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,
* Fish Culture and Fisheries Group,
and
Adaptation Physiology Group, and
and
Animal Nutrition Group, Department of Animal Science, Wageningen Institute of Animal Science, Wageningen Agricultural University,6700 AH Wageningen, The Netherlands and
and
Danisco Animal Nutrition, Marlborough, Wilts, U.K.
1 Correspondence:
PO Box 338 (phone: +31.317.483371; fax: +31.317.483937; E-mail:
Johan.Schrama{at}WUR.NL).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Betaine • Energy Requirements • Nutrition • Pigs
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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In studies focusing on the methionine-sparing effect of betaine (e.g., Matthews et al., 2001a), diets are often designed to be limiting in methionine (or protein). In other studies (e.g., Pettey et al., 2001), the dietary protein:energy ratio was formulated according to or in excess of NRC (1998). In fact, interactions between betaine supplementation and the dietary protein:energy ratio are shown in several studies (e.g., Matthews et al., 1998), but are not consistent. The variable response to betaine supplementation across studies is likely due to the different mode of action of betaine tested and/or the difference in animals’ health and stress status between studies.
Observations that the dietary betaine effect increases when feed intake is restricted (Casarin et al., 1997) or increases with decreasing dietary energy density (Cromwell et al., 1999) suggest that betaine can also affect energy metabolism. In broilers, betaine aids the birds’ response to a coccidia challenge (Kettunen et al., 2001; Klasing et al., 2001). This indicates that dietary betaine may be beneficial specifically under energy-limiting and/or stressful conditions.
This study aims to quantify the effect of betaine supplementation on energy partitioning of growing pigs under energy-restricted conditions and to assess its effect on the adaptation of pigs after mild housing and nutritional stress.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Two groups of 15 crossbred barrows (35 ± 0.36 kg; [Finnish Landrace x Yorkshire] x Yorkshire) per block were obtained from a commercial farm. Within each block, groups were assigned to one of two experimental diets: control or betaine (betaine was control diet supplemented with Betafin at 1.25 g/kg feed; Danisco Animal Nutrition, Marlborough, U.K.). Group was the experimental unit in this study. The study was conducted in accordance with the Dutch Law on Experimental Animals.
During the preliminary period, pigs were housed in typical growing facilities. Each group (15 pigs) was housed in a pen of 4.5 x 3.45 m with 20% slatted floor. At the start of the experimental period, each group was reduced to 14 pigs and was split into two equal subgroups based on live weight. From this moment onward, each group (i.e., two subgroups) was housed in one of two identical, large open-circuit, climate-controlled respiration chambers (Verstegen et al., 1987). In each chamber, the subgroups were housed in one of two pens of 8.3 m2 each (seven pigs per pen). In the chambers, ambient temperature was kept at 20°C, which was assumed to be above the lower critical temperature of pigs at the applied feeding level. Relative humidity was maintained at about 65%. Air velocity was below 0.2 m/s. Animals were exposed to 12 h of light (from 0700 to 1900) and 12 h of darkness.
Feeding
At the start of the preliminary period, groups of pigs were assigned to one of two dietary treatments: a control or a betaine supplemented diet. The pigs were kept on these treatments throughout both the preliminary and the experimental period. Control and betaine diets were identical except for the supplementation of betaine (Table 1
). At the start of the experimental period, all pigs were transferred to the climate-controlled respiration chambers, and exposed to a change in dietary composition (from the preliminary to the experimental diets, see Table 1). The pigs were changed to the experimental diets for the last morning feeding in the growing facilities (at 0800, 1 h before the transfer to the chambers). The experimental feeds were then offered until completion of the experimental period. The change in dietary composition from the preliminary to the experimental period involved a dilution of the diet by including 10% oat hulls. The net energy value of oat hulls (close to 600 g of poorly fermentable NDF/kg of DM) is close to zero, and its inclusion was considered to be a form of nutritional stress. In this way, the pigs were subjected to a combination of a mild housing and nutritional stress at the start of the experimental period. Measurements during the first two balance periods were expected to provide information on the effect of betaine on the time-related changes (i.e., adaptation) in energy metabolism due to change in dietary and housing conditions. The third balance period was assumed to reflect the effect of betaine on energy metabolism of pigs in a steady state.
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; CVB, 1999) and an assumed NE requirement for maintenance of 293 kJ/(kg0.75•d). Therefore, feed intake, but not NE intake, increased during the transition from the preliminary to the experimental period. All diets were formulated to be limiting in energy by increasing the content of ileal digestible lysine, tryptophan, methionine + cysteine, and threonine relative to the net energy content to at least 110% of the recommended requirements by the CVB (1999). Furthermore, 200 mg of choline chloride was added per kilogram of diet. The betaine content in betaine-supplemented diets was 1.33 and 1.29 g/kg during the preliminary and experimental periods, respectively. The betaine content of the control diets was below the detection limit (<0.01 g/kg). All diets were pelleted at low temperatures (about 65°C). During both the preliminary and experimental periods, pigs were fed restrictively according to their metabolic body weight (kg0.75). Because oat hulls have almost no nutritional value, intakes of amino acids, vitamins, and minerals were similar during both periods. The daily amount of feed per pen was based on the average body weight of the animals per pen adjusted for an expected daily weight gain of 600 g/d. Two similar portions were fed at 0800 and 1530, and pigs had ad libitum access to water.
Measurements
Throughout both the preliminary and the experimental period, pigs were weighed at the start and end of each week. Furthermore, ADFI was recorded daily per group.
During the experimental period, energy and nitrogen balances were measured per group of pigs (one chamber) during the three successive balance periods. During each balance period, the mixed feces and urine production was collected quantitatively per group of pigs, homogenized, and sampled for energy and nitrogen analysis. The GE content of the diet and of the mixed feces and urine samples was determined after freeze-drying by adiabatic bomb calorimetry (IKA-C700, Janke & Kunkel GmbH & CoKG, Staufen, Germany). Kjeldahl nitrogen was determined according to ISO 5983 (ISO, 1979) in feed, mixed feces and urine samples (fresh), and condensed water collected from the respiration chambers, as well as in acidified liquid samples through which out-flowing air from the chambers was directed to trap gaseous ammonia. The dietary betaine content was analyzed after extraction of the feed sample with 20% ethanol solution by HPLC with a cation exchange column and refractive index detector (Rajakylä and Paloposki, 1983). Dietary crude fat was determined according to ISO/DIS 6492 (ISO/DIS, 1996), dietary crude ash according to ISO 5984 (ISO, 1978), and dietary starch according to the procedures described by Goelema et al. (1998).
Intake of ME was calculated per group of pigs by subtracting the energy losses of feces, urine, and methane from the GE intake. Total heat production (HPtot) was measured in 9-min intervals by determining the exchange of oxygen, carbon dioxide, and methane as described by Verstegen et al. (1987). These gaseous exchanges were used to calculate HPtot according to the formula of Brouwer (1965). During the last 5 d of the first and the last 6 d of the other balance periods, HPtot measurements were made. Total energy retention (ER) was calculated by subtracting HPtot from ME intake. The retention of N was calculated from N intake, minus N losses in feces plus urine, in aerial NH3 and in NH4+ of water that condensed on the heat exchanger. Energy retention as protein (ERp) was calculated from N retention by assuming an N content of 160 g/kg of protein and an energy content of 23.7 kJ/g of protein. Energy retention as fat (ERf) was calculated by subtracting ERp from ER. Metabolizable energy required for maintenance (MEm) was calculated as:
 | [1] |
The values of 0.54 and 0.74 were used as the efficiency of energy utilization for protein and fat retention, respectively (ARC, 1981).
Statistical Analyses
Energy and N balance traits, as well as the mean daily values of HPtot per balance period, were analyzed for the effect of betaine supplementation and time (balance period) by means of F-tests using a split-plot model (GLM procedure of SAS; SAS Inst., Inc., Cary, NC), with weekly values within groups taken as repeated measurements, using the following model:
 | [2] |
where Yijkl = dependent variable, µ = overall mean; e1,i = error term 1, which represents the random effect of block i (i = 1, 2, 3); Dj = fixed effect of diet j (j = 1,2); e2,ijk = error term 2, which represents the random effect of group k within diet j and block i (k = 1,2); Tl = fixed effect of time (balance period l) (l = 1, 2, 3); and e3,ijkl = error term 3, representing the random effect within groups between balance periods. The effect of dietary betaine supplementation was tested against error term 2. The effect of balance period and the interaction between balance period and betaine supplementation were tested against error term 3. In addition to the mean daily HPtot, the mean HPtot during the light (from 0700 to 1900) and dark periods of the day was calculated per balance period and analyzed using Eq. [2].
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). In the beginning of the preliminary period, feed refusals occurred. Feed refusals were unaffected by dietary treatment (P > 0.10). Averaged over both diets, orts were 11.8, 0.9, and 0% of the offered feed during Wk -2, -1, and 0, respectively.
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). Averaged over diets, water consumption increased with time during the experimental period from 3.7 L/d in Wk 1 to 6.3 L/d in Wk 3 (P < 0.01, Table 2). Numerically, the water consumption was 1.0 L/d higher at the control diet compared to the betaine-supplemented diet (Table 2).
In accordance with the experimental design, GE intake (expressed per kg BW0.75) was equal between diets and did not change with time during the experimental period (P > 0.10, Table 3). Methane production increased with time during the experimental period (P < 0.001, Table 3). This increase of methane production with time was dependent on dietary betaine supplementation (P < 0.10). The difference in methane production between the betaine and the control diet decreased with time (Table 3). These changes in methane production were, however, small and did not result in significant effects of diet and time on metabolizability (ME/GE) and ME intake during the experimental period (Table 3).
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Figure 1 presents the hourly means of HPtot for each diet during Wk 3. HPtot peaked after feeding (at 0800 and 1530). Throughout the dark phase of the day (from 1900 to 0700), HPtot was reduced by betaine supplementation (P < 0.05). This effect occurred predominantly from 2100 to 0200 (Figure 1). However, during the light period of the day (from 0700 to 1900), HPtot was not different between diets, except between 1700 and 1800 when betaine supplementation resulted in a decreased HPtot (P < 0.05).
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). Energy retention (ER) was increased by betaine supplementation averaged over the total 3-wk experimental period (325 vs. 341 kJ/[kg0.75•d], P = 0.053). Averaged over both experimental diets, ERp declined from 159 to 150 to 145 kJ/(kg0.75•d) during Wk 1, 2 and 3, respectively (P < 0.01). The decrease in ERp with time was, however, affected by betaine supplementation (week x feed interaction, P < 0.05) and was larger for the control diet compared with the betaine-supplemented diet. In contrast to ERp, fat retention (ERf) was not affected by time or betaine supplementation (P > 0.1). Averaged over the total experimental period, both ERp and ERf were numerically increased by betaine supplementation (148 vs. 154 and 177 vs. 186 kJ/[kg0.75•d] for the control vs betaine supplemented diets for ERp and ERf, respectively).
The MEm was decreased by betaine supplementation (P = 0.08). The effect of betaine supplementation, however, reduced maintenance requirements of the pigs by 1, 15, and 25 kJ/(kg0.75•d) in Wk 1, 2 and 3, respectively (week x feed interaction, P < 0.05, Table 3) compared with those fed the control treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). The effects of betaine supplementation on energy digestibility and metabolizability as well as these effects on heat production and protein to lipid deposition ratio are discussed below.
Effects on Energy Digestibility and Metabolizability.
In the present study, the ME:GE ratio was unaffected by dietary betaine supplementation, implying either that both digestibility (DE/GE) and metabolizability of digestible energy (ME/DE) were unaffected or that the effects at the levels of digestion and metabolizability are opposite. Literature data on the effect of dietary betaine supplementation on energy digestibility and metabolizability are limited. Øverland et al. (1999) suggested no effects of betaine supplementation on total-tract nutrient digestibility or on nitrogen balance. Their balance data were, however, hampered by a lack of experimental power.
Effects on Heat Production.
The observed decrease in heat production of those restrictively fed pigs offered the betaine treatment may be caused by either a decrease in MEm or by an improvement in the energetic efficiency with which protein and fat are deposited. When assuming that these efficiencies were not influenced by dietary betaine supplementation, the observed decline in heat production (Table 3) reflected a reduction in MEm of 25 kJ/(kg0.75•d) during Wk 3 (5.5%). Possible causes of the reduction in MEm included effects on physical activity and on energy expenditure of the gastrointestinal tissues, as discussed below.
Dietary factors have been shown to be capable of modifying the energy expenditure on physical activity in growing pigs (e.g., Schrama et al., 1998). It is therefore interesting to question whether the observed reduction in heat production due to betaine supplementation was related to an effect of betaine on the physical activity of the pigs. In the current study behaviour and physical activity were not recorded. However, the within day variation in heat production gave an indication on effects on physical activity. During the dark phase of the day (1900 to 0700) heat production is usually less influenced by physical activity (Schrama et al., 1996). Figure 1 illustrated that the effect of betaine on heat production was mainly present during the dark period of the day. Consequently, this suggested that the energy-sparing effect of betaine was not due to a reduction in physical activity of the pigs.
In growing 50-kg pigs, the empty weight of the gastrointestinal tract is approximately 4 to 5% of the total body weight (Bikker, 1994; Rijnen et al. 2001). Due to their disproportionately high rate of metabolic activity, however, these tissues consume a relatively high proportion of the animals’ oxygen need. In general, oxygen consumption, and thus energy utilization of portal drained viscera (PDV, gastrointestinal tissues plus pancreas and spleen), may total up to 25% of whole-body oxygen consumption in ruminants (review of McBride and Kelly, 1990); between 10 and 20% of the energy requirements for maintenance in neonate pigs (calculated from Ebner et al., 1994); and 25% in pigs of 55 kg of BW (calculated from Darcy-Vrillon et al., 1999). Moreover, energy consumption by PDV was increased in the fed state and affected by feeding level and diet composition (Ebner et al., 1994; Darcy-Vrillon et al., 1999). Maintenance of ionic homeostasis (ion pumping) accounted for a major portion of the PDV oxygen consumption (35%, see review by Summers et al., 1986). By acting as an osmolyte (Simon, 1999), betaine could potentially reduce energy expenditure of ion pumping, particularly in gastrointestinal tissues. Although not statistically significant, the approximately 20% reduction in water consumption of the pigs fed the betaine supplemented diets (1 L pig-1•d-1) illustrated this mode of action (Table 2). Osmotic regulation was closely related to electrolyte balance, and thereby to the animals’ acid/base balance. Recently, it was demonstrated that an increased dietary cation-anion difference increased the weight of viscera, in particular the liver and the large intestine in young pigs (Dersjant-Li et al., 2001). Accordingly, an increase in the energy requirements for maintenance was observed (Dersjant-Li et al., 2002). However, the possible influence of betaine on osmotic regulation (and acid/ base balance), and thereby on the energy metabolism of pigs, requires further research.
Changes in the Lipid:Protein Deposition Ratio.
In addition to the reduction of heat production by betaine supplementation, there was also an effect on the energy partitioning in Wk 3. Although the ME:GE ratio in the present study was not different, the difference in protein retention between experimental diets increased with time (P < 0.05, Table 3), whereas fat retention was unaffected. During Wk 3, protein retention was higher in the betaine supplementation diet (153 vs 136 kJ/[kg0.75d]). As discussed above, in relation to heat production, the dietary energy-saving effect of betaine supplementation, under a dietary energy-limiting conditions, probably contributed to the numerically improved ratio between protein and fat retention in Wk 3 (Table 3). This "slimming effect" was supported by data on improved gain and increased carcass leanness in pigs fed adequate protein diets, as observed by Matthews et al. (1998; 2001c) and Pettey et al. (2001), but not by Matthews et al. (1998) and Øverland et al. (1999). However, as already mentioned by, for example, Matthews et al. (1998, 2001a,c) and reviewed by Simon (1999), responses of feed intake, rate of gain, and carcass leanness to dietary betaine supplementation were often inconsistent.
Effects of Betaine Supplementation on Adaptation After Exposure to Mild Housing and Nutritional Stress
A secondary aim of this study was to test the hypothesis that the effect of betaine is greater or even only present when animals are not in a steady state. Therefore, the effects of betaine on time-related changes (i.e., adaptation) in energy metabolism due to changes in diet composition and housing conditions were measured. Except for the effect of betaine on methane production, however, none of the energy metabolism traits supported this hypothesis (Table 3). On the contrary, the effects of betaine increased with time, with the largest effect measured in Wk 3. The effect of betaine on heat production, protein retention, and MEm increased with time.
The effect of betaine supplementation on the increase of methane production with time was clear, but quantitatively not important (Table 3). It is interesting, however, and it can be speculated that betaine aids the intestinal microflora in its adaptation to poorly fermentable carbohydrates originating from oat hulls. As reviewed by Kettunen (2001), bacteria could accumulate betaine to prepare for or deal with specific circumstances (e.g., thermal or osmotic stress). However, thermal stress could be ruled out in the case of gastrointestinal microflora. Another reason for the observed difference in methane production could be the demethylation of betaine by the gastrointestinal microflora, which might have been related to the inclusion of oat hulls to the diet. It is not clear what the mechanism is behind the observed effect.
During the non-steady state, the effect of betaine on heat production, protein retention, and maintenance energy requirements of pigs were apparently overruled by other factors related to the exposure to changes in environmental conditions (including dietary composition). The current observation paralleled the finding of Matthews et al. (2001b) that the effect of betaine on performance and carcass characteristics was not dependent on the stocking density of the pigs (i.e., a factor used to induce suboptimal husbandry conditions). Information on the effectiveness of betaine in aiding in the adaptation process to changes in environmental conditions is, however, limited.
Inconsistent Responses to Dietary Betaine Supplementation Across Studies
There is abundant conflicting information on the effectiveness of dietary betaine on feed intake, growth rate, and carcass quality. These studies have been conducted from a particular scientific or practical angle. The variable response of animals to betaine supplementation across studies is likely to be a consequence of the different modes of action of betaine tested in different studies and the variation in environmental circumstances of animals tested in different studies. The different modes of action can be grouped into 1) the capacity of betaine to replace methionine and/or choline and 2) betaine’s osmolytic properties. When researching the former, diets were usually designed to be limiting in protein and/or methionine, whereas the latter was usually researched under energy-limiting conditions. Even though several studies were performed under energy-limiting conditions, the degree of energy limitation varied between studies. For example, in the study of Matthews et al. (1998), the protein-adequate treatment for gilts of 55 to 75 kg contained 0.60 g of lysine/MJ of ME. Matthews et al. (2001c) used 0.62 g of lysine/MJ of ME for growing (65 to 88 kg BW) and 0.47 g of lysine/MJ of ME for finishing (88 to 115 kg BW) barrows and gilts, respectively. Øverland et al. (1999) used 0.68 g of lysine/MJ of ME for barrows and gilts of 20 to 100 kg BW. In the present study (Table 1), we used 0.87 g of lysine/MJ of ME. This wide range in lysine:ME ratios used reflects in part a real difference in requirements due to age, sex, and genotype. When feeding to specific requirements, however, there is always a risk that energy has not been limiting protein retention in all treatments or throughout the entire growth range studied and certainly not in all animals in a group. Furthermore, the level of feed intake contributes to variation in response. As observed by Casarin et al. (1997), the effect of betaine supplementation was more pronounced when feed intake was restricted indicating the effect was stronger when dietary energy was limiting.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Received for publication February 5, 2002. Accepted for publication January 6, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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) and betaine-supplemented diets (
). Lights were on between 0700 and 1900. Symbols above means indicate significant differences between the experimental diets for that time period: * = P < 0.05; # = P < 0.01.