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ANIMAL NUTRITION |
Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada N1G 2W1
| Abstract |
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0.10) TID of most essential AA or EPL (10.4 g/kg of DM intake). Including pectin in the diet did not influence TID of AA (P
0.10) but increased EPL (15.6 g/kg of DM intake; P
0.01). Inclusion of WS in the diet reduced TID of most essential AA (P < 0.01). The TID values for most essential AA, however, were the same (P
0.10) for both dietary WS levels, except for Lys and Met, which were further reduced at the greatest dietary WS level. Increased EPL (P < 0.01) was only observed for WS3 (16 g/kg of DMI). We concluded that (1) the effects of dietary protein source on AID of AA can be attributed both to reduced TID of AA and increased EPL, (2) the impact of dietary WS level on TID of AA and EPL does not seem to be linear, (3) soluble NSP from pectin or WS exerts a greater effect on EPL than insoluble NSP, and (4) because of the metabolic cost associated with EPL and the impacts of feed composition on microbial fermentation in the gut lumen, the effects of feed ingredients on the use of ileal digestible AA for protein deposition should be investigated further.
Key Words: amino acid digestibility pig wheat shorts
| INTRODUCTION |
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Wheat shorts (WS), a by-product of the wheat milling industry, are used extensively as a source of protein and energy in pig diets (Patience et al., 1995
). A portion of the protein and AA in wheat shorts, however, is structurally linked to the fiber fractions (Schulze et al., 1995
; Huang et al., 2001
). Therefore, reduced TID of AA and increased EAAL may contribute to the relatively low AID of AA in WS fed to pigs (Huang et al., 1999
; 2001
). The purpose of this study was to determine the effect of level of dietary inclusion of WS and casein (CS; a control) on TID of AA and EAAL in growing pigs. In a subsequent study, use of digestible AA intake from these 2 protein sources for body protein deposition was evaluated in growing pigs (Libao-Mercado et al., 2006
).
| MATERIALS AND METHODS |
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Five Yorkshire barrows with a BW of 17.5 ± 1.5 kg were obtained from the University of Guelph Arkell Swine Research Farm. Pigs were housed in large adjustable metabolic crates in a temperature-controlled room at 20 to 22° C (Nyachoti et al., 1997b
). After a 1-wk adaptation period, pigs were surgically fitted with a simple T-cannula at the distal ileum, following the procedures described by Nyachoti et al. (1997b)
. During the 1-wk recovery period, pigs were given increasing amounts of a pig starter diet. During the experiment, pig BW increased from 20.8 ± 1.2 kg to 54.8 ± 1.8 kg. After the study, the pigs were killed to determine if cannulation had caused any intestinal abnormalities. The use of animals in this experiment was reviewed and approved by the Animal Care Committee of the University of Guelph.
Experimental Diets
A basal diet was formulated to contain 0.27 g of SID Lys per MJ of DE (Lys level 1) by including CS and cornstarch (Corn Products, Etobicoke, ON, Canada) as sources of protein and energy, respectively (Table 1
). Other diets were formulated to contain additional SID Lys from WS (WS2, +0.1 g of SID Lys per MJ of DE, Lys level 2; WS3, +0.2 g of SID Lys per MJ of DE, Lys level 3) or from CS (CS3, +0.2 g of SID Lys per MJ of DE, Lys level 3). A fifth diet was formulated to be similar in protein level and source as CS3 but in which 6% commercial pectin (CPKelco, Wilmington, DE; Zhu et al., 2005
) was included at the expense of cornstarch (CS3 + pectin). Diets were formulated based on estimated AA contents and SID according to NRC (1998)
and CVB (1998)
, respectively.
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To estimate TID of AA and endogenous EAAL, diets were prepared in which 50% of the protein source was replaced with guanidinated material for which part of the Lys was converted to homoarginine (HA; Nyachoti et al., 1997b
). In addition, titanium dioxide at 0.10% was used in place of chromic oxide as a marker unique for the guanidinated diets. Guanidination of CS and WS was conducted as described by Nyachoti et al. (1997b)
.
After the 6-d guanidination process, the reaction was stopped by precipitating the guanidinated proteins at their isoelectric pH. This was done by lowering the pH to 4.6 for CS and 5.8 for WS, using 1 M HCl. Guanidinated proteins were recovered by centrifugation at 4,000 x g and 4° C and were then freeze-dried before incorporation into the guanidinated diets. Limitations of the HA technique have been discussed in detail elsewhere (e.g., Nyachoti et al., 1997b
). Key assumptions are that a representative fraction of Lys in the ingredient is converted to HA, that guanidination does not interfere with normal digestion and absorption, and that the AA profile of EAAL is constant.
General Conduct of the Study
After recovering from surgery, pigs were randomly allocated to 1 of the 5 experimental diets in a 5 x 5 Latin Square design with pigs and feeding periods as blocking factors. Each period lasted for at least 14 d, with more than 10 d of adaptation to the dietary treatments. The pigs were offered their assigned diets at 0830 and 1630 as wet mash, with a water:feed ratio of 2:1. An unlimited supply of drinking water was provided from low-pressure drinking nipples. The feeding level, based on 2.6 times the maintenance energy requirement (NRC, 1998
), was adjusted for every experimental period based on projected average BW.
During each experimental period, ileal digesta was collected continuously for 24 h on d 12 for determination of AID values of AA, CP, and DM. On d 14, pigs were given the guanidinated diets during the morning meal (0830) and at the beginning of the second 24-h collection of ileal digesta, for determination of TID of AA and EAAL, as well as AID of AA. Digesta was collected in plastic bags attached to the cannulas; bags were replaced at least every hour. Ten milliliters of 10% (vol/vol) formic acid was placed in the bag before collection to minimize microbial activity. After collection, digesta was poured into aluminum trays and immediately frozen at 20° C until further processing.
Sample Preparation and Chemical Analysis
Digesta samples were pooled per pig and 24-h collection period. Digesta samples were freeze-dried and, along with diet samples, were ground using a Wiley mill through a 0.5-mm screen and thoroughly mixed before chemical analysis. The DM, CP, GE, fat, ash, and titanium dioxide contents in diets and digesta were determined according to standard AOAC procedures (AOAC, 1997
). Chromic oxide was determined using the atomic absorption technique according to Saha and Gilbreath (1991)
, whereas fiber components (NDF and ADF) were analyzed using the Ankom filter bag method (Ankom Technology Corporation, Fairport, NY). The reported NDF and ADF values were corrected for the ash content. Analyses of AA, including HA, were performed at Degussa AG (Hanau, Germany) according to Llames and Fontaine (1994)
. Total NSP analyses of diets and WS were carried out at Massey University (Palmerston North, New Zealand) following the Englyst method (Englyst et al., 1994
). Neutral sugars were quantified through gas chromatography, whereas uronic acid was analyzed by spectrophotometry after acid hydrolysis of the polysaccharides. Total NSP was then calculated as the sum of the neutral sugars and uronic acid content (Englyst et al., 1994
). Pectin content of diet samples was also determined using the colorimetric procedure of Taylor (1993)
.
Calculations and Statistical Analyses
Lysine conversion to HA was calculated as
![]() | [1] |
where MCHA and MClys were the molar contents (mol/kg of DM) of HA and Lys, respectively. Apparent ileal digestibility of AA, CP, and DM in the experimental diets was calculated using the marker indicator method (Nyachoti et al., 1997b
). The AID values for AA in the experimental diets given at d 14 were calculated using the same method, but chromic oxide and AA concentrations were taken as the weighted average of the regular diet and the guanidinated diet because both diets contributed to the AA intake of the pigs on d 14. Relative contributions of regular and guanidinated diets to the d-14 collection were calculated based on the ratio of titanium dioxide to chromic oxide in the digesta and in the diets. Contribution (%) of regular and guanidinated diets (HA diets) to digesta collected on d 14 was calculated using the following equations:
![]() | [2] |
![]() | [3] |
where Crregular diet and TiHA diet were the marker contents (g/kg of DM) in the regular and guanidinated diets, respectively, whereas Tidigesta and Crdigesta were the marker contents (g/kg of DM) in the digesta collected at d 14.
Reported AID values for AA were pooled from d 12 and 14 measurements; repeated measures analysis (day as repeated variable) showed no effect of collection day as well as no interaction (P
0.10) of treatment and collection day, indicating that the process of guanidination did not interfere with protein digestion. The TID of Lys (TIDlys) in the experimental diets was calculated as the AID of HA (AIDHA) in the guanidinated diets.
Endogenous Lys loss (Endolys) at the terminal ileum (g/kg of DMI) was calculated using the formula
![]() | [4] |
where lysdiet, TIDlys, and AIDlys were the Lys content (g/kg of DM) and the TID (%) and AID (%) of Lys in the diet, respectively.
Endogenous ileal losses of other AA were calculated based on the ratio of specific AA to endogenous Lys as reported by Jansman et al. (2002)
, but Pro and Gly were taken from de Lange et al. (1989b)
because values for these AA seem overestimated by Jansman et al. (2002)
. The TID of other AA in the experimental diets were then calculated according to Nyachoti et al. (1997b)
:
![]() | [5] |
where AID, EndoAA, and AAdiet were the apparent ileal digestibility (%) of AA, endogenous ileal AA loss (g of DMI/kg), and AA content in the diet (g of DM/kg), respectively. The SID of AA in the experimental diets were calculated using the same formula, except EndoAA referred to basal EAAL according to Jansman et al. (2002)
instead of actual EAAL. Both TID and SID values of AA in CS and WS were determined following the difference method of Fan and Sauer (1995)
.
To calculate TID values of AA in WS, the TID values of AA in CS were estimated from observations on the CS3 diet and assuming that the TID values were 100% for synthetic AA added to the diets (Equation 6):
![]() | [6] |
where TIDdiet, TIDsynthetic, and TIDcasein were the TID values of AA in the WS diets (WS2 or WS3), synthetic AA (100%), and CS, respectively; and Ssynthetic, Scasein, and Swheat shorts were the percent contribution of synthetic AA, CS, and WS, respectively, to the total AA content of the diet. The SID values of AA in WS were calculated using the same equation.
Data were subjected to ANOVA using the GLM procedure of SAS v.8 (SAS Inst. Inc., Cary, NC). The statistical model included diet, experimental period, and animal effects, whereas metabolic BW (BW0.75) was used as a covariate. Treatment means represented least square means. The following contrasts were evaluated for significance: linear and quadratic relationships for WS inclusion level (basal, WS2, WS3); CS3 vs. WS3; CS3 vs. CS3 + pectin; basal vs. CS3. Contrasts, mostly nonorthogonal, were evaluated following the Bonferroni procedure. The type I error rate for individual comparisons was set at P < 0.01. The Corr procedure of SAS was used to relate dietary fiber components (NDF + pectin and NSP) to TID.
| RESULTS AND DISCUSSION |
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All animals seemed healthy and readily consumed their feed allowances. However, pigs assigned to WS3 showed reduced appetite at the beginning of the adjustment period but consumed their full allowance at d 3 of each experimental period. In period 4, the pig fed WS3 did not consume its feed allowance. This pig was changed to a different diet resulting in a missing observation in this period. Moreover, one pig refused to consume the guanidinated WS3 diet on d 14 resulting in another missing observation. There were no signs of intestinal abnormalities observed due to cannulation.
Dietary Nutrient Composition
Crude protein contents of the experimental diets were within 10% of the values calculated using published CP contents in feed ingredients (NRC, 1998
), except for the basal diet in which actual CP content was 20% greater than calculated (Table 2
). Similarly, total dietary Lys (g/kg) contents were within 5 to 10% from the anticipated dietary Lys content for all diets. The conversion of Lys to HA was 83% for CS and 62% for WS, indicating that a reasonably representative fraction of Lys was guanidinated in both ingredients. The basal and CS3 diets contained 2.7 and 2.1% NSP, respectively, which may be attributed to some resistant starch present in cornstarch (Englyst et al., 1994
). Analyzed NSP content in WS2 and WS3 diets (6.4 and 12.5%, respectively) were similar to anticipated values (5.6 and 11.2%) calculated from determined total NSP content in WS. Soluble NSP content in WS2 and WS3 diets, calculated from analyzed total NSP content of the diets and analyzed soluble NSP content of WS, were 1.22 and 2.4%, respectively. Analyzed NSP content of CS3 + pectin (4.4%) was 30% greater than anticipated value of 3.3%, which may be attributed to resistant starch present in cornstarch.
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At similar dietary SID Lys to energy ratio (CS3, WS3, CS3 + pectin), AID of AA were greatest for the CS3 diet and lowest for the WS3 diet (P < 0.01; Table 3
). This is consistent with the lower reported AID of AA in WS than CS (CVB, 1998
; NRC, 1998
). The result also demonstrates that the addition of pectin reduces AID of AA (P < 0.01). These results supported the observations of other researchers who demonstrated reduced AID values of AA when 5 to 7.5% pectin is added to the diet (Mosenthin et al., 1994
). The AID of AA in the WS3 diet generated from this study were about 10% greater than those reported by Huang et al. (1999)
. However, in the study of Huang et al. (1999)
, 17.53% soybean meal and 44.08% WS were included as dietary protein sources; it has been well documented that the AID of AA in soybean meal is lower than in CS (NRC, 1998
). In addition, differences in terms of location, variety, or processing method applied during wheat milling may have influenced AID of AA in the WS-containing diets.
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In pigs fed the basal and CS3 diets, 94% of the DM intake was digested before the distal ileum (Table 3
). The inclusion of WS in diets substantially reduced ileal DM digestibility (P < 0.01) reflecting the low digestibility of the large NDF fraction present in WS. As mentioned earlier, NDF in WS represent largely cellulose and arabinoxylans that are closely associated with lignin and that are not easily fermented by the gastrointestinal microbes (Bach Knudsen and Canibe, 2000
). Inclusion of 6% pectin in the diet decreased ileal DM digestibility by about 7%, suggesting that the ileal digestibility of pectin is rather low (de Lange et al., 1989a
). In addition, pectin may reduce digestibility of other diet components and induce endogenous gut nutrient losses.
Endogenous Ileal Amino Acid and Protein Losses
Increasing the diet CS level did not influence endogenous ileal Lys losses (Table 4
), reflecting that there were no specific components in CS that influence endogenous ileal Lys losses, and likely EAAL (Nyachoti et al., 1997b
), and that estimates obtained when feeding these diets can be considered basal EAAL. The mean basal endogenous ileal protein losses (EPL) for the basal and CS3 diets (11.4 and 9.5 g of DMI/kg) observed in this study are close to the wide range of values (10.5 to 17.1 g of DMI/kg) reviewed by Jansman et al. (2002)
. Experimental method and environmental conditions are known to influence EPL (Nyachoti et al., 1997a
; Sève et al., 2001
; Jansman et al., 2002
). The low diet NDF levels in the basal and CS3 diets (12 g of DM/kg), compared with the NDF content of diets (< 80 g of DM/kg) used in the review of Jansman et al. (2002)
, likely contributed to the low EPL observed in this study.
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The pectin- or WS-induced increase in EPL above the basal level (specific EPL) can be attributed to either increased endogenous secretion or reduced reabsorption of EPL. In particular, the ileal flows at the terminal ileum of Gly, Pro, and Cys are influenced by pectin level (Table 3
). Mucins coming from stomach and intestines are known to be rich in Pro along with Ser, Thr, and Cys (van Klinken et al., 1998
), whereas Gly can be found predominantly as a conjugate of bile acids aside from taurine (de Lange et al., 1989a
). Soluble NSP such as pectin can also create viscous digesta apart from enhancing mucus production, which in turn can increase thickness of unstirred water layer. Both processes can potentially restrict reabsorption of endogenous ileal AA (Smits and Annison, 1996
). For WS3, the increased EPL may also be attributed to adsorption of endogenous protein to the fiber and lignin, abrasion of the intestinal mucosal surface, or increased pancreatic, mucosal, or biliary secretions (Smits and Annison, 1996
). The presence of large quantities of undigested material in the gut, which is the case when feeding a WS-containing diet, can exert mechanical force that can cause an increase in desquamation of the intestinal mucosa (de Lange et al., 1989a
). The sloughed off cells and mucin proteins are known to be highly resistant to enzymatic digestion, thereby reducing reabsorption of endogenous protein (Nyachoti et al., 1997a
).
True Ileal Amino Acid Digestibility
At similar AA to energy ratio (CS3, WS3, or CS3 + pectin), TID of AA in the CS3 diet were greater (P < 0.01) than TID values observed for the WS3 diet (Table 5
). Values were high at 99% for most essential AA in CS, and inclusion of 6% pectin in the diet did not influence TID. High TID observed in CS3 diet was consistent with observations by others (Nyachoti et al., 1997b
; Yin et al., 2004
) and indicates that proteins in CS can be easily hydrolyzed and absorbed. To our knowledge, TID of AA in WS have not yet been established.
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True ileal digestibilities of AA in WS, calculated by the difference method, were influenced by the inclusion level of WS in the diet. For Lys and Met, TID was reduced (P < 0.05), but it increased for Thr and Ile (P < 0.05) with increasing WS level. Obviously, these calculations are sensitive to the estimated TID values of AA in CS and synthetic AA, which have a large impact on indirectly calculated TID values in WS at the lower dietary WS inclusion level. The calculations are also sensitive to the assumed AA composition of EPL as discussed earlier. Because the effect of dietary WS inclusion level on indirectly calculated TID values of AA in WS was not consistent among AA, the effects of dietary WS inclusion level are likely more reflective of methodological and analytical inaccuracies than actual effects of dietary WS inclusion level on TID. Values for TID of AA in WS, therefore, are approximately 12 to 17% lower than in CS.
The high TID observed for CS was as expected as there are no known compounds that can hinder physical access of digestive enzymes to proteins. In contrast, most of the proteins found in WS are physically bound to fiber fractions (Huang et al., 2001
). Approximately 80% of the proteins found in wheat are present in the endosperm, and the rest are present in the germ and bran fractions of the seed (Cornell and Hoveling, 1998
). Access of digestive enzymes to proteins in the bran fraction might be lower than those in endosperm fraction; the former includes cell walls that consist predominantly of insoluble and highly lignified cellulose and arabinoxylans (Bach Knudsen and Canibe, 2000
). Huang et al. (2001)
showed that the digestibility of proteins associated with purified NDF is about 60% in wheat fractions largely because a substantial fraction of the protein is physically inaccessible to digestive enzymes. This implies that as NDF content of the diet increases, TID of AA decreases. This is supported by the current observations as both dietary NDF and NSP contents are negatively correlated with the TID of AA. For instance and based on the 5 treatment means, TID of Lys was highly correlated with NDF and NSP content of the diet (r = 0.94; 0.90, respectively). Another factor that might influence TID of AA in WS diets is the faster digesta passage rate, which reduces time available for interaction between hydrolytic enzymes and protein. Le Goff et al. (2002)
observed that diets containing NSP from bran fractions have shorter mean retention time in the digestive tract compared with diets containing soluble fiber such as pectin.
Considering the AID, EPL, and TID results, this study clearly showed that dietary inclusion of WS reduced AID of AA not only because of lower TID of AA but also because of greater EPL. Inclusion of 6% pectin, on the other hand, reduced AID of AA mainly by increasing EPL.
Standardized Ileal Digestibility of Amino Acids
Because of the difficulty in routine measurement of total or specific EPL in pigs fed protein-containing diets, AID digestibility values may be corrected for basal EPL losses only (Jansman et al., 2002
). The resulting SID values are more likely to be additive in mixtures of pig feed ingredients but should be differentiated clearly from TID values for feed ingredients that induce specific EPL. In ingredients that induce specific EPL, SID values will be lower than TID values.
Calculated SID content of CS-containing diets (basal, CS3) were very close to actual SID observed in the current study. Calculated SID of WS-containing diets were 2 to 5% lower than observed SID values, whereas calculated SID of CS3 + pectin diet was 3% greater than actual SID value. At the greatest level of SID Lys/MJ of DE (CS3, CS3 + pectin, WS3), SID values were greater for the CS3 diet than for the WS3 and CS3 + pectin diets (P < 0.01). The SID of AA were lowest in the WS3 diet and greatest in the CS3 diet (Table 6
), which is in agreement with diet effects on AID (Table 3
).
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Indirectly calculated SID values of AA in WS were not influenced (P
0.10) by the dietary inclusion level of WS for most AA, except for Lys and Met, which were 7 and 6% greater (P < 0.05; Table 7
) at the lower dietary WS level. Similar to TID values, calculated SID values for AA in CS and WS are sensitive to the estimated SID in CS and synthetic AA as well as the assumed AA composition of EPL. The SID values of AA in WS, means of WS2 and WS3, were very similar to values published by NRC (1998)
except for Lys (84.5 vs. 77%) and Ile (70 vs. 81%; current study vs. NRC, 1998
, respectively).
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Conclusions
In this study, diet CS level (4.5 vs. 8.5%) did not influence TID of most essential AA and EAAL in pigs fed CS and cornstarch-based diets. Inclusion of 22.5 or 45% WS in the CS-based diet reduced TID of most essential AA (P < 0.01). The TID values for most essential AA, however, were the same for both levels of dietary WS inclusion (22.5 vs. 45%), except for Lys and Met, which were further reduced at the greatest dietary WS inclusion level. An increase in EAAL was only observed at the greatest dietary WS inclusion level. Addition of 6% pectin did not influence TID of AA but increased EAAL.
Effects of dietary protein source or addition of dietary pectin on AID of AA can be attributed to decreased TID or increased EAAL losses. Wheat shorts effects on EAAL losses seem to be largely attributed to the soluble NSP content in WS. The impact of diet WS level on the various aspects of digestion such as TID of AA and endogenous ileal protein losses does not seem to be linear. This needs further evaluation, especially with greater number of dietary WS inclusion levels. The impact of increased EAAL on dietary AA requirements should be explored further and may provide an important argument to routinely measure total EAAL.
| Footnotes |
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2 The animal utilization protocol was reviewed and approved by the Animal Care Committee of the University of Guelph. Technical assistance provided by Eric Jeaurond, Linda-Trouten Radford, and Julia Zhu is greatly appreciated. ![]()
3 Current address: Cargill Animal Nutrition Philippines, Inc., Dampol 1st, Pulilan, Bulacan, Philippines. ![]()
4 Current address: Changsha Institute of Agricultural Modernization, The Chinese Academy of Science, Hunan, Changsha 410125, P.R. China. ![]()
5 Former address: Agribrands International Inc., St. Louis, MO; Current address: Global Animal Nutrition Solutions, 12 Résidence Le Clos Baron, 78112 Fourqueux, France. ![]()
6 Corresponding author: cdelange{at}uoguelph.ca
Received for publication June 6, 2005. Accepted for publication January 24, 2006.
| LITERATURE CITED |
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This article has been cited by other articles:
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A. J. Libao-Mercado, S. Leeson, S. Langer, B. J. Marty, and C. F. M de Lange Efficiency of utilizing ileal digestible lysine and threonine for whole body protein deposition in growing pigs is reduced when dietary casein is replaced by wheat shorts J Anim Sci, June 1, 2006; 84(6): 1362 - 1374. [Abstract] [Full Text] [PDF] |
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