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* Unité Mixte de Recherches sur le Veau et le Porc, Institut National de la Recherche Agronomique, Ecole Nationale Supérieure Agronomique de Rennes, 65 rue de Saint Brieuc, CS 84215, 35042 Rennes Cedex, France
Station de Recherches Avicoles, Institut National de la Recherche Agronomique Nouzilly, 37380 Monnaie, France
Corresponding author:
L. Montagne; e-mail:
montagne{at}roazhon.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Abbreviation key: HSPI = partially hydrolyzed soybean protein isolate, Mr = molecular mass, PPC = potato protein concentrate, SMP = skim milk powder, SPC = soybean protein concentrate
Key Words: calf nutrition • protein digestion • protein and peptide fractions
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Improving digestive utilization of plant protein in diets for veal calves helps to improve knowledge of the biochemical process of the digestion in the small intestine. There are, to our knowledge, no data for calves comparable to those for pigs and chickens. The aims of this study were, therefore, to examine the progress of digestion and absorption along the small intestine of veal calves by measuring the Mr profiles of proteins and peptides in digesta. Calves were fed diets based on SMP and differing by their CP content in experiment 1, or diets differing in the protein source (SMP, unhydrolyzed or partially hydrolyzed soybean and potato) in experiment 2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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. Briefly, in experiment 1, a diet with extremely low level of protein (14 g CP/kg DM, called protein-free diet in the rest of the text) and three diets based on SMP containing 104, 205 and 279 g CP/kg DM were formulated. In experiment 2, four experimental diets differing in the source of protein were formulated, with the control diet based on SMP. The soybean protein concentrate (SPC, Danproveal, Central Soya, Aarhus, Denmark) had been treated with hot aqueous ethanol in order to remove sucrose and 
-galactosides and to reduce antigenicity. The partially hydrolyzed soybean protein isolate (HSPI, Nurish 1500 from Protein Technologies International, St. Louis, MO) was also hypoantigenic. The potato protein concentrate (PPC, Lysamine, Roquette, Lestrem, France) was obtained by thermocoagulation. The three plant protein-containing diets contained a mixture of SMP and plant products (1:1, on a digestible CP basis), except the SPC diet for which this ratio was lower (2:3) due to an error in SPC mixing. As previously reported (Montagne et al., 2000a), all these products were virtually devoid of antinutritional factors and proved to be well digested by calves (Lallès et al., 1996). All the eight diets were balanced for digestible energy (5 Mcal/kg DM) using lactose. Different mixes that took into account the contribution of the protein sources for vitamins and minerals were formulated in order to have a constant level of vitamins and minerals in all the diets. The level of feed intake was fixed at 55 g DM/kg of BW0.75/d), which is close to the level (57 g DM/kg of BW0.75/d) that would allow a daily BW gain superior to 1000 g in (nonfistulated) veal calves (Lallès et al., 1995). Individual amounts of feed intake were adjusted weekly to animal BW. Chromium (Cr)-EDTA was used as a liquid phase marker and added to the reconstituted milk (Lallès and Poncet, 1990). Sodium citrate was introduced into the diets in order to prevent clotting in the abomasum, as an attempt to get a regular abomasal emptying for protein and fat, comparable between the diets based on SMP only and those containing plant protein sources, that do not coagulate in the abomasum of the calf (Toullec et al., 1974). Clotting prevention is considered only to affect nutrient flow and not nutrient digestibility or performance in calves aged one month or more (Longenbach and Heinrichs, 1998), as those used in the present study.
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Animals
The experiments were conducted under the guidelines of the French Ministry of Agriculture for animal research. Briefly, Holstein male calves were obtained at 2 d of age from the experimental dairy herd of INRA Rennes, France. They were kept on straw in individual pens (1.5 x 2 m) throughout the experiment. At 65 to 75 kg of BW (approximately 6 wk of age), the calves were fitted with a catheter placed at the limit between the fundic and antral regions of the abomasum, (20 to 30 cm from the pylorus), and T-piece cannulae in the proximal duodenum (immediately below the pancreatic duct), midjejunum, and terminal ileum (Montagne et al., 2000a). The calves were then allowed to recover from surgery for 2 wk. After the end of the experiments, the calves were euthanized and the actual location of the cannulae was measured. They were positioned on the duodenum and the ileum at 3 ± 0.2 and 99 ± 0.1% of the length of the small intestine, respectively (means ± SEM for all the calves of the two experiments). The jejunal cannula was positioned at 40 ± 15 and 54 ± 4%, in experiments 1 and 2, respectively.
Experimental Design
Experiment 1 was carried out on six calves that received each of the four diets (the protein free diet and the three diets based on different levels of SMP) at random. Each diet was tested during an experimental period lasting 9 d. During the experimental periods, the milk was infused continuously through the abomasal catheter, using a peristaltic pump (Masterflex, Cole-Parmer Instrument Co., Vernon Hills, IL), in order to reach a steady state for digestion process and digesta flow along the small intestine. The infusion rate comprised between 6 and 10 g/min, depending on the BW of the calves. In a previous study in which the SMP-based milk replacer was continuously infused in the duodenum to reach such a steady state, we found satisfactory fecal digestibility data of 0.95 and 0.91 for organic matter and N, respectively, with calves aged 5 to 6 wk (Lallès and Poncet, 1990). Between two experimental periods (5 d), the calves were bucket fed a commercial diet.
Experiment 2 was carried out on 10 calves, different than those used in experiment 1. The design of experiment 2 was similar to that of experiment 1. However, abomasal infusion of HSPI, and to a lesser extent SPC, generated successive episodes of constipation and diarrhea, thus preventing the calves from completing the experimental design. Therefore, SPC, HSPI, and PPC diets were tested separately on 5, 3, and 8 different calves, respectively, that also received the control diet randomly. Therefore experiment 2 was analyzed as three independent trials, each involving the comparison of data between a diet containing a plant protein source and the respective control diet based on SMP only.
Ileal, jejunal and duodenal digesta were collected on d 6, 7, and 8, respectively, after the beginning of diet infusion. Approximately 150 g of digesta, were sampled five times a day at 0800, 1200, 1600, 2000, and 2400 h at each anatomical site, representing altogether approximately 6.5% of milk replacer intake. A mixture of preservatives [15 mM EDTA, 1 mM sodium azide, 2 mM phenylmethylsulfonyl fluoride] was then added to the digesta in order to limit enzymatic and bacterial activities. The samples were vigorously shaken and frozen at -20°C prior to freeze-drying. Afterwards, representative average samples were prepared per experimental diet and intestinal site for this fractionation study. Aliquots of these representative samples at the duodenum were also filtered (mesh size 500 µm) in order to estimate the contribution of straw particles. This was found to be negligible (<2% DM), suggesting straw consumption had been limited in these experiments with veal calves.
Chemical Analysis
The diet and digesta samples were analyzed as previously described (Guilloteau et al., 1986; Lallès and Poncet, 1990). Briefly, DM and ash were determined by drying in an oven at 105°C for 16 h and 600°C, respectively. Chromium was extracted from ash residue and measured by atomic absorption spectrometry (Perkin-Elmer 2380 spectrometer). Nitrogen was measured using the Reardon method (Reardon et al., 1966) and data expressed on a CP basis using 6.25 as conversion factor.
Determination of Protein/Peptide Molecular Mass Profiles in Diets and Digesta
Sample preparation.
Freeze-dried samples of feed and digesta were homogenized (60 mg/ml) in 40 mM phosphate buffer, pH 7, containing 1% SDS and 0.2 M 2-mercapto-ethanol. Total CP were then extracted by heating at 100°C, for 20 min. After cooling, extracts were centrifuged (15 min, 24000 x g) and supernatants were filtered (Minisart Sartorius Filter 0.20 µm, Goettingen, Germany). The filtrates were then frozen at -20°C until chromatography fractionation.
Chromatography fractionation.
Separation of proteins and peptides according to Mr was carried out by gel filtration on a TSK PW precolumn and a TSK PW XL G3000 column measuring 30 cm length and 7.8 mm internal diameter (Tosa Hass, Montgomeryville, PA), as described previously (Crévieu et al., 1997b). Each filtrate (200 µl representing approximately 800 µg of CP) was applied on the top of the column and eluted at a rate of 0.6 ml/min with 40 mM phosphate buffer, pH 7, containing 0.1% SDS. The void volume was determined with thyroglobulin (Mr = 330,000). The column was calibrated using BSA (Mr = 66,000), carbonic anhydrase (Mr = 29,000), cytochrom C (Mr = 12,400), insulin (Mr = 5750), and glucose (Mr = 180) as Mr standards. All were purchased from Sigma (St Louis, MO). Six fractions were collected. Fraction 1 corresponded to proteins with a Mr > 20,000; fraction 2 to proteins with 20,000 
Mr > 10,000; fraction 3 to proteins or peptides with 10,000 Mr > 4500; fraction 4 to peptides with 4500 Mr > 2000; fraction 5 to peptides with 2000 Mr > 400; and fraction 6 to oligopeptides and free AA with Mr 
400.
Analysis of protein and peptide fraction.
A 150-µl aliquot of each fraction was hydrolyzed with 50 µl of 8 M NaOH for 2 h at 130°C. Samples were then neutralized with 100 µl of 40% acetic acid. Next, -amino nitrogen was estimated by reaction with o-phtaldialdehyde according to the method of Frister et al. (1988). A standard curve was prepared with graded concentrations of leucine (0 to 1 mM) and the results were expressed as mM of leucine equivalents according to Crévieu et al. (1997b).
Expression of Results
The concentration of -amino nitrogen in each fraction from a sample was multiplied by the volume of this fraction. Then, the percentage of CP (% CP Fi) in each fraction i was calculated as follows:
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where -amino N Fi was the content of -amino nitrogen in the fraction i in mM equivalent leucine.
The flow of CP from each fraction in digesta was calculated using the following equations. First, the flow of digesta was calculated individually from the dilution factor of the indigestible marker as previously described (Montagne et al., 2001):
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where the flow of digesta is in kg/d, Cr intake = the amount of Cr intake in g/d and Cr digesta = the Cr concentration in digesta in g/kg. The flow of DM and total CP was next calculated as follows:
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where the flow of DM and CP are in g/d, DMdigesta = concentration of DM in digesta in g/kg, flow of digesta is in kg/d, and CPdigesta = concentration of CP in digesta in g/g DM.
The flow of CP was also calculated per kg DMI per d for comparison with literature data. Then, the flow of CP in each fraction (flow of CP in Fi in g/kg DMI/d) was calculated by multiplying the flow of CP (in g/kg DMI/d) by the percentage of CP in each fraction (% CP Fi) as follows:
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Statistical Analysis
As described previously (Montagne et al., 2001) data of flow from experiment 1 and individual trials of experiment 2 were analyzed as repeated measures for each calf-diet combination, at each intestinal site, using the Repeated statement of the general linear model procedure of SAS (SAS, 1990). Significant intestinal site effects were partitioned into single degrees of freedom orthogonal comparisons, i.e., into linear and quadratic effects of the distance of the cannula from the duodenum. When the effect of diet was significant, differences between means were separated by Bonferroni’s test. Differences were declared significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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. In experiment 1, 55 to 60% of total CP of diets containing SMP had Mr > 20,000 whereas less than 6% had Mr 400. The protein-free diet contained only 14 g CP/kg DM because protein supplement was not included in the formula and so proteins came only from the milk cream as the source of fat. This diet contained less protein with Mr > 20,000 (45.5%) and more oligopeptides and free AA with Mr 400 (18.4%) than the diets containing SMP.
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400. Distribution of CP in the HSPI diet differed from the other diets containing plant protein sources. It contained less proteins with Mr > 20,000 (48%) due to partial hydrolysis and, consequently, more proteins and peptides with Mr < 20,000. The percentage of CP in fractions 2 to 6 was 46 to 59% higher with HSPI than with the other diets used in experiment 2.
Distribution of Proteins and Peptides in Digesta
Experiment 1.
Figure 1 illustrates the differences in Mr distribution of digesta CP between intestinal sites and according diets, in calves receiving diets varying in CP level. With the diets containing 14, 105, and 279 g CP/kg DM, fraction 1 (Mr > 20,000) at the jejunum represented a percentage higher, or similar to that at the duodenum. Conversely fractions 5 and 6 (Mr 2000) at the jejunum were inferior than or similar to that at the duodenum. By contrast, with the diet containing 205 g CP/kg DM, the contribution of proteins with Mr > 20,000 was lower at the jejunum than at the duodenum, and that of fraction with Mr 2000 was higher. Between the jejunum and ileum, the contribution of the fractions with Mr > 20,000 increased, and that with Mr 2,000 decreased, regardless of the dietary CP level.
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. The flow of total CP and CP in each fraction decreased linearly along the small intestine (P < 0.0001). With the protein-free diet, the total flow of CP and the flow of CP in fractions with Mr > 400 were quite similar at the duodenum and jejunum. The flow of total CP decreased between the jejunum and the ileum by approximately 40%. This was explained mainly by the decrease in the flow of CP from fractions 2 to 6 (Mr 20,000).
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Experiment 2.
Figure 2 illustrates the differences in Mr distribution of digesta CP between sites and diets in calves receiving diets differing in the protein source. The main observation was that the percentage of protein with a high Mr (fraction 1) decreased between the duodenum and jejunum and then increased between the jejunum and ileum, regardless of the diet. Conversely, the proportion of peptides, oligopeptides and free AA (fractions 5 and 6) increased between the duodenum and the jejunum and decreased between the jejunum and the ileum.
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. As observed in experiment 1, the flow of total CP and CP in each fraction decreased along the small intestine (P < 0.0001). With the control diet, the total flow of CP was, on average for the three trials, 228, 67, and 19 g/kg DMI/d, at the duodenum, jejunum and ileum, respectively. The flow of CP in fractions 1 to 6 was on average at the duodenum 79, 39, 28, 22, 36, and 23 g/kg DMI/d, respectively, and at the jejunum 13, 5.3, 6.1, 8.2, 19.7, and 14.1 g/kg DMI/d, respectively. At the ileum, it was on average 8.0, 2.5, 1.8, 1.7, 5.3, and 15.5 g/kg DMI/d.
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Mr > 4500) decreased when SMP was partially replaced by plant protein (P < 0.05 for fraction 2 with HSPI and PPC diets and for fraction 3 with SPC and PPC diets). By contrast, the flow of peptides, oligopeptides and free AA (Mr < 2000) increased when plant protein was incorporated into the milk replacers (P < 0.06 for fraction 5 with the SPC and HSPI diets, and for fraction 6 with the HSPI and PPC diets). The flow of total CP at the jejunum tended to be higher with plant proteins, as compared with the control diet (+66% and +40% with the SPC and PPC diets, respectively, P < 0.08). This was, or tended to, hold true for the flow of each fraction.
The flow of total CP in ileal digesta was nearly doubled with diet SPC (P = 0.07), three-fold higher with HSPI (P = 0.06), and two-fold higher with PPC (P < 0.001) compared with the control diet. The flow of CP significantly increased in most fractions when SMP was partially replaced by plant protein, but the magnitude of this increase differed between fractions. Especially, the flow of CP in fractions of Mr < 2000 increased more than in fraction of higher Mr (fractions 1 and 2). This lead to an accumulation of peptides, oligopeptides, and free AA in ileal digesta of calves fed with plant proteins compared with SMP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Effect of Dietary CP Level on Digestion of CP Along the Small Intestine
Digesta from calves receiving the protein-free diet contained protein from endogenous origin only. The flow of proteins and peptides with Mr 400 was globally similar at the duodenum and jejunum whereas it decreased between the jejunum and ileum. That of oligopeptides and free AA decreased along the small intestine.
At the duodenum, fraction Mr > 20,000 probably contained endogenous proteins from digestive secretions, including enzymes and mucus, and proteins sloughed-off in the lumen from the lining of anterior segments of the gut, as suggested by Montagne et al. (2000) and Montagne et al. (2002). Endogenous proteins flowing at the duodenum are hydrolyzed and absorbed proximo-distally by the same mechanisms as dietary proteins. In the present experiment, the similar flow of CP with Mr > 20,000 observed at the duodenum and jejunum could be explained by two phenomena. First, hydrolysis and absorption could be counterbalanced by secretion of other endogenous proteins, and second, proteins may be poorly hydrolyzed. Mucin especially has a high Mr (Mr > 10 6) and a high degree of glycosylation that protects it from proteolysis. As an example, exhaustive in vitro proteolysis of mucin from the small intestine of pig resulted in a loss of 5% of the total mucin molecule only (Mantle et al., 1981).
At the ileum, the contribution of protein with Mr > 20,000 was again high. It has recently been shown that mucin is a major contribution of ileal flow of CP (20%; Montagne et al., 2000a). As mucin is a high Mr glycoprotein, we suggest that it is the major component of fraction 1 with Mr > 20,000. The remainder was probably proteins from the mucosa and from bacteria that colonize the terminal ileum.
When the dietary CP level increased, the flow of CP in all fractions increased at the duodenum and the jejunum. This could be easily explained by the presence of milk proteins in digesta. They have been shown to be mostly intact at the duodenum of calves fed diets containing SMP (Caugant et al., 1992), and so probably partially degraded at the jejunum. Our results confirmed that the digestion of proteins from SMP was not completed at the jejunum (Montagne et al., 2000b). At the ileum, the flow of CP with Mr < 400 significantly increased with the dietary CP level. Oligopeptides and free AA represented on average 9% of total CP flowing at the ileum of calves fed the diet based on SMP. Accumulation of oligopeptides and free AA in ileal digesta was more pronounced in calves fed the diets based on SMP than the protein-free diet. Such an accumulation was also observed in other animal species. In pigs fed an enzymatically hydrolyzed casein, CP with Mr < 500 represented 11 to 21% of the CP in ileal digesta (Moughan et al., 1990; Butts et al., 1992) whereas in chickens fed intact casein, this fraction accounted for 29% of total CP (Sklan and Hurwitz, 1980).
Effect of Dietary Plant Protein Source on Digestion of CP along the Small Intestine
The incorporation of plant protein into milk replacers for calves had no effect on the flow of total CP in duodenal digesta but it increased this flow at the jejunum and ileum. Moreover, Mr distribution of CP was modified. The main effect was the accumulation of oligopeptides and free AA in ileal digesta. This was also observed in rats given severely heated protein (Zebrowska, 1968), and in chickens fed a diet containing pea protein (Crévieu et al., 1997a). A possible explanation is that the accumulation of undigested peptides saturated the absorption sites involved in the transport of AA, di- and tri-peptides across the mucosal barrier, and so blocked AA uptake, as first hypothesized by Zebrowska (1968) from rat studies. Absorption of AA, di- and tri-peptides might also be a limiting factor of plant protein digestion in the calf. This may explain why calves fed diets containing plant protein grow less than those fed SMP diets. In addition, it has been shown recently that protein catabolism in the muscle is higher when plant protein, as compared to SMP, is fed to pigs (Löhrke et al., 2001).
The origin of peptides accumulating in ileal digesta could be dietary or endogenous. Resistant dietary fractions have been evidenced in ileal digesta of calves fed soybean or potato proteins (Lallès et al., 1999; Branco-Pardal et al., 1995), pigs fed kidney bean (Begbie and Ross, 1993) and chickens fed pea protein (Crévieu et al., 1997a). Dietary peptides might interact with the gut, and be responsible for the increase of endogenous secretion observed with plant protein (Montagne et al., 2001). This hypothesis is supported by the observation of Santoro et al. (1999) in rat fed pure phaseolin, the storage globulin of kidney bean Phaseolus vulgaris. Native phaseolin and/or related fragments that escaped digestion bound to the small intestinal epithelium. It was suggested that they could act as natural secretagogues stimulating secretion of endogenous protein, and especially mucin, from the mucosa (Santoro et al., 1999). The increase in the flow of proteins with Mr > 20,000 in ileal digesta of calves fed diets containing plant protein, when compared with diets containing SMP, is in agreement with this hypothesis.
To conclude, partial replacement of SMP by plant protein sources lead to an accumulation of oligopeptides and free AA in ileal digesta of calves. Thus, the intestinal breakdown of oligopeptides and absorption may be a limiting step in the digestion process.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Received for publication July 12, 2002. Accepted for publication September 12, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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