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Chemical composition and relationship to in vitro gas production of Zimbabwean browsable indigenous tree species
ANlMAl.FEED SCIENCEAND TECHNOLOGY
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Animal Feed Science Technology 69 (1997) 121-129
Chemical composition and relationship to in vitro gas production of Zimbabwean browsable indigenous tree species L.R. Ndlovu*, F.V. Nherera Depaltment ofAnimal Science, University of Zimbabwe, PO Box MP 167, Harare, Zimbabwe
Abstract Seventeen Zimbabwean browse species were analysed for crude protein (CP), neutral and acid detergent fibre (NDF and ADF), acid detergent lignin (ADL), insoluble proanthocyani-
dins (IPAs) and protein precipitating polyphenolics (PPPs). Gas production of the species was measured over 96 h and gas-production constants estimated using the equation: gas produced = b(1 - epcf). The browse varied greatly in their CP, fibre and phenolic content. Crude protein ranged from 56 to 210 g kg-’ dry matter (DM), NDF from 294 to 835 g kg-’ DM and PAS from 14.2 to 389 Ass,, nm g-’ NDF. None of the polyphenolics assayed were related to gas-production constants (P > 0.05) but NDF, ADF and ADL were negatively correlated (P < 0.05) to rate and extent of gas production. The results indicate that the effect of polyphenolics on gas production is complex and varies across browse species and that the fibre fraction of browse may be more important than tannins in limiting fermentation in vitro. 0 1997 Elsevier Science B.V. Keywords: Indigenous
browse; Gas production;
Chemical composition;
Proanthocyanidins;
Polyphenolics
1. Introduction Browse is an important feed resource during the dry season in Zimbabwe, especially in marginal ecological regions where browse can act as sole source of * Corresponding author. Tel.: + 263 4 303211; fax: + 263 4 333407; e-mail: [email protected] 0377-8401/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII s0377-8401(97)00100-5
122
L.R Ndlovu, F. V. Nherera /Animal Feed Science Technology 69 (1997) 121-129
nutrients. Grass biomass and quality is low during the dry season; with crude protein (CP> content dropping to as low as 30 g kg-’ of dry matter (Sibanda, 1984). However, some browse species may contain anti-nutritive factors that reduce intake and protein and dry matter digestibility (Reed, 1986). An important group of these allelochemicals found in tropical browse species is polyphenolics, especially tannins (proanthocyanidins and hydrolysable tannins) (Reed et al., 1990). The effects of polyphenolics on the nutritive value of browse in ruminants may vary from: (1) affecting the species composition of the microflora in the rumen; (2) complexing with proteins, carbohydrates and minerals and thus reducing or completely preventing their availability; (3) complexing with and inhibiting extracellular microbial cellulolytic enzymes; and/or (4) being absorbed from the rumen and resulting in toxicosis at the tissue level (Butler et al., 1986; Mueller-Harvey and McAllan, 1992). On the other hand tannins are involved in the control of bloat (Waghorn et al., 1994) and at low to moderate concentration may increase protein utilization in ruminants (Reed, 1995). Current chemical analytical techniques do not reflect the biological effects of tannins; the use of in vitro techniques has been proposed instead (Nsahlai et al., 1994). The gas-production technique has proved to be efficient in determining the nutritive value of feeds containing anti-nutritive factors (Khazaal et al., 1993; Siaw et al., 1993). It has been shown to be positively related to intake (Blummel and Orskov, 19931, microbial protein synthesis (Krishnamoorthy et al., 1991) and in vivo digestibility (Khazaal et al., 1993). The objective of the present study was to assess gas production of indigenous browse species in relation to their chemical composition including tannin content.
2. Materials and methods 2.1. Samples Seventeen indigenous browse samples (leaves, petioles and thin twigs of less than 4 mm in diameter) were collected from tree species that were observed to be browsed by cattle in the late dry season (September-October) in Chinamhora communal area (longitude 17”15’ S and latitude 31” E). The area has predominantly infertile granitic sandy soils and receives a mean annual rainfall ranging from 750 to 1000 mm in the months of November-March. The samples were air-dried in a well ventilated room (25°C) for 10 days and then ground to pass through a l.O-mm sieve. 2.2. Chemical analysis The dry matter (DM) content was measured by drying the samples at 105°C overnight and organic matter (OM) was determined by difference after igniting the samples in a muffle furnace at 550°C for 8 h. Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined according
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123
to the method of Van Soest and Robertson (1980). Crude protein (CP) was estimated using the procedures of the Association of Official Analytical Chemists (AOAC, 1990). Proanthocyanidins insoluble in neutral detergent (IPAs) were determined by boiling 10 mg NDF for 1 h in 955 (v/v) n-butanol:HCl and absorbance was read at 550 nm (Reed, 1986) and results are reported as absorbance units (AU)/g of NDF (AU 550“,/g NDF). The radial diffusion method (Hagerman, 1987) was used to estimate protein precipitating polyphenolics (PPP). PPP values were expressed as the diameter of the protein-tannin band per gram of DM (mm/g DM). 2.3. Gas production Fermentation was carried out in 100 ml graduated glass syringes following the method described by Menke et al. (1979). Rumen liquor was obtained from the rumen of two steers receiving Katambora rhodes grass (Chloris gayana cv. Katambora) hay ad libitum. Approximately 300 mg of air-dry milled sample was weighed into the glass syringes after which the pistons, lubricated with silicone grease to ease the movement of the piston and to prevent escape of gas, were inserted. The syringes and pistons were prewarmed to 40°C prior to the addition of approximately 30 ml of incubation media prepared as described by Menke et al. (1979), followedby incubation in a water bath at 39°C. The syringes were shaken 30 min from the start and at every recording of gas production. Recording of gas production was done after 2, 4, 6, 8, 12, 24,48, 72 and 96 h of incubation. The piston was reset to the 35 ml mark by releasing the gas produced whenever it reached a level of 90 ml. All samples were incubated in triplicate together with three other syringes containing only incubation media (blank). Gas production from samples was calculated by subtracting the volume of gas produced in the blanks and was corrected to a weigh-out of exactly 300 mg air-dry sample. 2.4. Statistical analysis The non-linear equation u = b(1 - e-“) was fitted to gas-production data using the PROC NLIN procedures of the SAS computer programme (SAS, 1988) based on the assumption that no gas is produced from unfermented feed (Siaw et al., 1993; Nsahlai et al., 1994) where, u = volume of gas produced at time t, (ml/300 mg DM), and c = fractional rate of gas production.
b = potential gas production
The effect of browse species on the gas-production constants (b and c) was determined by a one-way analysis of variance (ANOVA) using PROC GLM procedures of SAS @AS, 1988). Multiple regression using the backward elimination procedure of SAS @AS, 1988) was used to establish which chemical components of browse had the most effect on gas-production constants (b and c>. The variable
124
L.R. Ndlovu, F.V. Nherera /Animal Feed Science Technology 69 (1997) 121-129
elimination criterion was set at the 0.1 probability level of significance. Correlation analysis was done to establish relationships between gas-production constants and chemical properties of browse using SAS (SAS, 1988).
3. Results Variation in the CP, NDF, ADF, ADL, PPPs and IPAs between species was large (Table 1). CP content was lowest in Ficus cupensis (56 g kg-i DM) and highest in Euphorbiu matubelensis (210 g kg-’ DM). Concentrations of IPAs were generally high (above 50 A,,, nm g-l NDF) except in Diplorhynchus condylocurpon, Pteroculpus rotundifolius, Jucurundu mimosifoliu and Sechnos cocculoides. The volumes of gases produced ranged from 30.2 to 92.7 ml, with an average of 53.9 ml, whilst the fractional rate of gas production varied between 0.6 and 3.6%/h with a mean of 1.6%/h (Table 2). CP content was not significantly correlated to any of the gas-production constants (P > 0.05) but cell-wall constituents were all negatively correlated to the gas constants (P < 0.1, Table 3). The concentration of proanthocyanidins was positively related to content of ADF, NDF and ADL (P < 0.01, Table 3). Samples high in cell-wall constituents were high in proanthocyanidin concentration. The protein precipitating phenolics were not related to cell-wall constituents nor to gas-production constants (P > 0.01, Table 3). CP content was negatively correlated to concentration of proanthocyanidins but not to level of PPPs (Table 3). NDF explained 23% of the variation in potential gas produced (B = 96.6 (18.5) - 0.08 (0.031) NDF, r2 = 0.23, P = 0.09) but only ADL was significant in the prediction of fractional rate of gas evolution (C = 0.03 (0.003) - 0.0001 (0.00002) ADL, r2 = 0.34, P = 0.006). For every unit increase in NDF, the potential gas produced decreased by 0.08 ml whilst every unit increase in ADL reduced rate of gas evolution by 10e4 units.
4. Discussion
Levels of CP (range 56-210 g kg-l DM) of the browse were lower than has been reported for West African browse (range 137.5-212.5 g kg-l DM) by Rittner and Reed (1992) and lower than in those Sesbuniu species growing in East Africa (156.3-275 g kg-’ DM) (Nsahlai et al., 1994). The wide variation in NDF, ADF, ADL and IPAs content has been observed in other studies on browse (Reed, 1986; Makkar and Singh, 1991; Rittner and Reed, 1992; Khazaal et al., 1994). Proanthocyanidins are chemically reactive and susceptible to change during sample drying. However, the effects of drying temperature and time on tannin extraction are not consistent. Makkar and Singh (1991) found no effect of time and temperature on PPP but Hagerman (1987) reported that PPPs could be altered by freeze drying or oven drying at 40°C. Dzowela et al. (1995) reported that sun air-drying for 3 days at 25°C did not affect quantity of phenolics in browse leaves but it reduced in vitro
compositiona
a PPP, protein acid detergent
precipitating lignin.
Brachystegia glauscens Brachystegia spiciformis Combretum imberbe Combretum molle Diplorhynchus condylocarpon Euphorbia matabelensis Ficus capensis Ficus glumosa Jacaranda mimosifolia Julbenardia globijlora Parinati curatellifolia Paropsia brazzeana Pterocarpus rotundifolius Strychnos cocculoides Terminalia sericea Uapaca kirkiana Ziziphus abyssnica
Species
Table 1 The chemical
phenolics;
901 914 868 914 934 878 892 858 893 892 897 901 929 897 881 884 825
g kg-’
DM
NDF, neutral
185 105 135 83 86 210 56 139 95 155 97 131 162 169 163 77 63
CP gkg-‘DM 52 54 49 49 56 80 85 91 38 38 58 57 27 63 96 52 105
766 489 773 634 491 505 542 835 294 556 655 570 404 479 647 769 586
NDF g kg-’
by cattle
665 218 597 388 365 475 446 631 152 538 548 443 340 340 496 625 523
ADF g kg-’ DM
proanthocyanidins;
DM
fibre; IPA, insoluble
ASH gkg-‘DM
browsed
detergent
of dried leaves of tree species
DM 175.0 71.0 271.0 65.8 14.2 151.0 389.0 270.0 15.0 85.6 76.1 336.0 26.7 22.0 260.0 300.0 220.0
‘ NDF
fibre; CP, crude
IPAs A5sunmg-
ADF, acid detergent
212 87 229 37 173 167 156 226 27 111 65 144 124 143 216 268 172
ADL g kg-’
protein;
0.90 3.73 2.90 15.56 0.00 14.23 0.00 2.63 2.76 13.92 12.56 5.29 9.00 0.42 13.52 4.02 6.14 ADL,
PPPS mm g-’ DM
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L.R. Ndlovu,F. K Nherera /Animal Feed Science Technology69 (1997) 121-129
Table 2 Gas production from dried leaves of tree species browsed by cattle Species
Gas-production constants
Brachystegiaglauscens Brachystegiaspiciforms Combretumimberbe Combretummolle Diplorhyncuscondylocatpon Euphorbia matabelensis Ficus capensis Ficus glumosa Jacaranda mimosifolia Julbenardiaglobijlora Parinaricuratellifolia Paropsiabrazzeana Pterocarpusrotundifolius Strychnoscocculoides Terminaleasericea Uapacakirkiana Ziziphusabyssinica Mean Effect of species Root M.S.E. Significance
b (ml 300-l mg air-dry sample)
c (h- ‘)
58.9 40.6 31.4 33.7 61.0 65.5 69.2 56.7 87.9 37.6 41.8 32.8 82.8 63.2 39.8 30.2 92.7 53.9
0.010 0.010 0.015 0.006 0.020 0.016 0.027 0.007 0.036 0.016 0.017 0.006 0.017 0.022 0.013 0.007 0.010 0.016
6.04 0.0001
0.009 0.006
b and c are constants in the experimental equation v = b(1 - emcf), where v = volume of gas evolved at time t; b = gas that will evolve in time; and c = fractional rate of gas production. M.S.E. = mean square error.
Table 3 The correlation coefficients between gas-production constants, phenolics and chemical properties of 20 indigenous browse species Extent of gas production (ml 300- 1 mg air-dry sample) Nitrogen (CP) Cell-wall constituents Acid detergent fibre Neutral detergent fibre Acid detergent lignin Tannins IPAS PPPS
0.16”’
Rate of gas production (h-l)
IPAs,,, “m g-r NDF
PPPS mm g-’ DM
-o.llns
-0.39**
- o.07”s
-0.49*** -0.48*** - 0.35*
-0.53*** -0.49*** -0.55***
0.50*** 0.50*** 0.56* **
- o.15ns -O.lPS
- 0.39* -0.11”s
o.02”s
Levels of significance: *, P < 0.1; **, P < 0.05; ***, P < 0.01; NS, P > 0.05. IPAs, insoluble proanthocyanidins. PPPs, protein precipitable phenolics.
0.04”s - o.02”5 0.001”’
L.R. Ndlovy F.lr Nherera /Animal Feed Science Technology 69 (1997) 121-129
121
digestibility. These authors recommended air-drying in the shed. Whilst formation of artifacts cannot be completely ruled out, the drying method used in the present experiment is unlikely to have affected the chemical composition of the leaves. There was a significant positive correlation between NDF, ADF and ADL content and level of insoluble proanthocyanidins, in agreement with observations by Reed (1986) and Nsahlai et al. (1994). The fractional rates of gas production observed in this experiment were slower than those previously reported (Blummel and Orskov, 1993; Siaw et al., 1993; Khazaal et al., 1994; Nsahlai et al., 1994). These studies used germplasm that had been selected for desirable agronomic traits whilst browse species used in the current experiment were subjected to environmental stress including droughts, herbivory and high temperatures - all of which would negatively affect gas production. Additionally, our incubation media was flushed with N, instead of CO,. The N, does not lower the pH of the culture media to the same extent as CO, and the elevated pH could have slowed microbial activity. Rate of gas production was negatively related to ADF, ADL and NDF content (Table 3). However, both IPAs and PPPs were not correlated to extent of gas production. The lack of correlation between PAS and gas production has been observed in previous studies (Khazaal et al., 1994). These results indicate that the polyphenolic assays used failed to account for some (phenolic) compounds which have an effect on gas production. Some browse species like Ziziphus abyssinica and Ficus sp. were high in PAS but also had high potential gas-production levels indicating that the phenolics measured had no effect on gas production. Ficus capensis, whilst high in PAS, failed to precipitate protein in the radial diffusion test and had high fractional rate of gas production implying that the type of IPAs present in this species did not interfere with fibre digestion. The results emphasize the importance of determining the chemical structures, in addition to the quantities, of polyphenolics in browse if chemical composition is to be used to predict biological effects of these compounds on rumen digestion. Changes in ADF and NDF accounted for approx. 25% of the variation in gas-production constants with differences in ADL accounting for 34% of the variation in rate of gas production. Nsahlai et al. (1994) reported that 70% of the variation in gas production in Sesbania could be explained by changes in either NDF, hemicellulose or lignin. The low predictive values in the current experiment could be due to species variation in quantity and quality of fibre content as well as variation in complexation of the fibre with polyphenolics, as the browse used was of varied genetic background.
5. Conclusion There was a low correlation between PA content and gas production whilst cell-wall constituents adversely affected gas-production constants in the 17 indigenous browse species studied.
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Acknowledgements This work was supported by the African Feed Resources Research Network, International Livestock Research Institute (ILRI), Addis Ababa, Ethiopia and the University of Zimbabwe Research Board.
References AOAC, 1990. Official methods of analysis, 15th edition. J. Assoc. Anal. Chem. 65, 496. Blummel, M., Orskov, E.R., 1993. Comparison of in vitro gas production and nylon bag degradability of forages in predicting intake in cattle. Anim. Feed Sci. Technol. 40, 109-119. Butler, L.G., Rogler, J.C., Mehansho, H., Carlson, D.M., 1986. Dietary effects of tannins. Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity Relationships. Alan R. Liss, New York, p. 141. Dzowela, B.H., Hove, L., Mafongoya, P., 1995. Effect of drying method on chemical composition and in vitro digestibility of multi-purpose tree and shrub fodders. Trop. Grassl. 29, 263-269. Hagerman, A.E., 1987. Radial diffusion method for determining tannin in plant extracts. J. Chem. Ecol. 13,437-449. Khazaal, K., Boza, J., Orskov, E.R., 1994. Assessment of phenolics-related antinutritive effects in Mediterranean browse: a comparison between the use of the in vitro gas production technique with or without insoluble polyvinylpolypyrrolidone or nylon bag. Anim. Feed Sci. Technol. 49, 133-149. Khazaal, K., Dentinho, M.T., Ribeiro, R., Orskov, E.R., 1993. A comparison of gas production during incubation with rumen contents in vitro and nylon bag degradability as predictors of the apparent digestibility in vivo and voluntary intake of hays. Anim. Prod. 57, 105-112. Khazaal, K., Markantonatos, X., Nastis, A., Orskov, E.R., 1993. Changes with maturity in fibre composition and levels of extractable polyphenols in Greek browse: effects on in vitro gas production and in sacco dry matter degradation. J. Sci. Food Agric. 63, 237-244. Krishnamoorthy, U., Steingass, H., Menke, K.H., 1991. Preliminary observations on the relationships between gas production and microbial protein synthesis in vitro. Arch. Tiererniihr. 41,521-526. Makkar, H.P.S., Singh, B., 1991. Distribution of condensed tannins (proanthocyanidins) in various fibre fractions in young and mature leaves of some oak species. Anim. Feed Sci. Technol. 32, 253-260. Makkar, H.P.S., Singh, B., 1991. The effect of drying conditions on tannin, fibre and lignin levels in mature oak (Quercus incana) leaves. J. Sci. Food Agric. 54, 323-328. Menke, K.H., Raab, L., Salewski, A., Steingass, A., Fritz, D., Schneider, W., 1979. The estimation of the digestibility and metabolisable energy content of ruminant feeding stuffs from the gas production when they are incubated with rumen liquor in vitro. J. Agric. Sci. Cambridge 93, 217-222. Mueller-Harvey, I., McAllan, A.B., 1992. Tannins: their biochemistry and nutritional properties. Adv. Plant Cell Biochem. Biotechnol. 1, 151-182. Nsahlai, I.V., Siaw, D.E.K.A., Osuji, P.O., 1994. The relationship between gas production and chemical composition of 23 browses of the genus Sesbania. J. Sci. Food Agric. 65, 13-20. Reed, J.D., 1986. Relationship among phenolics, insoluble proanthocyanidins and fibre in East African browse species. J. Range Manage. 39,5-7. Reed, J.D., 1995. Nutritional toxicology of tannins and related polyphenols in forage legumes. J. Anim. Sci. 73, 151661528. Reed, J.D., Soller, H., Woodward, A., 1990. Fodder tree and straw diets for sheep: intake, growth and digestibility and the effects of phenol& on nitrogen utilisation. Anim. Feed Sci. Technol. 30, 39-50. Rittner, U., Reed, J.D., 1992. Phenolics and in vitro degradability of protein and fibre in West African browse. J. Sci. Food Agric. 58, 21-28. SAS, 1988. User’s Guide for Personal Computers, Version 6 edition. SAS Institute, Cary, NC, USA. Siaw, D.E.K.A., Osuji, P.O., Nsahlai, I.V., 1993. Evaluation of multipurpose tree germplasm: the use of gas production and rumen degradation characteristics. J. Agric. Sci. Cambridge 120, 319-330.
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Sibanda, S., 1984. Composition of diet selected from veld by steers fistulated at the oesophagus and body mass of nonfistulated steers grazing the same paddocks. Zimbabwe J. Agric. Res. 22, 105-107. Van Soest, P.J., Robertson, J.B., 1980. Systems of analysis for evaluating fibrous feeds. In: Pigden, W.J., Belch, C.C., Graham, M. (Eds.), Standardization of Analytical Methodology for Feeds. International Development Research Centre, Ottawa, Canada, pp. 46-60. Waghom, G.C., Shetton, I.D., McNabb, W.C., McCuthcheon, S.N., 1994. Effects of condensed tannins in Lotus pedunculatw on its nutritive value for sheep, 2. nitrogenous aspects. J. Agric. Sci. Cambridge 123, 109-117.
r
k ELSEVIER
Animal Feed Science Technology 69 (1997) 121-129
Chemical composition and relationship to in vitro gas production of Zimbabwean browsable indigenous tree species L.R. Ndlovu*, F.V. Nherera Depaltment ofAnimal Science, University of Zimbabwe, PO Box MP 167, Harare, Zimbabwe
Abstract Seventeen Zimbabwean browse species were analysed for crude protein (CP), neutral and acid detergent fibre (NDF and ADF), acid detergent lignin (ADL), insoluble proanthocyani-
dins (IPAs) and protein precipitating polyphenolics (PPPs). Gas production of the species was measured over 96 h and gas-production constants estimated using the equation: gas produced = b(1 - epcf). The browse varied greatly in their CP, fibre and phenolic content. Crude protein ranged from 56 to 210 g kg-’ dry matter (DM), NDF from 294 to 835 g kg-’ DM and PAS from 14.2 to 389 Ass,, nm g-’ NDF. None of the polyphenolics assayed were related to gas-production constants (P > 0.05) but NDF, ADF and ADL were negatively correlated (P < 0.05) to rate and extent of gas production. The results indicate that the effect of polyphenolics on gas production is complex and varies across browse species and that the fibre fraction of browse may be more important than tannins in limiting fermentation in vitro. 0 1997 Elsevier Science B.V. Keywords: Indigenous
browse; Gas production;
Chemical composition;
Proanthocyanidins;
Polyphenolics
1. Introduction Browse is an important feed resource during the dry season in Zimbabwe, especially in marginal ecological regions where browse can act as sole source of * Corresponding author. Tel.: + 263 4 303211; fax: + 263 4 333407; e-mail: [email protected] 0377-8401/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII s0377-8401(97)00100-5
122
L.R Ndlovu, F. V. Nherera /Animal Feed Science Technology 69 (1997) 121-129
nutrients. Grass biomass and quality is low during the dry season; with crude protein (CP> content dropping to as low as 30 g kg-’ of dry matter (Sibanda, 1984). However, some browse species may contain anti-nutritive factors that reduce intake and protein and dry matter digestibility (Reed, 1986). An important group of these allelochemicals found in tropical browse species is polyphenolics, especially tannins (proanthocyanidins and hydrolysable tannins) (Reed et al., 1990). The effects of polyphenolics on the nutritive value of browse in ruminants may vary from: (1) affecting the species composition of the microflora in the rumen; (2) complexing with proteins, carbohydrates and minerals and thus reducing or completely preventing their availability; (3) complexing with and inhibiting extracellular microbial cellulolytic enzymes; and/or (4) being absorbed from the rumen and resulting in toxicosis at the tissue level (Butler et al., 1986; Mueller-Harvey and McAllan, 1992). On the other hand tannins are involved in the control of bloat (Waghorn et al., 1994) and at low to moderate concentration may increase protein utilization in ruminants (Reed, 1995). Current chemical analytical techniques do not reflect the biological effects of tannins; the use of in vitro techniques has been proposed instead (Nsahlai et al., 1994). The gas-production technique has proved to be efficient in determining the nutritive value of feeds containing anti-nutritive factors (Khazaal et al., 1993; Siaw et al., 1993). It has been shown to be positively related to intake (Blummel and Orskov, 19931, microbial protein synthesis (Krishnamoorthy et al., 1991) and in vivo digestibility (Khazaal et al., 1993). The objective of the present study was to assess gas production of indigenous browse species in relation to their chemical composition including tannin content.
2. Materials and methods 2.1. Samples Seventeen indigenous browse samples (leaves, petioles and thin twigs of less than 4 mm in diameter) were collected from tree species that were observed to be browsed by cattle in the late dry season (September-October) in Chinamhora communal area (longitude 17”15’ S and latitude 31” E). The area has predominantly infertile granitic sandy soils and receives a mean annual rainfall ranging from 750 to 1000 mm in the months of November-March. The samples were air-dried in a well ventilated room (25°C) for 10 days and then ground to pass through a l.O-mm sieve. 2.2. Chemical analysis The dry matter (DM) content was measured by drying the samples at 105°C overnight and organic matter (OM) was determined by difference after igniting the samples in a muffle furnace at 550°C for 8 h. Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined according
L.R. Ndlovu, F.V. Nherera /Animal Feed Science Technology 69 (1997) 121-129
123
to the method of Van Soest and Robertson (1980). Crude protein (CP) was estimated using the procedures of the Association of Official Analytical Chemists (AOAC, 1990). Proanthocyanidins insoluble in neutral detergent (IPAs) were determined by boiling 10 mg NDF for 1 h in 955 (v/v) n-butanol:HCl and absorbance was read at 550 nm (Reed, 1986) and results are reported as absorbance units (AU)/g of NDF (AU 550“,/g NDF). The radial diffusion method (Hagerman, 1987) was used to estimate protein precipitating polyphenolics (PPP). PPP values were expressed as the diameter of the protein-tannin band per gram of DM (mm/g DM). 2.3. Gas production Fermentation was carried out in 100 ml graduated glass syringes following the method described by Menke et al. (1979). Rumen liquor was obtained from the rumen of two steers receiving Katambora rhodes grass (Chloris gayana cv. Katambora) hay ad libitum. Approximately 300 mg of air-dry milled sample was weighed into the glass syringes after which the pistons, lubricated with silicone grease to ease the movement of the piston and to prevent escape of gas, were inserted. The syringes and pistons were prewarmed to 40°C prior to the addition of approximately 30 ml of incubation media prepared as described by Menke et al. (1979), followedby incubation in a water bath at 39°C. The syringes were shaken 30 min from the start and at every recording of gas production. Recording of gas production was done after 2, 4, 6, 8, 12, 24,48, 72 and 96 h of incubation. The piston was reset to the 35 ml mark by releasing the gas produced whenever it reached a level of 90 ml. All samples were incubated in triplicate together with three other syringes containing only incubation media (blank). Gas production from samples was calculated by subtracting the volume of gas produced in the blanks and was corrected to a weigh-out of exactly 300 mg air-dry sample. 2.4. Statistical analysis The non-linear equation u = b(1 - e-“) was fitted to gas-production data using the PROC NLIN procedures of the SAS computer programme (SAS, 1988) based on the assumption that no gas is produced from unfermented feed (Siaw et al., 1993; Nsahlai et al., 1994) where, u = volume of gas produced at time t, (ml/300 mg DM), and c = fractional rate of gas production.
b = potential gas production
The effect of browse species on the gas-production constants (b and c) was determined by a one-way analysis of variance (ANOVA) using PROC GLM procedures of SAS @AS, 1988). Multiple regression using the backward elimination procedure of SAS @AS, 1988) was used to establish which chemical components of browse had the most effect on gas-production constants (b and c>. The variable
124
L.R. Ndlovu, F.V. Nherera /Animal Feed Science Technology 69 (1997) 121-129
elimination criterion was set at the 0.1 probability level of significance. Correlation analysis was done to establish relationships between gas-production constants and chemical properties of browse using SAS (SAS, 1988).
3. Results Variation in the CP, NDF, ADF, ADL, PPPs and IPAs between species was large (Table 1). CP content was lowest in Ficus cupensis (56 g kg-i DM) and highest in Euphorbiu matubelensis (210 g kg-’ DM). Concentrations of IPAs were generally high (above 50 A,,, nm g-l NDF) except in Diplorhynchus condylocurpon, Pteroculpus rotundifolius, Jucurundu mimosifoliu and Sechnos cocculoides. The volumes of gases produced ranged from 30.2 to 92.7 ml, with an average of 53.9 ml, whilst the fractional rate of gas production varied between 0.6 and 3.6%/h with a mean of 1.6%/h (Table 2). CP content was not significantly correlated to any of the gas-production constants (P > 0.05) but cell-wall constituents were all negatively correlated to the gas constants (P < 0.1, Table 3). The concentration of proanthocyanidins was positively related to content of ADF, NDF and ADL (P < 0.01, Table 3). Samples high in cell-wall constituents were high in proanthocyanidin concentration. The protein precipitating phenolics were not related to cell-wall constituents nor to gas-production constants (P > 0.01, Table 3). CP content was negatively correlated to concentration of proanthocyanidins but not to level of PPPs (Table 3). NDF explained 23% of the variation in potential gas produced (B = 96.6 (18.5) - 0.08 (0.031) NDF, r2 = 0.23, P = 0.09) but only ADL was significant in the prediction of fractional rate of gas evolution (C = 0.03 (0.003) - 0.0001 (0.00002) ADL, r2 = 0.34, P = 0.006). For every unit increase in NDF, the potential gas produced decreased by 0.08 ml whilst every unit increase in ADL reduced rate of gas evolution by 10e4 units.
4. Discussion
Levels of CP (range 56-210 g kg-l DM) of the browse were lower than has been reported for West African browse (range 137.5-212.5 g kg-l DM) by Rittner and Reed (1992) and lower than in those Sesbuniu species growing in East Africa (156.3-275 g kg-’ DM) (Nsahlai et al., 1994). The wide variation in NDF, ADF, ADL and IPAs content has been observed in other studies on browse (Reed, 1986; Makkar and Singh, 1991; Rittner and Reed, 1992; Khazaal et al., 1994). Proanthocyanidins are chemically reactive and susceptible to change during sample drying. However, the effects of drying temperature and time on tannin extraction are not consistent. Makkar and Singh (1991) found no effect of time and temperature on PPP but Hagerman (1987) reported that PPPs could be altered by freeze drying or oven drying at 40°C. Dzowela et al. (1995) reported that sun air-drying for 3 days at 25°C did not affect quantity of phenolics in browse leaves but it reduced in vitro
compositiona
a PPP, protein acid detergent
precipitating lignin.
Brachystegia glauscens Brachystegia spiciformis Combretum imberbe Combretum molle Diplorhynchus condylocarpon Euphorbia matabelensis Ficus capensis Ficus glumosa Jacaranda mimosifolia Julbenardia globijlora Parinati curatellifolia Paropsia brazzeana Pterocarpus rotundifolius Strychnos cocculoides Terminalia sericea Uapaca kirkiana Ziziphus abyssnica
Species
Table 1 The chemical
phenolics;
901 914 868 914 934 878 892 858 893 892 897 901 929 897 881 884 825
g kg-’
DM
NDF, neutral
185 105 135 83 86 210 56 139 95 155 97 131 162 169 163 77 63
CP gkg-‘DM 52 54 49 49 56 80 85 91 38 38 58 57 27 63 96 52 105
766 489 773 634 491 505 542 835 294 556 655 570 404 479 647 769 586
NDF g kg-’
by cattle
665 218 597 388 365 475 446 631 152 538 548 443 340 340 496 625 523
ADF g kg-’ DM
proanthocyanidins;
DM
fibre; IPA, insoluble
ASH gkg-‘DM
browsed
detergent
of dried leaves of tree species
DM 175.0 71.0 271.0 65.8 14.2 151.0 389.0 270.0 15.0 85.6 76.1 336.0 26.7 22.0 260.0 300.0 220.0
‘ NDF
fibre; CP, crude
IPAs A5sunmg-
ADF, acid detergent
212 87 229 37 173 167 156 226 27 111 65 144 124 143 216 268 172
ADL g kg-’
protein;
0.90 3.73 2.90 15.56 0.00 14.23 0.00 2.63 2.76 13.92 12.56 5.29 9.00 0.42 13.52 4.02 6.14 ADL,
PPPS mm g-’ DM
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Table 2 Gas production from dried leaves of tree species browsed by cattle Species
Gas-production constants
Brachystegiaglauscens Brachystegiaspiciforms Combretumimberbe Combretummolle Diplorhyncuscondylocatpon Euphorbia matabelensis Ficus capensis Ficus glumosa Jacaranda mimosifolia Julbenardiaglobijlora Parinaricuratellifolia Paropsiabrazzeana Pterocarpusrotundifolius Strychnoscocculoides Terminaleasericea Uapacakirkiana Ziziphusabyssinica Mean Effect of species Root M.S.E. Significance
b (ml 300-l mg air-dry sample)
c (h- ‘)
58.9 40.6 31.4 33.7 61.0 65.5 69.2 56.7 87.9 37.6 41.8 32.8 82.8 63.2 39.8 30.2 92.7 53.9
0.010 0.010 0.015 0.006 0.020 0.016 0.027 0.007 0.036 0.016 0.017 0.006 0.017 0.022 0.013 0.007 0.010 0.016
6.04 0.0001
0.009 0.006
b and c are constants in the experimental equation v = b(1 - emcf), where v = volume of gas evolved at time t; b = gas that will evolve in time; and c = fractional rate of gas production. M.S.E. = mean square error.
Table 3 The correlation coefficients between gas-production constants, phenolics and chemical properties of 20 indigenous browse species Extent of gas production (ml 300- 1 mg air-dry sample) Nitrogen (CP) Cell-wall constituents Acid detergent fibre Neutral detergent fibre Acid detergent lignin Tannins IPAS PPPS
0.16”’
Rate of gas production (h-l)
IPAs,,, “m g-r NDF
PPPS mm g-’ DM
-o.llns
-0.39**
- o.07”s
-0.49*** -0.48*** - 0.35*
-0.53*** -0.49*** -0.55***
0.50*** 0.50*** 0.56* **
- o.15ns -O.lPS
- 0.39* -0.11”s
o.02”s
Levels of significance: *, P < 0.1; **, P < 0.05; ***, P < 0.01; NS, P > 0.05. IPAs, insoluble proanthocyanidins. PPPs, protein precipitable phenolics.
0.04”s - o.02”5 0.001”’
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digestibility. These authors recommended air-drying in the shed. Whilst formation of artifacts cannot be completely ruled out, the drying method used in the present experiment is unlikely to have affected the chemical composition of the leaves. There was a significant positive correlation between NDF, ADF and ADL content and level of insoluble proanthocyanidins, in agreement with observations by Reed (1986) and Nsahlai et al. (1994). The fractional rates of gas production observed in this experiment were slower than those previously reported (Blummel and Orskov, 1993; Siaw et al., 1993; Khazaal et al., 1994; Nsahlai et al., 1994). These studies used germplasm that had been selected for desirable agronomic traits whilst browse species used in the current experiment were subjected to environmental stress including droughts, herbivory and high temperatures - all of which would negatively affect gas production. Additionally, our incubation media was flushed with N, instead of CO,. The N, does not lower the pH of the culture media to the same extent as CO, and the elevated pH could have slowed microbial activity. Rate of gas production was negatively related to ADF, ADL and NDF content (Table 3). However, both IPAs and PPPs were not correlated to extent of gas production. The lack of correlation between PAS and gas production has been observed in previous studies (Khazaal et al., 1994). These results indicate that the polyphenolic assays used failed to account for some (phenolic) compounds which have an effect on gas production. Some browse species like Ziziphus abyssinica and Ficus sp. were high in PAS but also had high potential gas-production levels indicating that the phenolics measured had no effect on gas production. Ficus capensis, whilst high in PAS, failed to precipitate protein in the radial diffusion test and had high fractional rate of gas production implying that the type of IPAs present in this species did not interfere with fibre digestion. The results emphasize the importance of determining the chemical structures, in addition to the quantities, of polyphenolics in browse if chemical composition is to be used to predict biological effects of these compounds on rumen digestion. Changes in ADF and NDF accounted for approx. 25% of the variation in gas-production constants with differences in ADL accounting for 34% of the variation in rate of gas production. Nsahlai et al. (1994) reported that 70% of the variation in gas production in Sesbania could be explained by changes in either NDF, hemicellulose or lignin. The low predictive values in the current experiment could be due to species variation in quantity and quality of fibre content as well as variation in complexation of the fibre with polyphenolics, as the browse used was of varied genetic background.
5. Conclusion There was a low correlation between PA content and gas production whilst cell-wall constituents adversely affected gas-production constants in the 17 indigenous browse species studied.
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Acknowledgements This work was supported by the African Feed Resources Research Network, International Livestock Research Institute (ILRI), Addis Ababa, Ethiopia and the University of Zimbabwe Research Board.
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