The study was conducted to investigate the effects of graded dietary inclusion levels 

of betaine on ileal and total tract nutrient digestibilities and intestinal bacterial fermentation 

characteristics in piglets. A total of 8 barrows (BW 7.9 kg) was fitted with simple T-cannulas at the 

distal ileum. The animals were randomly allocated to 1 of the 4 assay diets with 2 pigs per treatment in 

4 repeated measurement periods. The assay diets included a basal diet based on wheat, barley and 

soybean meal alone, or supplemented with a liquid betaine product at dietary levels of 1.5, 3.0, or 6.0 

g betaine kg–1

 diet (as–fed). Ileal digestibilities of dry matter (DM) and neutral detergent fiber (NDF) 

increased both quadratically and linearly (P<0.05), and ileal digestibility of glycine increased linearly 

as dietary betaine level increased (P<0.05). Moreover, there were linear increases in the concentrations 

of ileal D–lactic acid (P<0.05), indicating intensified intestinal bacterial activities as dietary betaine 

level increased. At the fecal level, total tract crude protein (CP) digestibility increased quadratically 

(P<0.05), and digestibility of amino acids (AA) tended to increase quadratically (P=0.06 to P=0.11), 

except for proline (P>0.05), as dietary betaine level increased. The increased bacterial degradation of 

CP and AA in the large intestine coincides with the linear increase (P<0.05) in fecal diaminopimelic 

acid concentrations, indicating enhanced intestinal bacterial growth with increasing dietary betaine 

levels. In most cases, there was a response in the variables that were measured up to 3.0 g betaine per 

kg diet, whereas increasing the betaine level from 3.0 to 6.0 g betaine per kg diet had no additional 

effect. It can be concluded that dietary betaine stimulates microbial fermentation of fiber in the small 

intestine, leaving less fermentable fiber to reach the large intestine and therefore, increased microbial 

degradation of protein in the large intestine may occur.  

 Keywords: piglets, betaine, digestibility, bacterial fermentation, microflor

Eklund, M.

Jezierny, D.

Mosenthin, R.

Ratriyanto, A.

English

Sebelas Maret Institutional Repository

 398 Bulletin UASVM, Veterinary Medicine 66(1)/2009 ISSN 1843-5270; Electronic ISSN 1843-5378   Intestinal Effects of Dietary Betaine in Piglets   Rainer MOSENTHIN1), Adi RATRIYANTO1,2), Dagmar JEZIERNY1), Meike EKLUND1)  1) Institute of Animal Nutrition, University of Hohenheim, Emil-Wolff-Str. 10, 70599 Stuttgart, Germany; rhmosent@uni-hohenheim.de 2) Department of Animal Science, University of Sebelas Maret, Surakarta, Indonesia  Abstract. The study was conducted to investigate the effects of graded dietary inclusion levels of betaine on ileal and total tract nutrient digestibilities and intestinal bacterial fermentation characteristics in piglets. A total of 8 barrows (BW 7.9 kg) was fitted with simple T-cannulas at the distal ileum. The animals were randomly allocated to 1 of the 4 assay diets with 2 pigs per treatment in 4 repeated measurement periods. The assay diets included a basal diet based on wheat, barley and soybean meal alone, or supplemented with a liquid betaine product at dietary levels of 1.5, 3.0, or 6.0 g betaine kg–1 diet (as–fed). Ileal digestibilities of dry matter (DM) and neutral detergent fiber (NDF) increased both quadratically and linearly (P<0.05), and ileal digestibility of glycine increased linearly as dietary betaine level increased (P<0.05). Moreover, there were linear increases in the concentrations of ileal D–lactic acid (P<0.05), indicating intensified intestinal bacterial activities as dietary betaine level increased. At the fecal level, total tract crude protein (CP) digestibility increased quadratically (P<0.05), and digestibility of amino acids (AA) tended to increase quadratically (P=0.06 to P=0.11), except for proline (P>0.05), as dietary betaine level increased. The increased bacterial degradation of CP and AA in the large intestine coincides with the linear increase (P<0.05) in fecal diaminopimelic acid concentrations, indicating enhanced intestinal bacterial growth with increasing dietary betaine levels. In most cases, there was a response in the variables that were measured up to 3.0 g betaine per kg diet, whereas increasing the betaine level from 3.0 to 6.0 g betaine per kg diet had no additional effect. It can be concluded that dietary betaine stimulates microbial fermentation of fiber in the small intestine, leaving less fermentable fiber to reach the large intestine and therefore, increased microbial degradation of protein in the large intestine may occur.   Keywords: piglets, betaine, digestibility, bacterial fermentation, microflora  INTRODUCTION Over the last years, considerable research has been carried out to study the effect of supplemental betaine on growth performance, carcass composition and intestinal health of pigs. Supplementation of betaine to the diet improved weight gain and feed conversion in pigs (Dunshea et al., 2007; Wray-Cahen et al., 2004). Improvements in growth performance of pigs due to dietary betaine supplementation may, at least in part, be attributed to improved nutrient digestibilities (Eklund et al., 2006a, b; Xu and Yu, 2000).  399 Due to its osmoprotective properties, betaine supplementation to piglet diets has been shown to improve digestibility of fiber (Eklund et al., 2006a, b; Ratriyanto et al., 2007), whereas the effects on CP digestibility were equivocal (Eklund et al., 2006b). Higher fiber digestibility following betaine supplementation has been attributed to increased bacterial fermentation of dietary fiber. It has not yet been clarified, if higher ileal CP and AA digestibilities following betaine supplementation can be attributed either to improved enzymatic digestion and a higher absorption capacity of the intestinal epithelium or to intensified microbial degradation of protein (Eklund et al., 2006b). Furthermore, decreased digestibility of CP indicated intensified bacterial assimilation of CP due to betaine supplementation (Eklund et al., 2006b). Moreover, between studies, there exist considerable variations in the level of betaine supplementation to pig diets, ranging between 0.02 and 0.50% (e.g. Fernandez-Figares et al., 2002; Hur et al., 2007), and betaine effects between studies were of different magnitude. Thus, the present study aimed to determine the effects of graded levels of dietary betaine supplementation on ileal and total tract nutrient digestibilities and intestinal microbial fermentation characteristics in piglets. MATERIALS AND METHODS  The experiment was carried out with 8 five-week-old barrows (German Landrace × Piétrain). The piglets were surgically fitted with a simple T-cannula at the distal ileum on d 7 and 9 after arrival according to the principles described by Li et al. (1993), and adhesive collection bags were attached to the pigs’ anus for collection of feces. The pigs were individually housed in stainless-steel metabolic crates. The basal diet consisted of wheat, barley, soybean meal, a mineral and vitamin premix and titanium dioxide (TiO2) as an indigestible marker (Table 1). The diet was formulated to meet the nutrient requirements of NRC (1998) for piglets from 10 to 20 kg of BW. The liquid betaine product (25% betaine content) was added to the basal diet at the expense of cornstarch. Four experimental diets with graded dietary inclusion levels of betaine were formulated: CON : basal diet (‘Control’) BET 0.15 : basal diet plus 0.15% betaine (0.60% liquid betaine product) BET 0.30 : basal diet plus 0.30% betaine (1.20% liquid betaine product) BET 0.60 : basal diet plus 0.60% betaine (2.40% liquid betaine product) The animals were fed 2 equal meals daily at 07.00 and 19.00 h at a level of 4.5% of their individual BW. The meals were offered in mash form, and were mixed with water (1/1 w/v). The pigs had free access to water. Following a 7–d adaptation to the experimental diets after surgery, the collection of feces was initiated at 07.00 h on d 8 and continued for 72 consecutive hours. Ileal digesta were collected for 2 x 12 h, from 07.00 to 19.00 h on d 11 and from 19.00 to 07.00 h on d 12 of each period. Between each feces and digesta collection there were 5 days adaptations to the new feed allowances. The collection procedure for ileal digesta was adapted from Li et al. (1993) using plastic tubing attached to the barrel of the cannula by elastic bands. The plastic tubing was changed at least every 20 min. During digesta collection, 2 ml 2.5 M formic acid was added to the sampling tubing in order to minimise further bacterial fermentation in digesta, except for the samples for the determination of volatile fatty acid (VFA) and lactate concentrations. Ileal and fecal samples were pooled and mixed within animal and period, and were freeze-dried thereafter. Samples of diets, ileal digesta and feces were milled through a 1.0 mm mesh screen prior to analyses. Determination of DM, crude ash (CA), CP, ether extracts (EE), NDF, acid detergent fiber (ADF) and AA was performed as outlined by Naumann and Bassler (1997). The D– and  400 L– lactate concentrations were determined by means of a photometric test kit (Boehringer, No. 1 112 821). The VFA concentrations were measured by gas chromatography using 4–methyl–iso–valerianic acid as internal standard. Samples for VFA analyses were prepared according to the principles described by Zijlstra et al. (1977). The TiO2 contents in feed, ileal digesta and fecal samples were determined according to the procedures described by Brandt and Allam (1987).  Initially, the following linear model for selecting a repeated correlation structure was considered: yijk = µ + βj + δi + βj × δi + eijk where yijk = jth measurement on kth animal in ith betain level, µ = general term (fixed), βj = effect of jth period (fixed), δi = effect of ith betaine level (fixed), eijk = error associated with yijk (random). The errors eijk of repeated measurements on the same subject (animal within betaine level group) are assumed to be serially correlated. Different serial correlation structures were fitted by the REML method as implemented in the MIXED procedure of SAS (2003) and the best structure according to the Akaike Information Criterion was selected. The following models were considered for eijk: independent, independent + animal effect (compound symmetry), AR(1) and AR(1) + animal effect. Using the selected correlation structure, treatment effects were modeled by linear and quadratic regression on betaine levels. The significance level for all Wald–type F–tests was set at α = 0.05.  RESULTS AND DISCUSSION  Ileal digestibilities of DM and NDF increased, both quadratically (P<0.05) and linearly (P<0.05) as dietary betaine level increased (Tab. 1). The highest increase in ileal DM and NDF digestibilities was obtained when 3.0 g betaine per kg assay diet was supplemented, amounting to 1.9 and 11.2 percentage units for DM and NDF, respectively, compared with the control treatment. However, a further increase in dietary betaine level from 3.0 to 6.0 g betaine per kg assay diet had no additional effect on ileal digestibilities of DM or NDF. Tab. 1  Effect of graded dietary levels of betaine (g kg–1 diet, as–fed) on ileal nutrient digestibilities (%)*    P–values†  Dietary betaine levels Item 0 1.5 3.0 6.0 Linear effect Quadratic effect n (pigs) 2 2 2 2   Observations 8 8 7 8   Dry matter 68.9 ± 0.44 70.3 ± 0.44 70.8 ± 0.48 69.5 ± 0.44 0.039 0.027 Crude ash 32.3 ± 2.59 36.2 ± 2.59 36.9 ± 2.74 34.5 ± 2.59 0.630 0.246 Ether extracts 80.8 ± 0.60 81.2 ± 0.60 82.3 ± 0.64 82.0 ± 0.60 0.287 0.354 Crude protein 75.5 ± 1.07 77.4 ± 1.07 77.5 ± 1.13 76.6 ± 1.07 0.567 0.228 NDF 11.3 ± 2.02 18.9 ± 2.02 22.5 ± 2.18 15.2 ± 2.02 0.006 0.002 ADF   0.4 ± 5.36   7.3 ± 5.36 11.2 ± 5.42   2.5 ± 5.36 0.551 0.152 * LS mean values ± SEM. † P–values for Wald–type F–tests for treatment differences.  The improvement in fiber digestibility in this study is confirmed by the results of previous studies in piglets, according to which betaine originating from different sources increased considerably ileal and (or) total tract NDF and ADF digestibilities from 8.7 to 17.9 percentage units (Eklund et al., 2006a, b). Another study revealed a tendency for increased ileal or total tract crude fiber digestibilities in piglets following betaine supplementation  401 ranging between 4.7 and 6.5 percentage units (Mosenthin et al., 2007). As pigs in general lack fiber degrading enzymes, these results indicate that betaine has the potential to stimulate bacterial fermentation of dietary fiber in the gastrointestinal tract. It has been suggested that intestinal bacteria may have a requirement for compatible osmolytes such as betaine (Eklund et al., 2005, 2006a, b). Betaine supplementation may aid the intestinal bacteria to cope with the various osmotic conditions in the gastrointestinal tract, allowing for enhanced intestinal bacterial fermentation of dietary fiber as dietary supplementation of graded levels of betaine did not affect (P>0.05) ileal and total tract digestibilities of CA and EE. Moreover, there was no effect of betaine supplementation on ileal CP and AA digestibilities except for a linear increase (P<0.05) in ileal digestibility of glycine as dietary betaine level increased (data not shown). Tab. 2 Effect of graded dietary levels of betaine (g kg–1 diet, as–fed) on total tract amino acid digestibilities (%)*        P–values†  Dietary betaine levels  Item 0 1.5 3.0 6.0 SEM Linear effect Quadratic effect n (pigs) 2 2 2 2    Observations 8 8 8 8    Indispensable AA        Arginine 91.3 92.1 93.4 92.2 0.59 0.115 0.067 Histidine 90.2 91.1 92.3 91.1 0.61 0.120 0.063 Isoleucine 85.1 86.2 87.7 86.2 0.86 0.240 0.108 Leucine 86.8 87.8 89.1 88.0 0.71 0.188 0.097 Lysine 89.2 90.1 91.4 90.1 0.67 0.181 0.076 Phenylalanine 87.8 88.6 89.7 88.7 0.63 0.200 0.104 Threonine 86.6 87.7 88.6 87.6 0.66 0.224 0.092 Valine 84.9 86.1 87.7 86.2 0.89 0.197 0.092         Dispensable AA        Alanine 80.5 82.1 83.7 82.0 0.99 0.203 0.085 Aspartic acid 86.0 87.0 88.4 87.0 0.75 0.177 0.082 Glutamic acid 93.6 94.0 94.8 93.9 0.37 0.184 0.075 Glycine 83.8 85.0 86.0 84.8 0.76 0.260 0.106 Proline 92.6 93.1 93.8 93.3 0.48 0.458 0.281 Serine 88.2 89.2 90.1 89.0 0.56 0.144 0.058 Tyrosine 85.0 86.1 87.5 86.0 0.84 0.215 0.086 * LS mean values. † P–values for Wald–type F–tests for treatment differences. In contrast to ileal digestibilities of CP and AA, total tract CP digestibility increased quadratically (P<0.05), and digestibility of AA tended to increase quadratically (P=0.06 to P=0.11), except for proline (P=0.28), as dietary betaine level increased (Tab. 2). The highest increase in total tract CP and AA digestibilities was obtained when 3.0 g betaine per kg assay diet were supplemented, amounting to 2.3 and 3.2 percentage units for CP and AA, respectively, compared with the control treatment. These results confirm previous observations in piglets according to which betaine improved total tract digestibilities of CP and AA (Eklund et al., 2006a, b; Mosenthin et al., 2007). In pigs, AA are absorbed proximal to the distal ileum only, whereas digestion and disappearance of AA in the large intestine results from bacterial degradation (Mosenthin and Rademacher, 2003; Sauer and Ozimek, 1986). Therefore, the present data indicate, that betaine stimulates the bacterial degradation of CP and AA in the large intestine. Accordingly, there were linear increases in the  402 concentrations of ileal D–lactic acid and of fecal diaminopimelic acid (P<0.05), indicating intensified intestinal bacterial activities as dietary betaine level increased. Moreover, the results of this study revealed that betaine supplementation improved ileal but not total tract NDF digestibility. It remains still speculative, however, if due to the lower amount of NDF reaching the large intestine bacteria might have utilised AA as source of energy, thereby stimulating breakdown and disappearance of AA as suggested by Eklund et al. (2006b). CONCLUSIONS  In conclusion, bacterial fermentation of dietary fiber in the small intestine rather than in the large intestine increased as dietary betaine level increased. On the other hand, bacterial degradation of CP and AA in the large intestine increased as the dietary betaine level increased. Changes in bacterial metabolite concentrations and bacterial marker levels, both in ileal digesta and in feces, indicate a shift in the composition of the bacterial community due to dietary betaine supplementation. REFERENCES  1. Brandt, M. and S. M. Allam (1987). Analytik von TiO2 im Darminhalt und Kot nach Kjeldahlaufschluß. Arch. Anim. Nutr. 37:453–454. 2. Dunshea, F. R., D. J. Cadogan and G. G. Partridge (2007). Dietary betaine and rectopamine have additive effects on lean tissue deposition, particularly in restrictively-fed gilts. In: Manipulating Pig Production XI. (Ed. J.E. Paterson and J.A. Barker). Australasian Pig Science Association, Scott Print, Perth, Western Australia. pp. 120. 3. Eklund, M., E. Bauer, J. Wamatu and R. Mosenthin (2005). Potential nutritional and physiological functions of betaine in livestock. Nutr. Res. Rev. 18:31–48. 4. 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Pretreatment methods prior to gaschromatographic analysis of volatile fatty acids from faecal samples. Clinica Chimica Acta 78:243–250.    

(2006b). Effects of betaine and condensed molasses solubles on ileal and total tract nutrient digestibilities in piglets.

Betaine improves growth, but does not induce whole body or hepatic palmitate oxidation in swine (Sus scrofa domestica).

Caperna (2002). Effect of dietary betaine on nutrient utilization and partitioning in the young growing feedrestricted pig.

Die chemische Untersuchung von Futtermitteln. VDLUFA–Verlag,

Dietary betaine and rectopamine have additive effects on lean tissue deposition, particularly in restrictively-fed gilts. In: Manipulating Pig Production

Digestibility of amino acids in swine: results and their practical applications. A review.

Digestible amino acids in diet formulation for pigs. In:

Effect of betaine on digestive function of weaned piglets.

Effects of betaine on ileal and faecal digestibilities and intestinal microbial fermentation of crude fibre in piglets. In:

Effects of betaine supplementation on ileal and fecal digestibilities and intestinal microbial fermentation of nutrients in piglets.

Effects of dietary glycine betaine on pork quality in different muscle types.

Potential nutritional and physiological functions of betaine in livestock.

Pretreatment methods prior to gaschromatographic analysis of volatile fatty acids from faecal samples. Clinica Chimica Acta 78:243–250.

Research Council

SAS User's Guide: Statistics, Version 9.

The effect of dietary crude protein level on amino acid digestibility in early–weaned pigs.

Wamatu (2006a). Effects of betaine and condensed molasses solubles on nitrogen balance and nutrient digestibility in piglets fed diets deficient in methionine and low in compatible osmolytes.

Intestinal Effects of Dietary Betaine in Piglets

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Intestinal Effects of Dietary Betaine in Piglets

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