Ileal amino acid digestibility and reactive lysine content in peas (Pisum sativum) fed to growing pigs

Animal Feed Science and Technology 129 (2006) 210–223

Ileal amino acid digestibility and reactive lysine content in peas (Pisum sativum) fed to growing pigs M.J. Friesen, E. Kiarie, C.M. Nyachoti ∗ Department of Animal Science, University of Manitoba, Winnipeg MB, Canada R3T 2N2 Received 3 June 2005; received in revised form 19 December 2005; accepted 19 December 2005

Abstract Four barrows with an average initial BW of 24.4 ± 1.8 kg and fitted with a simple T-cannula at the distal ileum were used to determine the coefficients of ileal apparent (CIAD), standardized (CISD) and true (CITD) digestibility of crude protein (CP) and amino acids (AA) in four pea-based diets according to 4 × 4 Latin square design. Coefficients of ileal true digestibility of reactive lysine (CITDrlys ) and digestible reactive lysine content (Drlys ) were also determined. The CITD of lysine, CITDrlys , and Drlys were determined using the homoarginine (HA) method. The pigs were fed four pea-based diets formulated to contain 145 g CP/kg DM from three pea cultivars; Profi, Swing, Croma and a pea mixture of AC Melfort, CDC Mozart, and Eclipse. The diets were formulated to contain either of the three pea cultivars or pea mixture as the sole source of AA. Chromic oxide and titanium oxide (4 g/kg) were included as indigestible markers to determine CIAD of nutrients and homoarginine, respectively. Each experimental period lasted 8 days and ileal digesta were collected continuously for 24-h on day 6 for determining CIAD. On the morning of day 8, pigs were fed a diet in which half of the pea was replaced with the respective guanidinated pea to determine CITD of lysine. Part of the digesta collected on day 6 was guanidinated for determining CITDrlys and Drlys . The CIAD, CISD and CITD of CP were similar (P>0.05) among the diets. The CIAD of indispensable AA, except phenylalanine were different (P<0.05) among the diets. The CITD of AA other than lysine were estimated using observed endogenous lysine flows and their published ratios relative to lysine. The CISD and CITD of AA were similar (P>0.05) among the diets. The Drlys were different (P<0.05) among the diets. The values of CITDrlys (range: 0.964–0.982) were similar (P>0.05) to the CITD of lysine (range: 0.981–0.993) within and among the diets. The CIAD of AA obtained in this study were more variable ∗

Corresponding author. Tel.: +1 204 474 7323; fax: +1 204 474 7628. E-mail address: Martin [email protected] (C.M. Nyachoti).

0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.12.010

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compared with CISD and CITD. The results also indicate that the CITDrlys and CITD of lysine provide close values, validating suitability of either method in determining true ileal digestibility of lysine. © 2006 Elsevier B.V. All rights reserved. Keywords: Amino acids; Digestibility; Pea; Homoarginine; Reactive lysine; Pigs

1. Introduction Pea (Pisum sativum) production in Canada has increased considerably over the last few years. Peas are potentially an excellent source of energy and protein (CP) for swine (Jansman and Verstegen, 2002). The average CP content in pea cultivars is 239 g/kg DM-basis, ranging from 208 to 264 g/kg DM-basis (Fleury, 2004). The content of lysine in peas, as g per kg of CP, is relatively high (66 g/kg CP as is), the content of the sulfur-containing amino acids (AA), as in many other grain legumes, is relatively low (21 g/kg CP as is, NRC, 1998). There is a considerable variation in CP and AA content among pea cultivars (Castell et al., 1996), which can be attributed to differences in environmental and genetic factors (Savage and Deo, 1989), especially the proportion of the three major protein fractions; legumins, vicilins, and albumins (Daveby et al., 1993). The nutritional value of peas has been assessed through performance and apparent nutrient digestibility studies in pigs (Fan and Sauer, 1999; Owusu-Asiendu et al., 2002; Stein et al., 2004). However, for accurate formulation of pig feeds in terms of AA supply, true as opposed to apparent ileal digestibilities should be used because true digestibilities are additive in a mixture of feed ingredients (Nyachoti et al., 1997a; NRC, 1998; Nyachoti and Stein, 2005). For determining true ileal AA digestibilities, the undigested AA present in the ileal digesta should be differentiated from endogenous AA. The homoarginine (HA) and reactive lysine methods, which involves the transformation of dietary and digesta lysine to its amino analogue, homoarginine, in a guanidination reaction with methylisourea, are promising methods for determining true lysine digestibilities and available lysine in pig feed ingredients, respectively (Moughan and Rutherfurd, 1996; Nyachoti et al., 1997a, 2002). However, these methods have not been evaluated with most commonly used pig feed ingredients. Furthermore, the true ileal CP and AA digestibilities in pea cultivars grown in Manitoba fed to pigs have not been reported. The objectives of the study described herein were to determine the coefficients of ileal apparent digestibility (CIAD) of AA, coefficients of ileal true digestibility (CITD) of lysine and coefficients of ileal true digestibility of reactive lysine (CITDrlys ) in pea-based diets. The coefficient of ileal standardized digestibility (CISD) and CITD of AA other than lysine were estimated as well. 2. Materials and methods 2.1. Animals and housing Four Cotswold barrows with an average initial body weight of 24.4 ± 1.8 kg (mean ± S.D.) were obtained from Glenlea Research Farm. Pigs were housed in individual

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adjustable metabolism crates (118 cm × 146 cm) with smooth transparent walls and plastic covered woven metal flooring in a temperature controlled (20–22 ◦ C) room. After a 7-day adjustment period, pigs were surgically fitted with a simple T-cannula at the terminal ileum following the procedures described by (Sauer et al., 1988). After surgery, pigs were immediately returned to their metabolism crates and allowed a 17-day recovery period. During this period, they were fed thrice daily with increasing amounts of a commercial grower diet and had unlimited access to water from a low-pressure nipple drinker. The use of animals in the present study was reviewed and approved by the animal care committee of the University of Manitoba, and pigs were cared for according to the guidelines of the Canadian Council on Animal Care (CCAC, 1993). 2.2. Pea cultivars and processing Three pea (Pisum sativum) cultivars namely, Swing, Croma and Profi were sourced from a local farm; three other cultivars AC Melfort, CDC Mozart, and Eclipse were sourced from Agriculture and Agri-Food Canada, Research Station, Morden. The three cultivars from Agri-Food Canada were thoroughly mixed together to give a pea mixture with CP content of 237 g/kg DM. Thus, the three cultivars Swing, Croma, and Profi and pea mixture constituted the four ingredients tested in this study. Peas were ground through a 3-mm screen in a hammer mill prior to diet mixing. In order to determine the CITD of AA and endogenous AA flows, samples of the four pea–protein sources were guanidinated. The guanidination reaction was carried out following the procedures described by Imbeah et al. (1996) with modifications (Nyachoti et al., 1997a, 2002). Briefly, methylisourea (MIU) solution was prepared by reacting 86.13 g of O-methylisourea hydrogen sulfate with 157.75 g of barium hydroxide octahydrate in distilled water that had been preboiled for 10 min to remove carbon dioxide. The solution was then centrifuged at 1500 × g for 10-min and the supernatant decanted into a 1-L volumetric flask. The precipitate was washed, centrifuged, and the supernatant added to the contents of the volumetric flask. The total volume was brought to mark with distilled water to give 0.5 M solution of MIU. Each of the ground pea sample, was soaked in distilled water, and then thoroughly mixed with 0.5 M MIU solution. The pH of the mixture was adjusted to 10.5 using 1 M NaOH and thoroughly mixed to ensure uniform conditions before incubating the mixture at 4 ◦ C for 6 days. The pH was checked and adjusted accordingly once daily during incubation. After 6 days, the guanidination reaction was stopped by lowering the pH to the isoelectric point (4.5) of pea protein using 1 M HCl. Samples were then centrifuged at 1500 × g and 4 ◦ C to recover the guanidinated protein. The samples were then washed and centrifuged three times with water whose pH was adjusted to the isoelectric point of pea protein to remove excess MIU. Washed samples were freeze-dried and stored in a cool dry place until required for diet preparation. The average conversion rate of lysine to homoarginine for the four peas samples was 96%. 2.3. Experimental diets and feeding Four semi-purified, iso-nitrogenous (145 g/kg CP DM) diets were formulated to contain pea as the sole source of protein (Tables 1 and 2). The diets contained Profi, Swing, Croma,

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Table 1 Composition and calculated chemical analysis of experimental diets (as-is basis) Item

Diet Profi

Swing

Croma

Mixturea

Ingredients (g/kg) Peas Cornstarch Vegetable oil Vitamin premixb Sucrose Chromic oxide

793 23 30 50 100 4

733 83 30 50 100 4

681 135 30 50 100 4

613 203 30 50 100 4

Calculated nutrient composition Crude protein (g/kg) Crude fat (g/kg) Lysine (g/kg) Ca:P Digestible energy (MJ/kg)

145.1 38.8 11.0 1.4 14.6

145.1 38.2 10.2 1.4 14.8

145. 39.5 11.9 1.4 14.5

145.0 37.4 9.2 1.5 14.9

a

Mixture of peas contained AC Melfort, CDC Mozart, and Eclipse cultivars. Premix provided per kg of diet: 180 g calcium, 85 g phosphorus, 60 g salt, 24 g sodium, 2.5 g magnesium, 35 mg manganese, 152.5 mg iron, 137.5 mg zinc, 125 mg copper, 0.75 mg iodine, 11,750 IU Vitamin A, 1500 IU Vitamin D3, 50 IU Vitamin E, 1.75 mg Vitamin K, 750 mg choline chloride, 38 mg niacin, 35.75 mg calcium pantothenate, 10 mg riboflavin, 1 mg thiamine, 1 mg pyridoxine, 27.5 mg Vitamin B12, 100 mcg biotin, 0.5 mg folic acid, and 0.3 mg selenium. b

Table 2 Analyzed composition of experimental diets (g/kg, DM-basis) Item

Diet Profi

Swing

Croma

Mixturea

921.39 150.20

921.36 141.60

921.15 156.65

911.67 141.60

Indispensable amino acids Arginine Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Valine

14.6 2.6 6.6 13.4 13.7 8.4 7.8 7.6

12.9 4.8 6.7 12.9 13.1 7.9 7.7 7.4

14.9 4.8 6.7 13.2 13.8 8.1 7.4 7.6

5.4 5.1 3.2 10.2 10.4 6.1 5.5 4.1

Dispensable amino acids Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine

8.4 21.9 31.8 8.2 9.7 9.8 5.2

7.9 19.9 31.2 7.7 8.4 9.7 4.7

8.1 21.7 30.9 7.9 7.2 9.5 5.1

6.7 20.2 28.4 7.0 7.7 8.6 5.3

Dry matter Crude protein

a

Mixture of peas contained AC Melfort, CDC Mozart, and Eclipse varieties.

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and pea mixture at concentration of 793, 733, 681, and 613 g/kg (as fed basis), respectively, as the only AA-containing ingredients. The guanidinated diets were prepared by replacing 50% peas in each diet with respective guanidinated material. Sucrose was included in the diets to improve palatability. Vitamins and minerals were supplemented to meet or exceed requirement estimates for growing pigs (NRC, 1998). Chromic oxide (4 g/kg) was included in both the unguanidinated and guanidinated diet as an indigestible marker for determining CIAD of AA. An additional marker, titanium oxide (4 g/kg), was included at the expense of sucrose in the guanidinated diet for determining the contribution of the guanidinated diet to digesta collected on day 8 and for determining CITD of AA (Nyachoti et al., 1997a,b, 2002). Feed was supplied in a daily amount equal to 2.6 times the energy requirement for maintenance (444 kJ ME/kg0.75 ; NRC, 1998) in three equal meals at 0800, 1600 and 2400 h as a wet mash with a water-to-feed ratio of 2:1. Additional drinking water was available from low-pressure drinking nipples. Feed refusals and spillage were recorded and used to determine actual DMI. 2.4. Experimental design and general conduct of study The experiment was designed and conducted according to a 4 × 4 Latin square design. The experimental periods lasted 8 days each. Pigs were allowed to acclimatize to their respective experimental diets for 5 days, on day 6, a 24-h continuous ileal digesta collection was conducted for determining apparent digestibilities in the regular non-guanidinated diets. On day 8, a meal of the diets with guanidinated protein was fed only at 0800 h followed by a 24-h continuous digesta collection for determining endogenous AA losses and true AA digestibilities. Digesta were collected into transparent plastic bags, which were attached to the barrel of the T-cannulas with a hose clamp and contained 10 mL of 10% (v/v) formic acid to minimize bacterial activity. Part of day 6 digesta was guanidinated for determining CITDrlys and Drlys following the procedure previously described for the diets. The unguanidinated and guanidinated digesta were then immediately frozen at −20 ◦ C until further processing. 2.5. Sample preparation and chemical analysis Digesta samples were pooled per pig and per collection period prior to freeze-drying. Dried digesta and diets were ground in a Wiley mill through a 1-mm screen and thoroughly mixed prior to analyses. All samples were assayed for dry matter (DM), CP, chromic oxide, and AA; titanium oxide and HA were only assayed in guanidinated diets and digesta. Dry matter content was analyzed according to AOAC procedures (Procedure 4.1.06; AOAC, 1998) while CP (N × 6.25) content was determined using Leco NS 2000 Nitrogen Analyzer (LECO Corporation, St. Joseph, MI., USA). The AA including HA were analyzed on a LKB 4151 Alpha analyzer (LKB Biochrom, Cambridge, UK). Prior to analysis, samples were hydrolyzed with 6N HCL for 24 h at 110 ◦ C (Procedure 4.1.11 alternative 3; AOAC, 1998). The sulfur-containing amino acids and tryptophan were not determined.

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Chromium concentration was determined according to Williams et al. (1962) on 0.44 of digesta and or 1 g of feed sample. Titanium concentration was determined according to AOAC (Procedure 965.06; AOAC, 1990) on 1 g of digesta or feed sample. All analyses were conducted in duplicate. 2.6. Calculations and statistical analysis The coefficients of ileal apparent digestibility (CIAD) for AA and CP were calculated using the observations made on day 6 and chromic oxide as indigestible marker using the following equation (Nyachoti et al., 1997a):
  [AA]digesta × [Cr]diet CIAD = 1 − (1) [AA]diet × [Cr]digesta where CIAD is the coefficient of ileal apparent digestibility of an AA or CP, [AA]diet and [AA]digesta the concentrations (mg/kg DMI) of AA and CP in the diet and digesta, respectively, and [Cr]diet and [Cr]digesta are the concentrations (mg/kg DMI) of chromic oxide in the diet and digesta, respectively. The total flow (mg/kg DMI) of CP, AA, and HA at the terminal ileum was calculated using the following equation (Nyachoti et al., 1997a):  
[Marker]diet AAflow = [AA]digesta × (2) [Marker]digesta where AAflow is the flow of an AA at the terminal ileum and [Marker]diet and [Marker]digesta are the concentrations of the appropriate indigestible marker (chromic oxide for day 6 and titanium oxide for day 8 observations) in the diet and digesta, respectively. The coefficient of ileal true digestibility of lysine (CITDlys ) was calculated using the following equation (Nyachoti et al., 1997a, 2002):   [HA]diet − [HA]flow CITDlys = (3) [HA]diet where CITDlys is the coefficient of ileal true digestibility of lysine, [HA]diet and HAflow are the homoarginine concentration in the diet (mg/kg DM) and the flow of homoarginine at the terminal ileum, respectively. The flow of endogenous lysine (LYSeflow ) was calculated as the total flow of lysine at the distal ileum (LYS flow ), determined with Eq. (2) and from observations made on day 6, minus the flow of unabsorbed dietary lysine using the following equation (Nyachoti et al., 1997a): LYSeflow = LYSflow − ([LYS]diet × (1 − CITDlys ))

(4)

where LYSeflow is the flow of endogenous lysine (mg/kg DMI) and [LYS]diet is the concentration of lysine in the diet. The endogenous flow of AA other than lysine (AAeflow ) was calculated from the observed flow of endogenous lysine and the amounts of other AA relative to lysine as reported by Boisen and Moughan (1996), except for proline and glycine, for which ratios from de Lange et al. (1989) were used. The values for glycine and proline were abnormally high in the Boisen and Moughan (1996) than in de Lange et al. (1989).

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The coefficients of ileal true digestibility (CTIDAA ) of AA other than lysine were then calculated using the following equation: CITDAA =

[AA]diet − (AAfolw − AAeflow ) [AA]diet

(5)

where CTIDAA is the coefficient of ileal true digestibility of an AA other than lysine and AAeflow is the endogenous flow of an AA other than lysine at the terminal ileum. Endogenous flow of CP was determined by multiplying CP flow at the distal ileum with the ratio of lysine in endogenous CP and the results employed in determining the CITD of CP using Eq. (5). The CISD of AA and CP were determined by correcting CIAD (Eq. (1)) for basal endogenous losses (ENL) of specific AA and CP (mg/kg DMI) using the following equation according to Rademacher et al. (2000): 

ENL CISD = CIAD + [AA]diet

 (6)

where, ENL is an average of published values for a specific AA or pea protein (Rademacher et al., 2000). The coefficients of true ileal reactive lysine digestibility (CITDrlys ) and digestible reactive lysine (Drlys ) were calculated according to Rutherfurd and Moughan (1997) using the following equations:  CITDrlys =

[RLYS]diet − (RLYSflow − LYSeflow ) [RLYS]diet

Drlys = CITDrlys × [RLYS]diet

 (7) (8)

where, [RLYS]diet is the concentration of reactive lysine (mg/kg DMI) in the guanidinated diets and RLYSeflow is the flow of reactive lysine at the distal ileum based on the guanidinated day 6 digesta. The CIAD, CISD, CITD of CP and AA and CITDrlys and Drlys in each of the four diets were compared statistically using the Proc Mixed procedure of SAS (Littell et al., 1996). An analysis of variance was conducted with pig, period, and diets as the main effects. Pig was the experimental unit. Treatment means were separated using the LSMeans statement and the PDIFF Adjust = Tukey option of proc Mixed, and an alpha level of 0.05 was used to assess significance between means. The CITDrlys and CITD of lysine means of respective diets were compared with a t-test.

3. Results All four pigs remained healthy, readily consumed their daily feed allowance, and grew normally throughout the study. There were no significant (P>0.05) effects of pig or period for any of the parameters studied.

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Table 3 Coefficients of ileal apparent digestibility (CIAD) of CP and AA acid in experimental diets by growing pigs Item

Diet

S.E.M.

Profi

Swing

Croma

Mixturea

Crude protein

0.706

0.692

0.707

0.694

0.0210

Indispensable amino acids Arginine Histidine Isoleucine Lysine Leucine Phenylalanine Threonine Valine

0.887a 0.618b 0.761a 0.834ab 0.786a 0.785 0.701a 0.728a

0.910a 0.801a 0.779a 0.840a 0.791a 0.799 0.716a 0.738a

0.892a 0.768a 0.745a 0.813ab 0.764ab 0.758 0.662ab 0.706a

0.713b 0.787a 0.526b 0.776b 0.723b 0.728 0.567b 0.528b

0.0223 0.0268 0.0328 0.0148 0.0219 0.0233 0.0272 0.0275

Dispensable amino acids Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine

0.723 0.787 0.821 0.678 0.742a 0.687 0.768

0.731 0.787 0.846 0.691 0.745a 0.743 0.789

0.691 0.770 0.809 0.646 0.671b 0.723 0.738

0.642 0.776 0.808 0.628 0.680b 0.701 0.777

0.0259 0.0168 0.0149 0.0312 0.0180 0.0444 0.0224

Data represents least square means of four observations per diet. Means within row lacking a common letter differ (P<0.05). a Mixture of peas contained AC Melfort, CDC Mozart, and Eclipse varieties.

3.1. Chemical composition of the pea-based diets The calculated and analyzed CP and AA profiles in the four diets are shown in Tables 1 and 2, respectively. The calculated (145.2, 145.2, 145.1, and 145.3 g/kg DM) CP profiles were slightly different from the analyzed (150.2, 141.6, 156.7, and 141.6 g/kg DM) CP profiles for Profi, Swing, Croma, and pea mixture, respectively. The AA profiles of Profi, Swing, and Croma diets were similar, while that of pea mixture diet had a slightly different AA profile. 3.2. Digestibilities of crude protein and amino acids 3.2.1. Coefficients of ileal apparent digestibility (CIAD) The CIAD of CP were not different among diets (P>0.05) and ranged from 0.692 for pea mixture to 0.707 for Profi and Croma (Table 3). Of the indispensable AA, the CIAD of arginine, isoleucine and valine were similar (P>0.05) between Profi, Croma, and Swing diets, which were in turn different (P<0.05) from pea mixture diet. Within each diet with exception of the pea mixture diet, compared with other indispensable AA, the CIAD of arginine and lysine were usually relatively high (Table 3). The CIAD of indispensable AA ranged from 0.526 for isoleucine and valine to 0.910 for arginine (Table 3). Among the dispensable AA, significant differences were only observed for proline with Profi and

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Table 4 Coefficients of ileal standardized (CISD) CP and AA in experimental diets fed to growing pigs Item

Diet

S.E.M.

Profi

Swing

Croma

Mixturea

Crude protein

0.766

0.792

0.772

0.783

0.0243

Indispensable amino acids Arginine Histidine Isoleucine Lysine Leucine Phenylalanine Threonine Valine

0.916 0.839 0.827 0.840 0.819 0.816 0.796 0.805

0.939 0.845 0.849 0.863 0.829 0.842 0.822 0.829

0.903 0.813 0.808 0.826 0.798 0.794 0.784 0.789

0.904 0.819 0.836 0.849 0.819 0.831 0.813 0.834

0.0168 0.0201 0.0201 0.0104 0.0148 0.0136 0.0186 0.0142

Dispensable amino acids Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine

0.776 0.819 0.863 0.765 0.769 0.808 0.775

0.806 0.8838 0.884 0.799 0.819 0.812 0.833

0.758 0.801 0.850 0.747 0.790 0.786 0.761

0.781 0.831 0.857 0.774 0.778 0.812 0.785

0.0195 0.0116 0.0122 0.0258 0.0168 0.0168 0.0166

Data represents least square means of four observations per diet. a Mixture of peas contained AC Melfort, CDC Mozart, and Eclipse varieties.

Swing diets having higher (P<0.05) values (0.742 and 0.745, respectively) than Croma and pea mixture diets (0.671 and 0.680, respectively). 3.2.2. Coefficients of standardized ileal digestibility (CISD) The CISD of CP were not different among diets (P>0.05) and ranged from 0.766 for Croma and Profi to 0.792 for Swing (Table 4). Like CIAD, CISD for arginine and lysine were relatively high compared with other indispensable AA within each diet. There were no differences (P>0.05) in CISD for any of the indispensable and dispensable AA among the diets. The CISD values of indispensable and dispensable AA ranged from 0.784 for threonine to 0.939 for arginine and 0.747 for glycine to 0.884 for glutamic acid, respectively. 3.2.3. Coefficients of true ileal digestibility (CITD) The total and endogenous lysine flows at the distal ileum are presented in Table 5. There were no differences (P>0.05) in the total and endogenous flows of lysine at the distal ileum of pigs fed the four experimental diets. The values of total and endogenous lysine flows (mg/day) ranged from 2220.8 for Swing to 2738.9 for Croma and 1661.6 for Swing to 2068.4 for Profi, respectively. The CITD of CP were not different among diets (P>0.05) and ranged from 0.915 for Profi and Croma to 0.939 for Swing (Table 6). There were no differences (P>0.05) in CITD for any of the indispensable and dispensable AA among diets. The CITD values of indispensable

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Table 5 Total and endogenous lysine flows at the distal ileum of pigs fed pea-based diets Item

Diet

S.E.M.

Profi

Swing

Croma

Mixturea

Total flow DMI (mg/kg) mg/day

2379.3 2517.3

2100.8 2220.8

2579.8 2738.9

2303.3 2436.8

199.98 209.06

Endogenous flow DMI (mg/kg) mg/day

1955.0 2068.4

1574.0 1661.6

1805.3 1922.7

1860.8 1968.7

218.94 227.84

Data represents least square means of four observations per diet. a Mixture of peas contains AC Melfort, CDC Mozart, and Eclipse varieties.

and dispensable AA ranged from 0.912 for histidine to 1.056 for threonine and 0.922 for aspartic acid to 1.041 for glycine, respectively. 3.2.4. Coefficients of ileal true digestibility of reactive lysine (CITDrlys ) and digestible reactive lysine (Drlys ) There were no differences (P<0.05) in CITDrlys in the four pea sources (Table 7). Within diet, compared to CITD of lysine values determined using the HA method, CITDrlys determined using the reactive lysine method were similar (P>0.05). There were significant Table 6 Coefficients ileal true digestibility (CITD) CP and AA in experimental diets by growing pigs Item

Diet

S.E.M. a

Profi

Swing

Croma

Mixture

Crude protein

0.916

0.939

0.915

0.934

0.0224

Indispensable amino acids Arginine Histidine Isoleucine Lysine Leucine Phenylalanine Threonine Valine

1.001 0.945 0.978 0.981 0.972 0.968 1.056 1.021

0.992 0.922 0.961 0.993 0.942 0.969 1.013 0.977

0.983 0.912 0.952 0.978 0.928 0.952 1.022 0.970

0.975 0.923 0.994 0.991 0.958 0.991 1.046 1.021

0.0123 0.0163 0.0191 0.0183 0.0144 0.0112 0.0181 0.0152

Dispensable amino acids Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine

0.967 0.943 0.945 1.041 0.967 0.979 1.022

0.938 0.934 0.954 1.000 0.949 0.934 1.014

0.933 0.922 0.942 0.994 0.952 0.948 0.982

0.951 0.943 0.947 1.023 0.944 0.968 1.019

0.0132 0.0101 0.0089 0.0193 0.0258 0.0144 0.0145

Data represents least square means of four observations per diet. a Mixture of peas contained AC Melfort, CDC Mozart, and Eclipse varieties.

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Table 7 Coefficients of ileal true digestibility of reactive lysine (CIADrlys ) and digestible reactive lysine (Drlys ) content (g/kg DM) in pea-based diets fed to growing pigs Diet Item

Profi

Swing

Croma

Mixturea

SEM

CITDrlys

0.978

0.982

0.964

0.982

0.0034

Digestible reactive lysine DM (g/kg) 6.39b

5.97c

6.54a

6.05c

0.022

Data represents least square means of four observations per diet. Means within row lacking a common letter differ (P<0.05). a Mixture of peas contained AC Melfort, CDC Mozart, and Eclipse varieties.

differences (P<0.05) in Drlys of pigs fed the four experimental diets (Table 7). The Drlys was lowest (P<0.05) in the Swing and pea mixture diets compared to the other diets. Croma had the highest Drlys , and the value of Profi was intermediate (Table 7).

4. Discussion The differences in AA profile observed between Profi, Swing, and Croma on one hand and pea mixture on the other hand could have been due to environmental and genetic factors. This is because the three cultivars were grown in the same year under the same environmental conditions, while the pea mixture diet contained pea cultivars grown in a different year and at a different location. It is well established that both genetic (e.g., variety) and environmental factors (e.g., climate) have significant effects on the chemical composition of pea cultivars (e.g., Savage and Deo, 1989; Castell et al., 1996). Furthermore, the pea mixture comprised of three pea cultivars, which may have had different proportions of the major protein fractions; legumins, vicilins, and albumins compared to Swing, Profi, and Croma. The AA composition in pea has been found to be dependent upon the proportion of the major protein fractions (Daveby et al., 1993). The CIAD of CP obtained in the current study were within the range of values previously reported for three European pea cultivars (Mariscal-Land´ın et al., 2002; 0.708–0.724) and slightly higher than the CIAD value reported for American pea cultivar (Stein et al., 2004; 0.683). However, the values for the CIAD of CP in the present study were somewhat lower than reported for six Canadian pea cultivars (Fan and Sauer, 1999; 0.749–0.797). The CIAD of arginine, isoleucine, and valine were similar between Profi, Croma, and Swing diets and different from pea mixture diet. Although not assessed in the current study, differences in AA digestibility among pea cultivars has been attributed to variations in trypsin inhibitor activity, fiber levels, proportions of major protein fractions and environmental conditions (Fan and Sauer, 1994). The fact that Croma, Profi, and Swing were grown under similar field conditions may explain why the CIAD of arginine, isoleucine, and valine, in these cultivars were similar. The high CIAD observed for lysine and arginine relative to the other indispensable AA concurs with findings on six Canadian pea cultivars fed to growing pigs (Fan and Sauer, 1994). These observations support the hypothesis that enzyme specificity is

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an important determinant of apparent AA absorption in the small intestine (Fan and Sauer, 1994). Arginine and lysine would be expected to appear first after enzymatic hydrolysis based on known specificity of the proteases and peptidases (Low, 1980). Compared to the CIAD, the CSID values for CP and AA were higher, which is essentially due to the correction for basal endogenous losses in the latter. The observed CISD values of CP and AA are comparable to those reported in literatures (Rademacher et al., 2000; Mariscal-Land´ın et al., 2002; Owusu-Asiendu et al., 2002; Stein et al., 2004). The endogenous lysine flows observed in the current study were similar among the diets, indicating that the pea cultivars used in the present study had similar influence on endogenous lysine flows. This observation is surprising given that the differences in CIAD of CP and AA among feedstuffs are usually attributed to differences in the amount of endogenous losses recovered at the distal ileum when different feedstuffs are fed (Nyachoti et al., 1997b). Using the nitrogen free method, Leterme and Th´ewis (2004) reported endogenous nitrogen losses of 710, 780, and 1030 mg/day in pigs of different BW (24, 62 and 105 kgs, respectively) fed diets containing isolated pea fibre. This clearly shows that the nitrogen free method underestimate the amount of endogenous nitrogen losses as pointed by Nyachoti et al. (2002). The recovery of endogenous nitrogen losses at the distal ileum of growingfinishing pigs represents a balance between secretion and reabsorption (Nyachoti et al., 1997b). Secretion and (or) reabsorption of endogenous nitrogen losses is influenced by such factors as BW, dietary fiber content, DMI, and the presence of anti-nutritive factors in the diet as reviewed by Nyachoti et al. (1997b) and Jansman et al. (2002). A limitation of the HA method is that only endogenous lysine flow is determined. Flows of AA other than lysine were determined based on an assumption that the AA composition of endogenous gut protein is relatively constant (de Lange et al., 1989; Boisen and Moughan, 1996). In the present study, the CITD of arginine, threonine, valine, glycine, and tyrosine were greater than 1, suggesting overestimation of endogenous flow of these amino acids for some diets. Even though we assume in our calculations of CITD of AA other than lysine that AA composition of endogenous gut protein is constant, we suggest that the effect of diet on AA be further evaluated (e.g. Boisen and Moughan, 1996). Other studies have estimated endogenous losses of AA other than lysine using the same procedure (e.g. Nyachoti et al., 1997a,b, 2002). The CTID of CP found in the current study agrees with the values obtained by Huisman et al. (1992: 0.93–0.95) in a study using the 15 N-dilution technique to estimate endogenous nitrogen losses. The HA method and the 15 N-isotope dilution techniques provide endogenous nitrogen losses amounts that are ingredient specific and the two methods generally provide similar estimations (Nyachoti et al., 1997b). The observed CITD of AA were higher than those reported by NRC (1998). This is to be expected because NRC (1998) values were determined by feeding a protein-free diet to estimate endogenous AA losses, a technique known to underestimate endogenous AA flow when feeding practical diets (Boisen and Moughan, 1996; Nyachoti et al., 1997b). In general, CITD of CP and AA were higher than CISD, which was in turn higher than CIAD, indicating the relevance of utilizing CITD in evaluating protein value in feedstuffs for swine. The use of CITD values of peas in formulating swine diets is particularly important, given that pea is quite variable in its AA profiles. The reactive lysine method has been proposed as a simpler method for estimating the bioavailability of lysine in feed ingredients. In this method it is assumed that the proportion of lysine that reacts with methylisourea to form HA is chemically available for use by the animal

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(Moughan and Rutherfurd, 1996). The CITDrlys obtained in the present study were quite high ranging from 0.964 to 0.978 thus indicating high bioavailability of lysine in the peas tested. The CITDrlys and CITD of lysine determined in the current study were quite similar, which is consistent with previous suggestions indicating this to be the case for ingredients that have not been heat damaged or stored for a prolonged period of time (Moughan and Rutherfurd, 1996). The Drlys reported in the present study (range 5.97–6.54 g/kgDM) was lower than that reported (13.1 g/kgDM) for raw pea fed to rats (Rutherfurd and Moughan, 1997). The differences between the two studies could have been due to guanidination conditions used in the two experiments. In the present study, the guanidination conditions were: 0.5 M MIU solution, pH 10.5 and temperature 4 ◦ C for 6 days, whereas Rutherfurd and Moughan (1997) used 0.6 M MIU solution, pH 11 and temperature 21 ◦ C for 7 days. It is well established that guanidination conditions affect the conversion of lysine to HA (Nyachoti et al., 1997a,b, 2002). 5. Conclusions and implications There were significant variations in the CIAD of some indispensable AA among the pea cultivars studied. Correcting for minimum endogenous AA losses (CISD) and specific endogenous losses (CITD and CITDrlys ) can eliminate the variability. The CITD and CITDrlys , however, give higher values than CISD and would therefore give better estimates of true digestibility of AA and lysine, respectively, and thus more accurate diet formulation. However, the procedure for determining CITD and CITDrlys are more complex and expensive for routine use. The CISD are simple and less expensive and could as well be applicable for routine evaluation of pea cultivars for swine feeds. Acknowledgments We gratefully acknowledge the funding for this project from the Manitoba Pork Council, Agri-Food Research and Development Initiative (ARDI), and the Manitoba Pulse Growers Association. Thanks to K. Carette, T. Davies, and Robert Stuski for technical support. References AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical chemists, Arlington, VA. AOAC, 1998. Official Methods of Analysis, 16th ed. Association of Official Analytical chemists, Arlington, VA. Boisen, S., Moughan, P.J., 1996. Dietary influences on endogenous ileal protein and amino acid loss in the pig — a review. Acta Agric. Scand. A: Anim. Sci. 46, 154–164. Castell, A.G., Guenter, W., Igbasan, F.A., 1996. Nutritive value of peas for non-ruminant diets. Anim. Feed Sci. Technol. 60, 209–227. CCAC, 1993. Guide to the Care and Use of Experimental Animals, vol. 1. Canadian Council on Animal Care, Ottawa, Ont. Canada. Daveby, Y.D., Abrahamson, M., Aman, P., 1993. Changes in chemical composition of three different types of peas. J. Sci. Food Agric. 63, 21–28. de Lange, C.F.M., Sauer, W.C., Souffrant, W., 1989. The effect of protein status of the pig on the recovery and amino acid composition of endogenous protein in digesta collected from the distal ileum. J. Anim. Sci. 67, 755–762.

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