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a Finca Experimental La Poveda, CCMA, CSIC. Ctra. de Campo Real km 1.300, 28500 Arganda del Rey, Madrid, Spain
b Dep. de Producción Animal. Fac. De Veterinaria, Univ. Complutense, Ciudad Universitaria, 28040 Madrid, Spain
* Corresponding author (rcaballero{at}iai.csic.es)
Received for publication September 22, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATION SUMMARYREFERENCES |
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Abbreviations: ADC, acid-detergent cellulose • ADF, acid-detergent fiber • ADIP, acid-detergent insoluble protein • ADL, acid-detergent lignin • CHO, carbohydrates • CNCPS, Cornell Net Carbohydrate and Protein System • CP, crude protein • DM, dry matter • EAA, essential amino acids • NDF, neutral-detergent fiber • NDIP, neutral-detergent insoluble protein • NEAA, nonessential amino acids • NFC, nonfiber carbohydrates • SC, structural carbohydrates • SEAA, semiessential amino acids • TC, total carbohydrates
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATION SUMMARYREFERENCES |
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The application of the Cornell Net Carbohydrate and Protein System (CNCPS) for prediction of metabolizable energy and protein requires accurate estimates of feed composition carbohydrate and protein fractions and additional inputs. The metabolizable energy and metabolizable protein derived from forages are highly sensitive to variations in forage content of NDF, lignin, CP, and protein solubility (Fox et al., 1995).
The CNCPS has recently received considerable interest by the scientific community, but only a few temperate grasses and legumes such as tall fescue (Festuca arundinacea Schreb), bromegrass (Bromus biebersteinii Roem and Schult.), and alfalfa (Medicago sativa L.) have been tested with this nutrition model, and mostly for CP fractions (Agbossamey et al., 1998; Elizalde et al., 1999). Reports of both the carbohydrate and protein fractions are less common (Masoero et al., 1999), and analytical parameters based on the CNCPS for annual forage legumes have not been reported.
Unlike alfalfa, some authors (Hintz and Albrecht, 1994; Caballero et al., 1996) recommend harvesting annual legumes, such as soybean [Glycine max (L.) Merr.] and common vetch, well beyond bloom when the seed fraction accounts for a higher proportion of the harvested biomass. A maturity factor was thus included to test the CNCPS in comparing fresh and field-cured common vetch forage. The objective of this research was to assess the impact of maturity and field curing on vetch carbohydrate and protein fractions used in the CNCPS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATION SUMMARYREFERENCES |
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Within harvest forms, vetch plants were harvested at three stages: flowering (more than 50% of plants with flowers), and two seed filling stages defined by progressively higher dry matter concentration in the seed (mean of 280 and 380 g DM kg-1 for seed-filling phase 1 and 2, respectively). More advanced stages of growth were discarded as vetch reached full pod growth before 500 g DM kg-1 seed, and losses of quality and biomass of vegetative plant parts occurred thereafter (Caballero et al., 1996). Maturity and harvest form treatments were allocated to single plots measuring 40 m by 20 m in a completely randomized design with three replications.
Plots assigned to the flowering stage were harvested with a rotary mower on 28 May 1997 and 8 May 1998, respectively. Twenty individual plants were selected randomly within single plots and separated into leaves and stems. Mean leaf/stem ratios at this stage, on a DM basis, were 1.64 and 1.23 in 1997 and 1998, respectively. Dry matter content of fresh forage (yearly mean of 173 g DM kg-1) and plant parts were determined by oven-drying at 60°C for 22 h and subsequently at 80°C for 2 h.
Plots assigned to seed filling phase 1 were harvested on 12 June 1997 and 20 May 1998 and plots assigned to seed filling phase 2 were harvested on 21 June 1997 and 4 June 1998, respectively. In these cases, 30 randomly selected plants were separated into leaves, stems, and full pods. Whole fresh forage and plant parts were oven-dried in a similar way. For seed filling phase 1 and seed filling phase 2, mean yearly DM contents of fresh forage were 267 and 298 g DM kg-1, respectively. Leaf, stem, and pod proportions (on a DM basis) were, respectively, 430, 420, and 150 g kg-1 in 1997 and 310, 350, and 340 g kg-1 in 1998 at seed filling phase 1, and 310, 380, and 310 g kg-1 in 1997 and 210, 190, and 600 g kg-1 in 1998 at seed filling phase 2.
Samples of field-cured vetch were obtained from the swathes and just before pressing. A tined-circle rake was used on field-cured plots. The drying periods lasted <5 d. Yearly mean maximum air temperature during the three field curing periods (flowering, seed filling 1, and seed filling 2) were 24.5, 28.6, and 30.1°C, respectively. Corresponding mean minimum air temperatures were 10.2, 13.7, and 12.8°C, respectively. No rain fell during field curing. Morphological composition of field-cured samples was similar to that of fresh samples as field-cured forage was collected from the swathes, which avoided pod-shattering that can be produced by baling. Dry matter content of field-cured samples at harvest was between 790 and 820 g kg-1.
Approximately 3 kg of fresh and field-cured vetch were randomly collected from each plot for chemical analysis. Fresh forage samples were put into freeze-bags, frozen at -20°C, and then freeze dried. Samples of field-cured vetch were oven dried at 60°C for 22 h and subsequently at 80°C for 2 h. Both type of samples were milled to pass a 1-mm screen.
Fractionation of Carbohydrates and Chemical Analyses
Fractionation of CHO was performed according to the CNCPS (Sniffen et al., 1992). Total carbohydrates (TC) were calculated by subtracting from 1000 the CP, crude fat, and ash contents, and expressed as g kg-1 DM. Structural carbohydrates (SC) and nonfiber carbohydrates (NFC) represented, respectively, the carbohydrates that are insoluble and soluble in neutral detergent. Carbohydrate fraction A includes soluble sugars and represents the CHO fraction that is degraded rapidly in the rumen. Carbohydrate fraction B1 has an intermediate rate of degradation and represents mainly starch and nonstarch polysaccharides soluble in neutral-detergent (pectic substances, beta-glucans, galactans, gums) and has an intermediate rate of degradation. Carbohydrate fraction B2 is available cell wall and its degradation rate is slow. Carbohydrate fraction C represents unavailable cell wall and is undegradable and undigestible. These CHO fractions were expressed as g kg-1 of TC and calculated as follows:
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Crude protein, crude fat, ash, and DM were determined by standard methods (AOAC Int., 1995). Conventional fractionation of structural carbohydrates and lignin was carried out by the detergent system. Neutral-detergent fiber was analyzed following Van Soest et al. (1991) using a heat-stable 
-amylase (Termamyl 120, Novo, Denmark), and acid-detergent fiber (ADF), acid-detergent cellulose (ADC), and acid-detergent lignin (ADL) were determined by the methods of Goering and Van Soest (1970) as modified by Robertson and Van Soest (1981). Soluble sugars were obtained by extraction with aqueous ethanol (80%, v/v) and measured colorimetrically by the anthrone method (Yemm and Willis, 1954). Starch was quantified from the amount of glucose released after its gelatinization and enzymatic hydrolysis by amyloglucosidase (EC 3.2.1.3.; Boehringer no. 102857) as described by Longstaff and McNab (1991).
Fractionation of Crude Protein and Chemical Analyses
Fractionation of CP was carried out by the Cornell Net Carbohydrate and Protein System (Sniffen et al., 1992). According to this system, CP is partitioned into three fractions: CP fraction A is nonprotein nitrogen (NPN) x 6.25; CP fraction B is true protein and CP fraction C is unavailable protein. Crude protein fraction B is further divided into three subfractions B1, B2, and B3 of rapid, intermediate, and slow rates of ruminal degradation, respectively. Crude protein fractions A and B1 are soluble in borate-phosphate buffer, CP fraction B2 is insoluble in the buffer but soluble in neutral detergent solution, and CP fraction B3 is insoluble in the buffer and in neutral detergent, but soluble in acid detergent. Crude protein fraction C is the protein that is insoluble in acid detergent (acid detergent insoluble protein, ADIP); it contains protein associated with lignin, tannin–protein complexes, and Maillard products that are highly resistant to microbial and mammalian enzymes.
Precipitated true protein, buffer insoluble protein, neutral-detergent insoluble protein (NDIP), and ADIP were analyzed as described by Licitra et al. (1996). Crude protein fraction A was calculated as the difference between the total crude protein and precipitated true protein. True protein was determined by Kjeldahl analysis of the residue resulting after precipitation with tungstic acid followed by filtration. Crude protein fraction B1 was estimated as true protein minus buffer insoluble protein, CP fraction B2 as buffer insoluble protein minus NDIP, and CP fraction B3 by subtracting the ADIP (fraction C) from the NDIP.
Amino Acids
Amino acid analysis was done on the 1996 to 1997 samples by OPA (o-phtaldialdehyde) precolumn derivatization following hydrolysis in 6 M HCl. Amino acids were separated on a HPLC system (Hewlett-Packard 5890, Waldronn, Germany) according to the method of Jones et al. (1981). Cystine was determined as cysteic acid (Moore, 1963).
Statistical Analysis
Variables determined were the cell-wall components (NDF, ADF, ADC, and ADL); starch; soluble sugars; crude fat; ash; TC, SC, and NFC; the concentrations of the four CHO fractions (on TC basis); CP concentration; and the concentrations of the five CP fractions (on total CP basis). A two-factorial model of fixed effects including interaction was used for statistical analysis:
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= the random error present in the ith on the jth; i = 1,2,3; and j = 1,2). The main effects were maturity (A) and harvest form (B). The mean squares of the main effects and those of the interaction were compared with that of the residual. Analyses of variance were done by year as differences between years were found, as tested by year x treatment interactions and heterogeneity of error. The 2V program of the BMDP statistical package was used (BMDP, 1992). |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATION SUMMARYREFERENCES |
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The results in Table 2 illustrate that SC including lignin, and NFC are evenly distributed in common vetch DM, structural carbohydrate being greater than NFC in the first season and lower in the second. This can be explained by the higher proportion of grain in the second growing season with corresponding higher starch content in the DM. The TC increased significantly (P < 0.001) from flowering to the seed filling 2 across seasons and harvest forms, but TC remained unchanged in the first growing season and decreased significantly from fresh to field-cured forage in the second.
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These results illustrate the combined effects of morphological changes and photosynthetic activity during seed filling. The general trend toward higher SC as maturity progressed was partially compensated for by higher proportion of seed in DM and starch accumulation on the seed fraction (Caballero et al., 1998). At seed filling phase 2, this compensatory effect was more apparent in the second (full pod proportion of 600 g kg-1 DM) than in the first (full pod proportion of 310 g kg-1 DM) growing season.
The biological significance of the conventional TC fractionation is not clear. Although the NFC fraction may represent the digestible fraction of TC, the rate of ruminal degradation of two of its main components (starch and total soluble sugars) is very different. Simple soluble sugars are rapidly fermented within the rumen but starch must first be degraded to simple sugars before it can be fermented (Nocek and Tamminga, 1991). In a protein-rich forage legume such as common vetch, the proportion of rapidly fermented soluble sugars relative to the CP content can affect the efficiency of microbial protein synthesis and forage CP utilization, as legumes presented a much lower soluble sugars/CP ratio than grasses (Givens, 1993).
The large difference found between NFC and sugars plus starch suggests a large soluble fiber fraction (nonstarch polysaccharides soluble in neutral detergent) in common vetch (Tables 1 and 2). With our fractionation system, organic acids were not discounted, but legume forage tend to be high in pectic substances (Van Soest, 1994). Our results showed that common vetch as fresh or field-cured forage contained a soluble fiber fraction higher than the starch or soluble sugars and unrelated to the maturity stage (means of 178 and 172 g kg-1 DM for fresh and field-cured samples across maturity and years, respectively). Pectic substances are rapidly and completely degraded within the rumen (White et al., 1993), but the rate of fermentation and utilization by rumen microorganisms of their degradation products cannot compare with soluble sugars (Dehority, 1993).
The CNCPS system stresses the availability of NFC fractions as estimated by the rate of fermentation. In forages, none of the soluble sugars escapes ruminal degradation (Sniffen et al., 1992). The rate of fermentation of CHO fraction B1 may reach 25 to 40% h-1, but two of its components (starch and pectic substances) may differ in functionality. The rate of digestion of legumes starch is lower than that of cereal grains and, while pectic substances are completely degraded in the rumen, some starch may escape ruminal fermentation (Nocek and Tamminga, 1991). Although pectic substances can make a high contribution to CHO fraction B1, it is questionable whether these components can act as substitutes of soluble sugars in their ability to provide a readily available source of energy to the rumen microorganisms. Many cellulolytic bacteria have pectinolytic activities but cannot utilize the resulting oligogalacturonides and galacturonic acid to support their own growth (Dehority, 1993). Our results showed that at seed filling phase 2, in the second growing season, starch concentration reached a mean of 151 g DM kg-1 across harvest forms (Table 1), but the concentration of CHO fraction B1 was 340 g DM kg-1 (Table 2). These results showed that even at the higher starch concentration, the common vetch plant may contain a substantial concentration of pectic substances. The diversity, amounts, and differences in functionality of the CHO fraction B1 stress the requirement of further investigation in vetch.
The CHO fraction A decreased (P < 0.001) with advancing maturity stages during both seasons. From flowering to the seed filling phase 2, CHO fraction A decreased by 31% in fresh forage and by 34% in field-cured forage in the first growing season. Corresponding values in the second growing season were 37 and 65%, respectively (Table 2). Caballero et al. (1998) reported decreasing values of soluble sugars as maturity advanced, within the seed filling phase, that were mostly attributable to the decreasing contents of the seed and stem plant parts.
The relative proportion of CHO fractions A and B1 in fresh and field-cured vetch were related, as CHO fraction B1 is derived from NFC by difference of CHO fraction A. However, this relation varied with maturity. At flowering and at seed filling phase 1, the proportion of CHO fraction A was higher for field-cured forage compared with fresh forage while the proportion of CHO fraction B1 decreased accordingly. At seed filling phase 2, however, the relative proportions of CHO fractions A and B1 may depend on the proportion of starch-rich seed on DM. The CHO fraction B1 was higher in the second than in the first growing season due to the effect of higher seed proportion in the former (Table 2). Higher starch concentration did not relate with proportional decrease of soluble sugars (CHO fraction A), because most starch is derived from photosynthesis and not redistributed from soluble sugars (Caballero et al., 1998).
The proportion of CHO fraction B2 on TC was not affected by drying in the first growing season, but it had a significant effect in the second (P < 0.01). As the proportion of plant parts did not vary between fresh and field-cured samples, this is probably a redistribution effect of other TC components, mainly starch. At flowering and at seed filling phase 1, CHO fraction B1 decreased significantly from fresh to field-cured forage and, accordingly, CHO fraction B2 increased. At seed filling phase 2, the differences between fresh and field-cured samples were not significant, (Table 2). During this latter phase, differences in SC content are more attributable to redistribution of components and less to accumulation (Caballero et al., 1998).
The undegraded and undigestible fraction of TC (CHO fraction C) represented 211 and 190 g kg-1 of TC in the first and second growing season, respectively, across maturity and harvest form (Table 2). The CNCPS model used this value for predicting the indigestible NDF using a linear equation. Traxler et al. (1998) have reported that this equation under-predicted daily gain in cattle (Bos taurus) because it overestimated the indigestible NDF fraction. Validation tests in sheep are much needed (Cannas, 2000).
Crude Protein Fractions
The CP concentration of fresh and field cured common vetch forage did not vary in the first growing season and differed (P < 0.001) in the second, being higher in the field-cured forage (Table 1). This can be the result of lower NFC in the field-cured samples of the second growing season and correspondingly higher relative proportion of other chemical components (CP and SC). In both seasons, the maturity effect was significant (P < 0.01) and the interaction effect (harvesting stage x harvest form) was not significant. Across maturities and seasons, the CP concentration of fresh and field-cured common vetch forage showed an average value of 200 g kg-1 of DM.
The harvest form did not affect the relative proportion of CP fraction A within maturity stages (Table 3). The field-cured forage always contained a higher concentration of CP fraction A than the fresh forage. This general trend was found across maturities and growing seasons. Harvesting common vetch at the recommended seed filling 2 maturity stage resulted in a lower proportion of CP fraction A in the second (higher seed proportion on DM), but not in the first growing season. Conversely, the CP fraction B1 showed a decreasing trend from fresh to field-cured forage and the effect of maturity was significant (P < 0.01). The relative proportion of CP fraction B1 was higher at seed filling 2 than at the two previous harvesting stages. As a whole, CP fraction A increased from fresh to field-cured forage and CP fraction B1 increased with maturity and decreased from fresh to dried. Both soluble fractions, the instantaneously fermented (CP fraction A) and the rapidly fermented (CP fraction B1), represented together between 410 and 530 g kg-1 of total CP.
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As CP fraction B3 represents CP insoluble in NDF but soluble in ADF, the proportion of CP fraction B3 is linked to NDF (Elizalde et al., 1999). For the same reason, values of CP fraction B3 are usually greater in grasses than in forage legumes at comparative growth stages and, within forage species, values of CP fraction B3 are usually lower in fresh forage than in the corresponding field-cured forage (Sniffen et al., 1992; Agbossamey et al., 1998). Our results with common vetch showed a similar pattern in the two growing seasons (Table 3). The compensatory effect of the grain proportion on CP fraction B3 was more apparent in the second growing season than in the first season. The CP fraction B3 increased at seed filling phase 2 but to a lesser extent in the second than in the first growing season. In the second growing season, the unavailable and indigestible CP fraction C remained unchanged with maturity. As a whole, a compensatory effect of the grain proportion was not found for this latter fraction (Table 3). Andrés (1990) indicated an estimated CP ruminal degradation from grain samples of common vetch of 75% to which would correspond to an effective CP degradability of common vetch grain of 77.7% (Rodríguez et al., 1999). These results may suggest that grain-rich samples had little influence on CP degradation. Higher CP fraction B3 at seed filling phase 2 in our samples seems to be the result of higher NDF in the vegetative components.
Amino Acid Pattern of Vetch Protein
Information on essential amino acid (EAA), semiessential amino acid (SEAA), and nonessential amino acid (NEAA) composition of vetch CP can be relevant for comparison with other studies on CP fractionation. Also, until EAA needs of sheep have been quantified, information about EAA requirement and optimality of EAA pattern of vetch CP must be inferred from comparison with the EAA profile of sheep products (milk, tissue, and wool) or from comparison with the amino acid profile of extensively cultivated forage such as alfalfa (NRC, 1982).
The vetch CP amino acid pattern is presented in Table 4. The mean vetch CP amino acid profile across maturity and harvest forms showed some differences with the alfalfa profile (Broderick, 1994). Within the EAA group, arginine was higher in vetch than in alfalfa (63 vs. 46 g kg-1 of CP) while the opposite occurred with lysine (41 vs. 51 g kg-1 of CP). Within the SEAA group, the cystine concentration of vetch CP was twofold (30 vs. 15 g kg-1 of CP). This fact can be of importance as cystine may spare the low content of both forage species in methionine (11 and 13 g kg-1 of CP, respectively). Within the NEAA group, the highest concentrations of individual amino acid in vetch CP were found for aspartic acid and glutamic acid (145 and 110 g kg-1 of CP, respectively).
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IMPLICATION SUMMARY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATION SUMMARYREFERENCES |
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The ratio of structural to nonfiber carbohydrates (SC/NFC) changed little with maturity in the first growing season (1.14 at flowering to 1.21 at seed filling phase 2). However, higher starch-rich seed proportion in the second growing season, determined a decrease of the SC/NFC ratio with maturity from 0.97 at flowering to 0.77 at seed filling phase 2.
The CP fraction B2 was the largest single CP fraction in fresh and field-cured vetch forage and the available CP fraction B3 escaping ruminal fermentation accounted for <100 g kg-1 of total CP. The CP fraction B3 increased with maturity, reinforcing the advantage of delaying harvesting if reduced ruminal protein degradation is an objective.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATION SUMMARYREFERENCES |
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-amylase (EC 3.2.1.1) in digesta and pancreas and on the digestion of lipid and starch by young chicks. Br. J. Nutr. 66:139–147.[ISI][Medline]
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