyl groups from 5,10-methylenetetrahydrofolate may be directed toward serine synthesis. In contrast, when adequate methyl groups to meet chicks requirement (NRC, 1994) are present, homocysteine remethylation may be favoured. In the presence of excess methyl groups in the system, in turn, serine metabolism may provide methyl groups for the production of more 5-methylenetetrahydrofolate, thereby resulting in a greater amount of remethylation through MS (Gregory et al., Reference GREGORY, III, CUSKELLY, SHANE, TOTH, BAUMGARTNER and STACPOOLE2000).

Changes in SAA, choline and betaine content of the diet have been shown to result in changes in the hepatic activity of BHMT in broilers (Saunderson and Mackinlay, Reference SAUNDERSON and MACKINLAY1990; Emmert et al., Reference EMMERT, GARROW and BAKER1996). Based on enzyme activity, most studies suggest a greater role for BHMT in homocysteine remethylation (e.g., Emmert et al., Reference EMMERT, GARROW and BAKER1996), especially under conditions of dietary methionine deficiency or when choline or betaine were fed in excess. For example, Saunderson and Mackinlay (Reference SAUNDERSON and MACKINLAY1990) reported that the activity of BHMT was higher than that of MS in avian liver, and methionine deficient conditions resulted in increased hepatic BHMT activity. Similarly, Emmert et al. (Reference EMMERT, GARROW and BAKER1996) pointed out that hepatic BHMT activity increased in broiler chickens in response to the addition of choline and betaine to diets deficient in methionine and cysteine, and that this response could be amplified by simultaneous addition of cysteine.

However, recent results (Pillai et al., Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a,b) suggest that MS-dependent remethylation of homocysteine clearly predominates and appears to be more responsive to dietary changes in poultry. In this respect, Pillai et al. (Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a) demonstrated a more substantial role for MS-dependent homocysteine remethylation in young broilers fed diets deficient in methionine and cysteine. They observed a lack of remethylation response to SAA level and an increased level of MS-dependent remethylation rather than BHMT-dependent remethylation. The relative importance of MS becomes more evident when looking at remethylation through the BHMT pathway as a percentage of total remethylation. Research in rats and humans (Mudd et al., Reference MUDD, LEVY and MORROW III1970; Finkelstein and Martin, Reference FINKELSTEIN and MARTIN1984) revealed that remethylation of homocysteine may be equally partitioned through the MS and BHMT pathways. However, in the study of Pillai et al. (Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a) the proportion of homocysteine remethylated by BHMT was, in most cases, below 40% and in some cases below 20%. Increasing the dietary methionine level did not affect the proportion of homocysteine that was remethylated through the BHMT pathway. Indeed, addition of excess choline or betaine to the diet tended to reduce the proportion of remethylation through the BHMT pathway; and the impact of betaine was more pronounced than that of choline when added to diets containing 0 or 0.07% supplemental methionine (Pillai et al., Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a). Supplementation of methionine at a level at which maximal growth performance can be achieved (dietary methionine, 0.28%, + supplemented methionine, 0.11%) and without supplementation of betaine and choline led to a proportion of homocysteine remethylated by MS or BHMT that was approximately equivalent (Pillai et al., Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a). Thus, for broilers fed diets containing apparently adequate SAA levels without excess methyl donors, homocysteine appears to be partitioned equally between the remethylation pathways. Similarly, Finkelstein et al. (Reference FINKELSTEIN, HARRIS, MARTIN and KYLE1982) observed increased BHMT activity when a methionine-free diet or a diet containing excess methionine was fed to rats. They reported that activity of BHMT increases with a higher intake of protein and methionine, whereas the activity of MS increases when the body is in need of methionine. They speculated that BHMT activity functions to conserve methionine under conditions of dietary deficiency.

The result of Pillai et al. (Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a) suggests a role for BHMT being in agreement with the observations of Frontiera et al. (Reference FRONTIERA, STABLER, KOLHOUSE and ALLEN1994), who studied regulation of methionine metabolism in human subjects and hypothesized that metabolic changes occur at situations of extreme excess or extreme deficiency of dietary methionine in an attempt to maintain or stabilise cellular methionine levels. In the study of Pillai et al. (Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a), in which actual homocysteine flux was assessed, only minimal changes in BHMT-dependent homocysteine remethylation were noted in response to dietary changes in methionine, choline or betaine levels that, based on previous research, would be expected to increase BHMT activity. Therefore, it does not appear that BHMT was acting to conserve cellular methionine levels when dietary methionine was deficient or to dispose of excess homocysteine when dietary methionine was in excess, regardless of dietary choline and betaine levels. Rather, MS appeared to be more responsive to dietary changes, but only when choline or betaine was fed in excess. The underlying mechanism behind the effect of excessive dietary choline and betaine on hepatic homocysteine remethylation through MS pathway in poultry is not clearly elucidated yet, but one of the by-products of BHMT-dependent remethylation is N,N-dimethylglycine, which is a feedback inhibitor of BHMT (Stipanuk, Reference STIPANUK2004). Thus, feedback inhibition of BHMT by N,N-dimethylglycine may prevent increased levels of BHMT in chicks when methionine deficient and surfeit choline and betaine diets are fed (Pillai et al., Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a,b). Moreover, sarcosine, a product of oxidative demethylation of N,N-dimethylglycine, may provide methyl groups to the folate pool, which make them available for MS-dependent homocysteine remethylation.

It is important to note that the basal diet in the study of Pillai et al. (Reference PILLAI, FANATICO, BEERS, BLAIR and EMMERT2006a) was deficient in methionine and cysteine, whereas the diets of Emmert et al. (Reference EMMERT, GARROW and BAKER1996) and Finkelstein et al. (Reference FINKELSTEIN, HARRIS, MARTIN and KYLE1982) contained adequate or excess levels of cysteine. Dietary cysteine levels have been shown to impact transsulphuration (Yamamoto et al., Reference YAMAMOTO, TANAKA and NOGUCHI1995); thus, cysteine level could potentially impact hepatic homocysteine remethylation as well. In mammals (Finkelstein and Mudd, Reference FINKELSTEIN and MUDD1967; Finkelstein, Reference FINKELSTEIN1974; Finkelstein et al., Reference FINKELSTEIN, MARTIN and HARRIS1988) and poultry (Pillai et al., Reference PILLAI, FANATICO, BLAIR and EMMERT2006b), diets containing excess cysteine combined with deficient or adequate levels of methionine have shown to decrease transsulphuration, most likely modulated by a decrease in cystathionine β-synthase activity (Yamamoto et al., Reference YAMAMOTO, TANAKA and NOGUCHI1995). The net result is an increase in the ratio of homocysteine transmethylation to transsulfuration (Finkelstein et al., Reference FINKELSTEIN, MARTIN and HARRIS1988) leading to more homocysteine being utilized for methionine synthesis.

betaine as potential substitute for methionine and choline

Methionine as an essential amino acid plays several crucial roles in the metabolism of all vertebrates and is the first limiting amino acid in poultry nutrition. It is required for protein synthesis and as a precursor of cysteine as well. Moreover, there is growing evidence that betaine is important for cellular and humoral immunity, and it has been shown that the methionine requirement during immune response is even higher than for growth (Tsiagbe et al., Reference TSIAGBE, STRAUB, COOK, HARPER and SUNDE1987). Finally, methionine acts as a key intermediate in the transfer of methyl groups of different origins to the acceptor molecules. SAM is involved in more than 100 methylation and transmethylation reactions, including the synthesis and metabolism of phosphatidylcholine, carnitine and creatine (Larbier and Leclerco, Reference LARBIER and LECLERCO1992). Methylation of phosphatidylethanolamine, the first step in choline biosynthesis, allows methionine to donate a methyl group via SAM and therefore spare choline in mammalian species (Kroening and Pond, Reference KROENING and POND1967). In contrast, avian species have limited capacity to carry out this initial biosynthetic step (Baker and Sugahara, Reference BAKER and SUGAHARA1970). Therefore, limited choline synthetic capacity translates into a relatively high dietary requirement for preformed choline in young chickens (Molitoris and Baker, Reference MOLITORIS and BAKER1976). Choline, as the precursor of betaine, is essentially required for a number of physiological functions including phosphatidylcholine (i.e., for cell membrane integrity) and acetylcholine formation (i.e., for neurotransmission). However, before choline can act as a methyl group donor, it needs to be converted to betaine in the mitochondria.

The supply of labile methyl groups in feed is a common problem in commercial broiler production. Therefore, choline and methionine are both used as feed additives in poultry nutrition. Research has stressed that betaine may serve as alternative source of methyl groups. Numerous studies have been conducted to evaluate the effect of betaine supplementation as substitute of methionine and choline on performance of poultry using methyl group donor adequate or deficient diets.

methionine replacement

The methionine-sparing effect of betaine has been subject of some controversy (e.g., Rostagno and Pack, Reference ROSTAGNO and PACK1996; Esteve-Garcia and Mack, Reference ESTEVE-GARCIA and MACK2000; Pillai et al., Reference PILLAI, FANATICO, BLAIR and EMMERT2006b) which may be related to the dietary supply of SAA and methyl group donors. For instance, the methionine, SAA and choline requirement of young chickens amounts to 0.50%, 0.90% and 1300 ppm of diet in the first week of life and declines to 0.32%, 0.60% and 750 ppm of diet at 6-8 weeks of age, respectively (NRC, 1994). Diets being severely deficient in methionine may alleviate the efficacy of betaine as a methyl donor due to a lack of homocysteine to remethylate because any methionine formed from methylation is taken for protein synthesis and not recycled back to homocysteine. Under these circumstances, the contribution of SAA including methionine to transmethylation processes is reduced, thus limiting protein synthesis in the bird (Wang et al., Reference WANG, XU and FENG2004). If cysteine is deficient in the basal diet this may also limit the response to betaine as a methyl donor, because homocysteine is completely used in the transsulphuration pathway. This consequently limits the remethylation pathway as the transsulphuration reaction is not reversible. Thus, any attempt to replace higher portions of methionine in the diet by betaine has not been successful (e.g., Rostagno and Pack, Reference ROSTAGNO and PACK1996; Schutte et al., Reference SCHUTTE, DE JONG, SMINK and PACK1997; Esteve-Garcia and Mack, Reference ESTEVE-GARCIA and MACK2000). In this context, Esteve-Garcia and Mack (Reference ESTEVE-GARCIA and MACK2000) fed a basal diet deficient in methionine (0.32, 0.28 and 0.25%) and cysteine (0.32, 0.36 and 0.34%) but adequate in choline and folate to broiler chickens (starter, finisher and withdrawal) which were supplemented with 3 levels of methionine (0, 0.06 and 0.12%) and 2 levels of betaine (0 and 0.05%). The authors concluded that betaine in diets deficient in methionine and adequately supplemented with choline to supply methyl groups cannot fully replace methionine to maintain similar growth and carcass yield. This finding is in agreement with results obtained by Rostagno and Pack (Reference ROSTAGNO and PACK1996), Schutte et al. (Reference SCHUTTE, DE JONG, SMINK and PACK1997), Pillai et al. (Reference PILLAI, FANATICO, BLAIR and EMMERT2006b) and Waldroup et al. (Reference WALDROUP, MOTL, YAN and FRITTS2006) in the sense that the responses to betaine were small and not comparable to those obtained by increasing the dietary level of methionine. Similarly, Simon (Reference SIMON1999) suggested that attempts to increase substrate levels for betaine- and folate-dependent homocysteine remethylation with the intent of generating methionine is of limited efficiency, even in young chickens.

Other authors have noted a response to supplemental betaine in diets marginally deficient in methionine, so that a portion of the methionine requirement appeared to be spared (Pesti et al., Reference PESTI, HARPER and SUNDE1980; Virtanen and Rumsey, Reference VIRTANEN and RUMSEY1996). In fact, Virtanen and Rumsey (Reference VIRTANEN and RUMSEY1996) concluded from the results of a field study that in chickens, kept on built-up litter, betaine is more efficient in promoting growth and feed efficiency than methionine. Accordingly, two parts of supplemental methionine could be replaced by one part of betaine, resulting in similar growth response. The reason for the discrepancy between the above described studies (e.g., Rostagno and Pack, Reference ROSTAGNO and PACK1996; Esteve-Garcia and Mack, Reference ESTEVE-GARCIA and MACK2000; Pillai et al., Reference PILLAI, FANATICO, BLAIR and EMMERT2006b) and that reported by Virtanen and Rumsey (Reference VIRTANEN and RUMSEY1996) is not clear since some of the studies reported by the latter investigators were done in the presence of coccidial challenge, which reportedly enhances the response to betaine (Matthews et al., Reference MATTHEWS, SOUTHERN and PONTIF1995). As methionine is involved in cellular and humoral immunity and tissue integrity, other stressors, such as built-up litter, can re-distribute methionine back from the use for protein synthesis towards SAM to support immunity and tissue maintenance.

However, when the diet is only marginally deficient in methionine, part of bird's methionine requirement may be replaced by betaine. In this respect, Virtanen and Rosi (Reference VIRTANEN and ROSI1995) fed a basal diet formulated to provide 75% of the NRC (1994) recommendation for methionine supplemented with 0.05, 0.10, or 0.15% methionine and betaine, respectively, to broilers. The authors demonstrated that betaine was more efficient than methionine in improving growth and feed conversion. Moreover, Florou-Paneri et al. (Reference FLOROU-PANERI, KUFIDIS, VASSILOPULOS and SPAIS A.V.1997) suggested that between 30 and 80% of supplemental methionine can be substituted by betaine without negative effects on performance. In a more recent study, Attia et al. (Reference ATTIA, HASSAN, SHEHATTA and ABD EL-HADY2005) showed that 0.07% betaine could replace 0.05% methionine in diets for slow growth type chickens in terms of growth performance and feed conversion. Similarly, Zhan et al. (Reference ZHAN, LI, XU and ZHAO2006) observed that betaine, at a supplementation rate of 0.05%, could spare methionine, and was as efficient as methionine in improving performance and carcass quality in growing broilers fed diets moderately deficient in methionine. In addition, when supplementing betaine to a diet for starter ducks (1-21 d of age), which was marginal deficient in methionine, betaine could replace part of the methionine, resulting in improved growth performance compared with control ducks (Wang et al., Reference WANG, XU and FENG2004). Abel et al. (Reference ABEL, LIBAL and ICKING1986) showed that 662 mg/kg betaine was more efficient in improving laying rate and feed efficiency in hens fed diets with reduced methionine content (0.25-0.35% methionine) than dietary supplementation of 1150 mg/kg choline, compared with the non-supplemented control diet. The added amounts of betaine and choline were methyl group equivalent. Therefore, the data of Abel et al. (Reference ABEL, LIBAL and ICKING1986) are in accordance with the results of Sketol et al. (1953) and Saarinen et al. (Reference SAARINEN, KETTUNEN, PULLIAINEN, PEURANEN, TIIHONEN and REMUS2001) according to which dietary betaine is twice to three times as efficient as the same mole equivalent level of choline.

The extent to which methionine can be substituted by betaine may depend on the dietary supply of cysteine as well. Methionine is exclusively used for protein synthesis when the animal's cysteine requirement is met, otherwise, part of the methionine will be irreversibly degraded to cysteine. Interactions between betaine and cysteine supplementation are confirmed by the observations of Firman and Remus (Reference FIRMAN and REMUS1999) showing that the combination of dietary cysteine and betaine had a positive impact on feed conversion in broiler chicks compared with the separate administration of cysteine and betaine.

Choline replacement

Initial work on the choline sparing effect of betaine goes back to the 1940s and 1950s (e.g., McKittric, Reference MCKITTRIC1948; Gillis and Noris, Reference GILLIS and NORIS1951); however, the efficacy of betaine to replace part of the dietary choline has not been considered for a long time. In recent years, this concept has gained more interest again. Miles et al. (Reference MILES, RUIZ and HARMS1987) demonstrated that betaine may substitute choline in diets for broilers and laying hens. This was confirmed by Hassan et al. (Reference HASSAN, ATTIA and EL-GANZORY2005). The authors reported that addition of 0.072 or 0.144% betaine to a basal diet containing 872 mg/kg choline yielded similar growth and FCR in slow-growing El-Salam broiler chickens compared to those fed the basal diet supplemented with 300 or 600 mg/kg choline. Hassan et al. (Reference HASSAN, ATTIA and EL-GANZORY2005) suggested an additive effect of choline and betaine in slow-growing chicks when betaine was supplemented to diets containing adequate levels of methionine and choline to meet chicks’ requirement (NRC, 1994). Similarly, previous work of Lowry et al. (Reference LOWRY, IZQUIERDO and BAKER1987) confirmed this observation according to which 25% of the choline requirement in poultry may be supplied as betaine. In laying hens, Hruby et al. (Reference HRUBY, OMBABI and SCHLAGHECK2005) showed that dietary supplementation of 350 mg/kg betaine maintained performance of hens fed diets without added choline and reduced in methionine content compared to hens fed the control diet with 400 mg/kg choline chloride added and adequate in methionine. A higher efficacy of betaine in poultry may be partly attributed to the use of ionophore coccidiostats in poultry diets, which inhibit the activity of choline oxidase (Tyler, Reference TYLER1977). As a result, additional betaine might be required for an adequate supply of labile methyl groups. Based on recent estimates of Dilger et al. (2007) in broiler chickens, 50% of the dietary choline requirement has to be supplied as choline per se, but the remaining 50% can be replaced by betaine. Generally, betaine and methionine have minimal choline-sparing activity in chicks fed purified diets devoid of choline. However, addition of betaine to diets containing minimal choline to meet the requirement of essential choline for physiological functions allowed a marked reduction in the total dietary choline supply (Dilger et al., 2007).

betaine effects on performance and carcass characteristics

Betaine effects on performance and carcass characteristics are presented in Table 1. Dietary supplementation of betaine to methyl group donor-adequate diets improved weight gain, feed efficiency, carcass yield and breast muscle yield by approximately 3-15% in poultry (Virtanen and Rosi, Reference VIRTANEN and ROSI1995; Augustine et al., Reference AUGUSTINE, MCNAUGHTON, VIRTANEN and ROSI1997; Waldenstedt et al., Reference WALDENSTEDT, ELWINGER, THEBO and UGGLA1999; Noll et al., Reference NOLL, STANGELAND, SPEERS, BRANNON and KALBFLEISCH2002; Hassan et al., Reference HASSAN, ATTIA and EL-GANZORY2005). Additionally, betaine has proven to be more efficacious in improving body weight gain and feed efficiency in broiler chickens than choline (Hassan et al., Reference HASSAN, ATTIA and EL-GANZORY2005).

Table 1

Betaine effects on bird performance and carcass characteristics.

^aBWG=Body weight gain; FCR=Feed conversion ratio; Met=Methionine; ALT=Alanine aminotransferase

As betaine is involved in the lipid metabolism, it has been often referred to as a ‘carcass modifier’. Hence it is often claimed that supplementation of betaine may offer an opportunity to the poultry industry to satisfy consumer requirements as well as improve feed conversion (Hassan et al., Reference HASSAN, ATTIA and EL-GANZORY2005). Several authors reported significantly reduced abdominal fat pad, whereas breast meat yield increased in broiler chickens (McDevitt et al., 2000; Waldroup et al., Reference WALDROUP, MOTL, YAN and FRITTS2006), tom turkeys (Remus, Reference REMUS2001; Noll et al., Reference NOLL, STANGELAND, SPEERS, BRANNON and KALBFLEISCH2002) and meat ducks (Wang, Reference WANG2000; Wang et al., Reference WANG, XU and FENG2004) fed betaine supplemented diets. Generally, breast meat yield was improved by 4-10%, while abdominal fat pad was reduced by 9-24% (Rostagno and Pack, Reference ROSTAGNO and PACK1996; Schutte et al., Reference SCHUTTE, DE JONG, SMINK and PACK1997; Wang et al., Reference WANG, XU and FENG2004; Waldroup et al., Reference WALDROUP, MOTL, YAN and FRITTS2006; Zhan et al., Reference ZHAN, LI, XU and ZHAO2006).

By contrast, other authors reported less pronounced responses of carcass characteristics to betaine supplementation (e.g., Esteve-Garcia and Mack, Reference ESTEVE-GARCIA and MACK2000). In this context, Waldroup and Fritts (Reference WALDROUP and FRITTS2005) did not find any improvements in breast meat yield of broilers fed from hatch a diet containing 0.1% of betaine. The authors suggested that the response of the birds to betaine supplementation may be age-dependent. In this study, birds at 42 days rather than at 49 days of age showed an improvement in carcass dressing percentage when betaine was added to the diet.

The mode of action of betaine as ‘carcass modifier’ appears to be related to its methyl group donor properties, thereby attributing to a leaner carcass due to a higher availability of methionine and cysteine for protein deposition in birds fed betaine supplemented diets (McDevitt et al., 2000). Consequently, improved utilisation of dietary amino acids for protein synthesis would leave less amino acids for deamination and eventual synthesis of adipose tissue (Wallis, Reference WALLIS1999). In addition, the effects of betaine on body fat may be related to its lipotropic activity. As a methyl donor, betaine interacts in the lipid metabolism by increasing the carnitine synthesis in liver and muscle (Xu and Zhan, Reference XU and ZHAN1998). Carnitine is a methylated component necessary for the transport of fatty acids through the inner mitochondrial membrane where fatty acid oxidation takes place (Stryer, Reference STRYER1988). Hence, enhanced synthesis of carnitine may have a positive effect on the reduction of carcass fat content as well. Another approach describes a choline-sparing effect of betaine in increasing the synthesis of very-low-density-lipoprotein and thus preventing deposition of fat in the liver and accelerating the removal of fat from the liver (Yao and Vance, Reference YAO and VANCE1990).

Moreover, there is growing evidence that betaine not only affects carcass characteristics but also the performance of laying hens (Table 2).

Table 2

Betaine effects in laying hens.

^aFSH=Follicle-stimulating hormone; LH=Luteinising hormone; VLDL=Very-low-density-lipoprotein; LDL=Low density lipoprotein.

For example, Zou and Feng (Reference ZOU and FENG2002) observed that 0.1% betaine significantly improved laying rate by 10% through stimulation of follicle-stimulating hormone and luteinising hormone secretion in the anterior pituitary. In addition, feed efficiency was improved by 6%, and the deposition of fat and protein into the egg was enhanced. The latter observation was confirmed by a more recent study of Lu and Zou (Reference LU and ZOU2006) showing that egg yolk precursors, i.e. very-low-density-lipoproteins and vitellogenin, were significantly enhanced by 0.06% betaine supplementation. In contrast, Harms and Russell (Reference HARMS and RUSSELL2002) did not observe any effects of betaine on performance of commercial layers when betaine was added to a corn-soybean meal based diet. However, in this study the basal diet was adequate in choline so that no further methyl group donors were needed to meet the animal's requirement. Similarly, Park et al. (Reference PARK, PARK and RYU2005b) proposed that betaine did not enhance laying hen performance, but may affect protein metabolism in hens due to observed increases in serum uric acid and hepatic amino acids.