Degradation in the rumen and nutritional value of lupin (Lupinus albus L.) seed proteins effect of extrusion

Animal Feed Science and Technology 105 (2003) 55–70

Degradation in the rumen and nutritional value of lupin (Lupinus albus L.) seed proteins effect of extrusion D. Rémond a,∗ , M.P. Le Guen b , C. Poncet a a

Institut National de la Recherche Agronomique de Theix, Unité de Recherches sur les Herbivores, Unité Nutrition et Métabolisme Protéique, 63 122 St Genès-Champanelle, France b GIE EURETEC II, 85 rue de St Brieuc, 35 000 Rennes, France

Received 17 January 2002; received in revised form 9 January 2003; accepted 9 January 2003

Abstract The objective of the present study was to assess, by in vivo measurements, the nutritive value of raw and extruded lupin seed proteins. The contribution of both particle associated and soluble proteins to the duodenal flow of lupin crude protein (CP) was determined. Furthermore kinetics of lupin CP degradation in the rumen was determined. A group of six fistulated sheep was used in a duplicated 3 × 3 Latin square design experiment. A control diet (diet C), in which urea and starch substituted for lupins, was included to enable determination of rumen escape of lupin CP. In the other two diets, raw (diet RL) or extruded (diet EL) lupin seeds (Lupinus albus, cultivar ‘Arès’) were used as the protein supplement, and both were ground to pass a 3 mm screen. Lupins provided 20% of the dietary dry matter, 50% of dietary CP, in both diets RL and EL. The undegraded CP fraction of lupins estimated by in situ measurements (i.e. particle associated outflow) was 4 and 25% for raw and extruded lupins, respectively. Lupin soluble CP not degraded in the rumen was 4.5 and 1.8% of lupin CP for raw and extruded seeds, respectively. By adding particle associated and soluble fractions, rumen degradability of raw and extruded lupin seeds was 92 and 73%, respectively. These values are consistent with calculated lupin CP flow to the duodenum, 92 and 74%, for raw and extruded lupins, respectively. The increase in dietary non-ammonia N (NAN) flow to the duodenum with lupin extrusion was balanced by a decrease in microbial flow, and total NAN flow did not differ between diet RL and EL. Apparent NAN digestion (% of N intake) in the small intestine was not affected by extrusion. Although dietary NAN contribution to duodenal flow increased from 16 to 24% between diet RL and diet EL, the profile of apparently absorbed amino acids did not differ. The in situ technique underestimates the nutritive value of lupin seed CP

∗ Corresponding author. Tel.: +33-4-7362-4074; fax: +33-4-7362-4273. E-mail address: [email protected] (D. R´emond).

0377-8401/03/$ – see front matter © 2003 Published by Elsevier Science B.V. doi:10.1016/S0377-8401(03)00040-3

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mainly from not considering solubilised lupin CP that escape ruminal degradation. By increasing rumen CP escape and intestinal CP digestibility, extrusion improved the nutritive value of lupin seed CP. © 2003 Published by Elsevier Science B.V. Keywords: Lupin; Digestion; Extrusion; Ruminant

1. Introduction In the context of the banning of meat and bone meal, and requests from consumers to avoid use of genetically modified crops in animal feeding, European countries must reconsider strategies for protein supply. Containing 35–40% crude protein (CP), lupin seed is a potential alternative to imported soya bean meal. However, because of the high in situ rumen degradability of its proteins, a low protein value is attributed to it in the French feeding system for ruminants (PDI system; Vérité and Peyraud, 1988). Nevertheless, production studies in lactating dairy cows have shown that lupin can replace soya bean meal without adverse effect on milk production (Emile et al., 1991; May et al., 1993; Robinson and McNiven, 1993; Singh et al., 1995). These studies suggest that, as reported for pea seeds (Poncet and Rémond, 2002), the protein value of lupin seeds is underestimated by standardised in situ techniques (Michalet-Doreau et al., 1987). Indeed, part of the protein leaving the in situ bags could be degraded less rapidly than previously believed, and so flow out of the rumen with the rumen liquid phase. Aufrère et al. (2001) showed that this rumen escape of lupin nitrogen could be twice that estimated by the in situ technique. However, this was observed in sheep fed an unbalanced diet (i.e. excess of N relative to energy), and it was shown that the significance of this escape of soluble N depended on energy availability in the rumen (Poncet et al., 1998). Data obtained with the in situ technique strongly suggest that the protein value of lupin seeds can be significantly increased by heat treatment (Cros et al., 1991; Kibelolaud et al., 1993; Singh et al., 1995; Aufrère et al., 2001) or by lowering the intensity of the mechanical treatment applied to the seed (Kibelolaud et al., 1991). Benchaar et al. (1994) confirmed in vivo, that extrusion of lupin seeds can lead to a 72% increase in dietary N flow to the duodenum of dairy cows. However, in this latter study, the lack of a control diet without lupin precluded a direct determination of in vivo rumen degradability of raw and extruded lupin protein. The objective of the present work was to assess, by in vivo measurement of lupin CP flowing to the duodenum, the nutritive value of raw and extruded lupin seed (L. albus L.) proteins. These values were compared to estimations obtained by the in situ method. The contribution of soluble lupin proteins undegraded in the rumen, to duodenal flow of lupin CP was determined to explain possible discrepancies between in vivo and in situ measurements. Furthermore, the dynamic nature of lupin protein degradation in the rumen was investigated.

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2. Material and methods 2.1. In vivo measurements 2.1.1. Animals, diets, and experimental design Six mature Texel weathers (51 ± 1.5 kg BW) were used in a duplicated 3 × 3 Latin square design experiment. They were surgically fitted with a ruminal cannula (silicone rubber; 50 mm i.d.), and T-shaped cannula (silicone rubber, 17 mm i.d.) in the proximal duodenum and distal ileum. The sheep were fed 1030 g of dry matter (DM) per day in two equal meals at 9:00 and 21:00 h. The three diets contained (% of DM) 44% chopped orchard grass hay, 21% chopped wheat straw, and 35% concentrate. The diets mainly differed in the source of N in the concentrate (Table 1): urea in the control diet (diet C), raw lupin (diet RL), or extruded lupin (diet EL). Diets RL and EL were formulated so that lupin seeds (370 g CP/kg DM) contributed approximately 50% of diet CP. In diet C, urea and starch substituted for lupin seeds. Urea and purified corn starch were chosen because they do not contribute to duodenal flow of non-ammonia dietary N. This diet was included in the experimental design to enable the determination of in vivo rumen degradability of lupin proteins. The amount of urea and starch in diet C was calculated to match energy and N microbial requirements according to the PDI system (Vérité and Peyraud, 1988). The chemical composition of the diets is in Tables 2 and 3. The lupins belonged to the species L. albus (cultivar ‘Arès’). Extrusion was performed on a single-screw extruder (162 ◦ C, specific mechanical energy delivery = 109 Wh/kg) after conditioning at 115 ◦ C for 1 h (12% moisture). Before pelleting of the concentrate, raw and extruded lupin seeds were ground to pass a 3 mm screen. Each experimental period lasted 35 days. The sheep were housed in a room under continuous lighting with controlled temperature (18–22 ◦ C). 2.1.2. Site and extent of nutrient digestion in vivo Total tract digestibility and intestinal flow measurement were simultaneously performed during a 6-day period (from day 25 to 30). Solute and particle markers were 51 Cr-EDTA Table 1 Ingredient composition of the concentrates (% of DM) Dieta

Beet pulp Raw lupin Extruded lupin Corn starch Animal fat (tallow) Urea Vitamin and mineral supplementb a

C

RL

EL

22.9

22.9 57.1

22.9

62.8 5.7 5.7 2.9

17.1

57.1 17.1

2.9

2.9

C: control diet; RP: raw lupin diet; EP: extruded lupin diet. Vitamins: vit. A, 1000 IU/kg; vit. D3, 200 IU/kg; vit. B1, 10 mg/kg; vit. B6, 0.5 mg/kg; vit. B12, 0.006 mg/kg; vit. PP, 1 mg/kg; folate, 0.13 mg/kg; vit. E, 15 mg/kg; vit. K3, 1.5 mg/kg. Minerals (mg/kg): Fe, 28; Cu, 0.1; Co, 0.16; Zn, 50; Mn, 40; Se, 0.22; I, 0.60; Mo, 0.48. b

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Table 2 Chemical composition of the dietsa Dietb

Dry matter (%) Organic matter (% DM) Neutral detergent fiber (% DM) Acid detergent fiber (% DM) Crude protein (% DM) a b

C

RL

EL

89.4 92.8 46.3 25.9 12.7

89.4 92.2 51.3 29.5 14.5

89.8 92.2 52.1 29.7 14.6

Values represent duplicate assays of duplicate samples. C: control diet; RP: raw lupin diet; EP: extruded lupin diet.

(25 ␮Ci/day/sheep), and 103 Ru-phenanthroline (6 ␮Ci/day/sheep), respectively. The two markers were infused continuously into the rumen, via separate lines, at a rate of 100 ml/day. Infusion started on day 20 with a priming dose of 100 ml of each infused solution and continued until day 30. For the determination of microbial protein synthesis, a continuous infusion of (15 NH4 )2 SO4 (35 mg of 15 N/day/sheep) into the rumen started on day 23 and ended on day 32. Total tract digestibility and recovery of markers were determined by total collection of urine and faeces. For intestinal flow measurements 12 samples were collected from the duodenum and ileum so that each 1 h interval of a 12 h feeding cycle (from 8:00 to 20:00 h) was represented (six sampling days, two sampling times/day). Each duodenal (160 ml) and ileal (80 ml) digesta sample was immediately subsampled under thorough Table 3 Amino acid (AA) composition of diets, and microbial biomass harvested in duodenal content (g/100 g AA)a Diet C

Diet RP

Diet EP

Bacteria

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

10.15 5.69 4.98 11.81 5.96 6.02 7.34 6.08 4.22 1.74 4.43 8.95 3.82 5.55 5.10 2.34 5.80

10.59 4.94 5.13 16.71 4.97 4.97 5.29 4.77 4.10 1.11 4.29 8.25 4.27 4.72 5.06 2.26 8.57

10.63 5.01 5.18 16.77 5.07 5.09 5.45 4.91 4.18 1.22 4.41 8.44 4.26 4.82 4.45 2.22 7.92

11.83 6.54 4.84 12.67 3.51 5.27 8.01 5.20 2.67 1.18 5.99 8.28 4.90 4.88 7.44 1.98 4.81

AA, % of DM

7.0

15.3

14.9

32.8

a

Values represent single assay of duplicate samples.

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homogenisation. One fraction (40 ml) was kept frozen as whole digesta. A second (40 ml) was squeezed dry through a nylon gauze (250 ␮m pore size), and both filtrate and particulate matter were frozen. For duodenal samples, the remaining fraction (80 ml) was frozen for bacterial separation (Poncet and Rémond, 2002). Fractions from the 12 sampling times were pooled to yield one sample. 2.1.3. Soluble N fractions in the rumen Rumen samples (100 ml) were collected on day 32, at 8:00, 10:00, 12:00, 14:00, 16:00 and 18:00 h. Samples were immediately strained through nylon gauze (250 ␮m) to remove the largest particles from the feed, the pH of the filtrate was recorded, and they were rapidly placed on ice, centrifuged at 800 × g for 10 min at 4 ◦ C to remove the remaining feed particles and protozoa, and the supernatant was centrifuged at 27,000 × g for 20 min at 4 ◦ C to remove bacteria. At each sampling time, the supernatant was subsampled for total N, ammonia N, and volatile fatty acid (VFA) determination. At 10:00, 14:00, and 18:00 h, the supernatant was further subsampled for nucleic bases, non-ammonia N (NAN) 15 N enrichment, and total amino acids (AA) determined after acid hydrolysis with 6N HCl at 115 ◦ C for 24 h. Another fraction was deproteinized by acid precipitation with sulfosalicylic acid (4%, wt/sample volume), followed by ultrafiltration (10,000 Mr cut-off filter; Centricon-10; Milliport, Bedfort, MA) as previously described for plasma samples (Rémond et al., 2000). The filtrates were analysed for AA composition before (i.e. free AA), and after acid hydrolysis with 6N HCl at 115 ◦ C for 24 h. The difference in AA concentration was attributed to peptides. The difference between total AA and the sum of free and peptide AA was attributed to proteins. 2.1.4. Rumen turn-over rates A pulse dose of CrEDTA and Yb labelled lupin was introduced into the rumen via the cannula 15 min before the morning meal of day 33. Rumen samples were collected 4 and 28 h after the dose delivery. Duodenal samples were collected 12 and 36 h after the dose delivery. 2.2. In situ incubation of lupin 2.2.1. Animals, diets Sheep were prepared as previously described but the duodenal and ileal cannula were replaced by cannula with an oval barrel of silicone rubber with a 327 mm2 opening to allow for easier introduction and withdrawal of mobile nylon bags. Four sheep receiving the RL diet were used for ruminal incubations and three for intestinal measurements. 2.2.2. In situ incubation The in situ procedure used for rumen effective degradability measurement was the same as that described by Poncet and Rémond (2002). Raw and extruded lupin were ground to pass a 3 mm screen before filling the bags. In situ intestinal digestibility of rumen undegraded CP was estimated using a mobile nylon bag technique (Poncet and Rémond, 2002).

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2.3. Laboratory analysis Most of the analytical procedures were completed as described by Poncet and Rémond (2002). Puric and pyrimidic bases were determined in lyophilised rumen fluid samples by HPLC after perchloric acid hydrolysis (Lassalas et al., 1993). 2.4. Calculations and statistics The double marker technique (Faichney, 1980) was used to estimate intestinal DM flows. Bacterial N flow at the duodenum was estimated from the bacterial and the duodenal digesta 15 N enrichment. Daily flows were estimated on the assumption that all 12 h periods were equivalent. Lupin N flow to the duodenum was calculated by difference with diet C. Pool size and turn-over rate of the rumen liquid were calculated assuming an exponential decrease against time in Cr concentration within ruminal contents. Lupin ruminal outflow rate (kp ) was estimated from the exponential decrease of Yb in the duodenal content. Ruminal outflow of non-ammonia N (NAN) components was calculated as average ruminal concentration over the feeding cycle (mg of N/l) × liquid outflow rate (l/day). Ruminal effective degradability (ED) of lupin N was estimated from the kinetics of in situ degradation of nitrogenous fractions (Ørskov and McDonald, 1979) using the kp measured in the experiment. The data were analysed by ANOVA using the GLM procedure of SAS (1988). When the treatment effect was significant (i.e. P < 0.05) means were compared by Duncan’s multiple-range test. The effect of time on rumen concentrations was analysed statistically using repeated measure ANOVA. The differences in instantaneous concentrations due to the treatment were tested by pairwise t-tests. 3. Results 3.1. Rumen turn-over rates Lupin passage rate and rumen liquid passage rate were not affected by cooking extrusion (i.e. P > 0.10), and were on average 0.040 ± 0.003 and 0.085 ± 0.003 h−1 , respectively. 3.2. In situ degradation measurements Cooking extrusion decreased the rapidly degraded fraction of lupin CP, and increased the potentially degradable fraction (Table 4). The degradation rate of the latter fraction increased with extrusion. As a consequence, the effective degradability of lupin CP decreased by 21% units. In situ intestinal digestibility of rumen undegraded lupin CP was 80 and 97% for raw and extruded lupin, respectively.

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Table 4 Effect of cooking extrusion on ruminal degradability of lupin crude protein

ab (%) bb (%) cb (h) EDb (%)

Raw lupin

Extruded lupin

S.E.M.a

P

78.8 20.6 0.20 96.1

49.9 43.1 0.06 74.8

6.2 5.2 0.04 3.3

0.005 0.013 0.040 0.001

a

S.E.M.: standard error of the mean. a: soluble fraction, n = 3; b: potentially degradable fraction, n = 3; c: degradation rate of b fraction; ED: ruminal effective degradability calculated with measured passage rate. b

3.3. Rumen traits Daily means of rumen fluid parameters are in Table 5. Cooking extrusion of lupin did not affect total VFA concentrations, but tended to decrease (P < 0.10) the branched chain VFA (i.e. isobutyrate and isovalerate) proportions. Total-N and NH3 –N concentrations were lower for diet RE than for RL. The average concentration of NAN was not significantly different between diets RL and EL. Nevertheless rumen NAN outflow tended to decrease with cooking extrusion (P < 0.10). By difference with the control diet, raw and extruded lupin resulted in a soluble NAN outflow of 0.5 and 0.2 g/day, respectively. From the effective degradability measured, lupin particulate NAN outflow from the rumen was 0.4 and 2.8 g/day for raw Table 5 Daily mean of ruminal pH, total VFA concentration and molar proportions of individual VFA, ammonia N and soluble non-ammonia N (NAN) concentrations, and on rumen liquid phase turn-over Item

Dieta C

pH

S.E.M.b RL

6.41 a

6.14 b

Total VFA (mM) Acetate (%) Propionate (%) Butyrate (%) Isobutyrate (%) Valerate (%) Isovalerate (%)

111.9 73.82 23.60 11.42 0.95 b 0.89 b 0.75 b

Soluble N (mg/l) Ammonia N (mg/l) NAN (mg/l)

360.6 b 223.3 b 137.3 b

Rumen liquid Volume (l) Fractional passage rate (per h) Rumen NAN outflow (g/day)

6.00 0.087 1.70 b

EL 6.22 b

0.07

123.7 82.62 24.46 12.63 1.20 a 1.42 a 1.00 a

118.0 78.17 24.51 11.84 1.09 ab 1.20 a 0.93 ab

3.29 2.22 0.80 0.47 0.04 0.08 0.05

451.9 a 262.3 a 189.7 a

379.2 b 203.1 b 176.1 a

5.53 0.087 2.18 a

(a, b) Means within a raw not sharing a common subscript differ (P < 0.05). a C: control diet; RL: raw lupin diet; EL: extruded lupin diet. b S.E.M.: standard error of the mean.

5.78 0.080 1.93 ab

13.88 11.7 7.8 0.15 0.0029 0.09

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and extruded lupin, respectively. With diets RL and EL, ingested lupin N was, on average, 11 g/day. By adding particulate and soluble NAN outflows, the rumen degradability was 92 and 73% for raw and extruded lupins, respectively. The dynamics throughout a feeding cycle of ammonia and NAN concentrations in the rumen fluid are in Fig. 1. The soluble NAN concentration with diet EL was lower (P < 0.05) than with diet RL at the end of the meal, but was higher (P < 0.05) before the meal and at 16:00 and 18:00 h. 3.4. Composition of rumen soluble NAN The nucleic bases concentration in the rumen fluid was 2.9, 3.3, and 3.7 mg of N/l (mean of the three sampling times) for the C, RL, and EL diets, respectively. Its contribution to soluble NAN was low, averaging 2%. The mean concentration in AA among the three sampling times was 147, 246, 201 mg of N/l for the C, RL, and EL diets, respectively. On average, amino acids (AA) in the rumen fluid accounted for about 50% of soluble NAN. For each diet, the highest AA concentration was observed at the end of the meal (Fig. 2a), whereas it did not differ between 14 and 18 h. Variations with time within the different fractions of AA are in Fig. 2b. Free AA (FAA) in the rumen fluid were only detectable at the sampling time at the end of the meal. At this time, FAA concentration was 0.5, 4.2, and 1.6 mg of N/l for diets C, RL, and EL, respectively. For each diet, protein and peptide AA concentrations were higher at the end of the meal (P < 0.05). On average, protein AA accounted for 88% of total AA with diet RL, and 73% with diet EL. Conversely peptide AA accounted for 11% of total AA with diet RL, and 26% with diet EL. 3.5. Origin of soluble NAN Because infusion of (15 NH4 )2 SO4 into the rumen was over a long period (10 days), and because bacterial AA accounts for more than 70% of the duodenal AA supply, it was assumed that endogenous N enrichment was close to that of bacterial N. From 15 N enrichment of NAN in rumen fluid and bacterial N, it was possible to estimate the contribution of feed N to NAN concentration in the rumen liquid phase (Fig. 3). The concentration of NAN derived from microbial turn-over and endogenous secretion is relatively constant within a feeding cycle. Conversely, NAN derived from feed degradation was affected by the diet (P < 0.001) and time of sampling (P < 0.001). One hour after the beginning of the meal this fraction accounted for 42, 72 and 53% of the soluble NAN for diets C, RL, and EL, respectively. For diet C and RL, this proportion fell to 20%, then to 15% at 4 and 8 h later, respectively. At these times, this contribution was slightly higher with diet EL, 30 and 25%, respectively. 3.6. Nutrient flow to the duodenum Parameters of OM digestion did not differ (P > 0.10) among the diets (Table 6). Dietary NAN flow to the duodenum was higher with diet EL (Table 7). Efficiency of microbial protein synthesis with the three diets was similar, when expressed as a percentage of OM apparently digested in the rumen (OMADR), but lower (P < 0.05) for diet EL when

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Fig. 1. Variations with time in ammonia: (a) and non-ammonia N; (b) concentration in the rumen fluid of sheep (n = 6) receiving urea; control (C), raw lupin (RL), or extruded lupin (EL) diet, at 9:00 h.

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Fig. 2. Composition of non-ammonia N in rumen fluid of sheep (n = 6) receiving urea control (C), raw lupin (RL), or extruded lupin (EL) diet, at 9:00 h. (a) Amino acid N and nucleic basis N content. (b) Partitioning of amino acid N between proteins, peptides, and free amino acids.

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Fig. 3. Variation with time in the origin of non-ammonia N (NAN) in rumen fluid of sheep (n = 6) receiving urea control (C), raw lupin (RL), or extruded lupin (EL) diet, at 9:00 h.

expressed as a percentage of the OM truly digested in the rumen. Lupin N that escaped ruminal degradation was 0.9 and 2.8 g of N/day for the raw and extruded seed, respectively. The in vivo ruminal degradability of lupin CP was therefore 92 and 74% for raw and extruded seeds, respectively. Although a small decrease in the apparent digestibility of NAN in the small intestine was observed between raw and extruded lupin (P < 0.10), the apparent total tract N digestibility was not affected. Table 6 Organic matter (OM) digestion as influenced by the protein supplement Dieta

S.E.M.b

C

RL

EL

OM intake (g/day)

904.1

912.3

904.7

4.69

OM apparently digested (g/day) Stomach Small intestine Large intestine

378.7 189.4 71.2

373.1 188.9 72.4

382.8 178.7 65.6

6.63 3.49 3.18

OM apparently digested, % of OM intake Stomach 41.9 Small intestine 21.0 Large intestine 7.9 Total tract 70.7

40.9 20.7 8.0 69.5

42.3 19.8 7.3 69.3

0.67 0.39 0.36 0.33

a b

C: control diet; RP: raw lupin diet; EP: extruded lupin diet. S.E.M.: standard error of the mean.

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Table 7 Nitrogen flow in the small intestine as influenced by the protein supplement Dieta C

S.E.M.b RL

EL

N intake (g/day)

21.2 b

23.2 a

23.2 a

0.30

NAN at duodenum (g/day) Microbial N (g/day) Feed Nc (g/day)

22.2 b 17.6 b 3.1 b

24.5 a 19.0 a 4.0 b

24.7 a 17.3 b 5.9 a

0.48 0.29 0.39

8.4 c

8.8 b

9.2 a

0.18

NAN apparently digested in the small intestine g/day % N intake % NAN duodenum

13.9 b 65.4 62.4 b

15.7 a 67.7 64.1 a

15.5 a 66.9 62.9 ab

0.34 1.13 0.39

Apparent total tract N digestibility (%)

69.6

70.9

70.4

0.43

291.9 197.0 a

319.6 203.3 a

284.1 189.5 b

8.98 3.71

NAN at ileum (g/day)

Microbial protein synthesis Gram of CP/kg of OMADRd Gram of CP/kg of OMTDRe

(a, b, c) Means within a raw not sharing a common subscript differ (P < 0.05). a C: control diet; RL: raw lupin diet; EL: extruded lupin diet. b S.E.M.: standard error of the mean. c Assuming an endogenous flow of nitrogen of 1.5 g/day. d OMADR: OM apparently digested in the rumen. e OMTDR: OM truly digested in the rumen (OMADR corrected for microbial OM).

Duodenal flow of AA followed the same pattern as NAN flow with lower values for the control diet and similar values for diets RL and EL (Table 8). Although dietary contribution to NAN duodenal flow increases between raw and extruded lupin, the duodenal AA profile was not affected. Similarly the net disappearance of AA from the small intestine, and the digestibility of individual AA in this compartment, were not affected by cooking extrusion (data not shown). 4. Discussion The effective ruminal degradability of raw lupin, at a kp of 0.06 h−1 , was identical to the value reported by Aufrère et al. (2001). The decrease in ruminal degradability with extrusion was consistent with previous data (Cros et al., 1991; Aufrère et al., 2001). By combining the data obtained in the present study, a good agreement was attained between lupin CP flow to the duodenum and its estimation by measurements at the ruminal level (i.e. particle associated + soluble CP outflow). As previously reported for pea seeds (Poncet and Rémond, 2002), soluble CP outflow from the rumen explained the largest part of the discrepancy observed between in situ and in vivo measurement of ruminal CP degradability. The present study showed that in the rumen, within a feeding cycle, soluble NAN derived from lupin was only significant during the first hours following the start of ingestion; the soluble NAN at this time entering as a supplement to a nearly constant pool of NAN

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Table 8 Duodenal flow (g/day) of amino acids (AA) as influenced by the protein supplement Dieta

Total flow Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Total AA EAA

S.E.M.b

C

RL

EL

15.8 b 8.3 b 6.8 b 17.0 b 5.7 b 7.8 11.1 8.0 3.8 1.3 b 8.0 11.7 b 5.9 b 7.1 b 10.1 3.1 b 6.6 b 137.9 b 64.1 b

17.4 a 9.1 a 7.7 a 19.3 a 6.4 a 8.6 11.8 8.8 3.9 1.4 a 8.8 13.3 a 6.7 a 8.0 a 10.8 3.4 a 6.4 a 153.3 a 71.3 a

18.3 a 9.2 a 8.1 a 20.3 a 6.8 a 8.7 11.7 9.2 3.2 1.4 ab 9.0 13.8 a 7.1 a 8.3 a 10.6 3.5 a 6.8 a 157.4 a 73.2 a

Bacterial flow Total AA EAA

96.3 44.5

Dietary + endogenous flow Total AA EAA

41.6 b 19.6 b

103.4 47.9 49.9 ab 23.4 ab

0.38 0.18 0.19 0.51 0.15 0.18 0.21 0.24 0.46 0.02 0.18 0.30 0.16 0.17 0.16 0.06 0.22 3.20 1.41

93.3 43.3

2.07 0.96

64.1 a 29.9 a

3.47 1.57

(a, b) Means within a raw not sharing a common subscript differ (P < 0.05). a C: control diet; RL: raw lupin diet; EL: extruded lupin diet. b S.E.M.: standard error of the mean.

supplied by bacterial and endogenous N. A preliminary study showed that soluble NAN accumulation in the rumen is affected by the balance between degradable N and fermentable energy of the diet (Poncet et al., 1998), making prediction of ruminal escape of this feed N fraction difficult. Indeed, in protein/energy unbalanced diets, Aufrère et al. (2001) observed a ruminal outflow of soluble NAN originating from raw and extruded lupin of 5.8 and 4.5 g of N/kg of lupin DM, respectively, whereas in the present study it was only 2.7 and 1.12 g of N/kg of DM, respectively. In the PDI system, the undegraded fraction of feed N is estimated to be 1.11 × (1 − ED). In the present work, comparison between in vivo data and ED, at a kp of 0.06 h−1 , showed that for raw lupin the coefficient of 1.11 is not high enough to explain the discrepancy between observed undegraded lupin N and its estimation by ED (i.e. it should have been 1.6), whereas it fits well for extruded lupin. As previously observed by Chen et al. (1987), soluble nucleic acids, which are rapidly degraded in the rumen liquid phase, gave a low contribution to the NAN pool in the rumen

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liquid phase. An important discrepancy was observed between AA + nucleic acid N and NAN. On a daily basis, it is estimated that the undetermined fraction accounted for about 50% of the NAN pool. Amino acid N accounted for 95 and 80% of the N in the lupin concentrates and the forage fraction, respectively. The dietary non-AA N could therefore have contributed to supply the NAN pool in the rumen. Furthermore, sialate, glucosamine and galactosamine of salivary mucoproteins may also contribute to the unidentified N in the ruminal NAN pool, mucus having a very slow rate of degradation in the rumen (Nugent et al., 1983). Part of the undetermined N could equally be explained by AA not accounted for in sample analysis. Within the amino acid N pool of the soluble NAN, protein N was the most important fraction, on average 80%. The highest protein–N concentration was recorded at the end of the meal with raw lupin. The presence of these proteins in the ruminal fluid, was previously shown by gel electrophoresis (Tai and Roy, 1997; Aufrère et al., 2001). At the end of the meal, the concentration in protein N observed with extruded lupin was lower than that observed with raw lupin. Similar observations were reported by Aufrère et al. (2001), probably related to the decrease in protein N solubility. Thereafter, protein N concentration remained stable and not affected by diet, suggesting that protein-N supply at this time mainly originated from other ingredients of the diet, or from endogenous and bacterial-N. The maximum peptide-N concentration in the rumen fluid was recorded 1 h after feeding. At this time, peptide-N concentrations were 40 and 60 mg of N/l for raw and extruded lupin, respectively. These values are in the range of those reported by Williams and Cockburn (1991; 20–160 mg of N/l) with dietary protein from varied origins. The increase in peptide concentration in the rumen with extrusion could probably be explained by establishment of chemical linkages between AA and other compounds of the seeds that made peptides resistant to microbial degradation. Benchaar et al. (1994) reported a 72% increase in dietary N flowing to the duodenum between raw and extruded lupin. In the present study, this increase was only 48%. Similarly, ruminal effective degradability decreased by 51% in Benchaar et al. (1994), whereas it decreased by only 22% in the present study. Differences in the extrusion process between studies probably explained this apparent discrepancy between the two studies. Consistent with Benchaar et al. (1994), lupin extrusion did not greatly impact efficiency of microbial protein synthesis, contrasting with previous data with pea seeds (Pisum sativum), for which an increase in microbial protein synthesis was observed with extrusion (Focant et al., 1991; Poncet and Rémond, 2002). Although dietary N contribution to duodenal NAN flow increased with extrusion, this difference in dietary AA profile did not affect the composition of the AA flowing to the duodenum. Small intestinal digestibility of rumen undegraded CP observed for raw lupin seed (80%) was in the range of reported post-rumen digestibility (63–94%; Cros et al., 1991; Goelema et al., 1998). In agreement with previous studies, heat treatment increased intestinal digestibility of rumen undegraded CP (Cros et al., 1991; Goelema et al., 1998). However, conversely to Benchaar et al. (1994), in the present study the apparent NAN digestibility in the small intestine observed in vivo slightly decreased. Because of the Maillard reaction with sugars, lysine and arginine availability generally decreases after heat treatment. However no differences in apparent intestinal digestibility of these two AA was detected between raw and extruded lupin.

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The PDIA value (dietary protein digested in the small intestine) of raw lupin, calculated from the in vivo ruminal degradability of lupin CP and the in situ intestinal digestibility of rumen undegraded lupin N, was 23 g/kg of DM. This value is higher than that in the PDI system (i.e. 13 g/kg of DM). Differences originate both from overestimation of the ruminal degradability of lupin CP by the in situ technique, and from underestimation of small intestinal digestibility of rumen undegraded lupin CP. Cooking extrusion raised the PDIA value of lupin seeds to 92 g/kg of DM. Cros et al. (1991) suggest that the increase in PDIA value would have been even higher with a higher extrusion temperature.

5. Implications The in vivo measurement of lupin CP degradability obtained in this study should help to correctly assess the nutritive value of lupin seed CP. This leguminous seed is currently penalised by feeding systems that use in situ measurements to predict rumen CP degradability, because a substantial fraction of solubilised CP escapes ruminal degradation. Because this escape depends on diet characteristics, quantitative prediction of this escape is difficult. Nevertheless, the nutritive value of lupins remains low when seeds are finely ground before pelleting, as generally achieved by feed manufacturers. In situ measurements and production studies suggest that this value is much higher for coarsely ground seeds. In vivo measurements should be undertaken to measure more precisely the effect of grinding on the ruminal escape of lupin CP. This study has confirmed that heat treatments, such as extrusion, can substantially increase the nutritive value of lupin seeds.

Acknowledgements This work was carried out in collaboration with the GIE EURETEC II (12 avenue George V, Paris) in the framework of EUREKA-EUROPROTEINS (EU623) project, with financial support from the French Research Ministry. The authors thank J.P. Chaise and E. Delval for technical assistance. References Aufrère, J., Graviou, D., Melcion, J.P., Demarquilly, C., 2001. Degradation in the rumen of lupin (Lupinus albus L.) and pea (Pisum sativum L.) seed proteins. Effect of heat treatment. Anim. Feed Sci. Technol. 92, 215–236. Benchaar, C., Moncoulon, R., Bayourthe, C., Vernay, M., 1994. Effects of a supply of raw or extruded white lupin seeds on protein digestion and amino acid absorption in dairy cows. J. Anim. Sci. 72, 492–501. Chen, G., Russel, J.B., Sniffen, C.J., 1987. A procedure for measuring peptides in rumen fluid and evidence that peptide uptake can be a rate-limiting step in ruminal protein degradation. J. Dairy Sci. 70, 1211–1219. Cros, P., Benchaar, C., Bayourthe, C., Vernay, M., Moncoulon, R., 1991. In situ evaluation of the ruminal and intestinal degradability of extruded whole lupin seed nitrogen. Reprod. Nutr. Dev. 31, 575–583. Emile, J.C., Huyghe, C., Huguet, L., 1991. Utilisation du lupin blanc doux pour l’alimentation des ruminants: résultats et perspectives. Ann. Zootech. 40, 31–44. Faichney, G.J., 1980. The use of markers to measure digesta flow from the stomach of sheep fed once daily. J. Agric. Sci. Camb. 94, 313–318.

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