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Determination of tannin levels in multi-purpose Kenyan trees and fodder crops, their variation and effect… Kangara, John N. N. 1993

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DETERMINATION OF TANNIN LEVELS IN MULTI-PURPOSE KENYAN TREES AND FODDER CROPS, THEIR VARIATION AND EFFECT ON PROTEIN DIGESTIBILITY IN RUMINANTS.  John Nduati Ngugi Kanga'ra B. Sci. (Agr.), University of Nairobi, 1985  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE  We accept this thesis as conforming to the requir•standard  THE UNIVERSITY OF BRITISH COLUMBIA July 1993 © John Nduati Ngugi Kang'ara  ^  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature  Department of  ^6-(1/11141—^gC/EAIC  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ^1/471-114)^3  ABSTRACT Energy and protein are the major limiting nutrients in dairy production on the small scale mixed farms in Kenya. Commercial feed supplements are expensive and therefore multipurpose fodder trees (MPT) and forage crops are advocated as the alternative supplements, because they are inexpensive, able to provide green forage even in dry season and have high protein content. These trees have tannins whose levels, seasonal and altitude distribution have not been established. Previous studies have indicated that tannins may have either beneficial effects like bloat control and increased protein bypass, or deleterious effects like the reduction feed intake and digestibility of protein in animals fed on tanniferous feed. The objectives of this study were to determine the tannin levels in the MPT, as influenced by altitude and season, and the effect of these tannins on ruminal degradation and intestinal digestion of the diet. Samples of four multipurpose fodder trees and four forage crops viz. leucaena, sesbania, gliricidia, calliandra, velvet bean, green leaf and silver leaf desmodium and cassava, were collected at Mombasa (low altitude, below 300 m ASL.) in the wet season and at Embu (high altitude, 1500 m ASL.) in both wet and dry season. Tannin and protein content were determined using gravimetric and wet oxidation nitrogen determination methods respectively. The effect of tannin on protein degradability was determined by comparing the polyethylene glycol (PEG) treated with untreated forage samples using the mobile nylon bag technique. The treated and untreated samples were incubated separately in the rumen of four Holstein cows with both rumen and duodenal cannula for 0, 6, 12, 24, 48 and 96 hours. Afterwards two sample of each species from time 12 and 24 hours were inserted into the intestine through duodenal cannula, and recovered from the feces. The dry matter (DM) and protein of samples recovered from the rumen and feces were determined and these values were fitted in a non linear regression equation P = a + b(1— ")  The results indicated that the MPT had ytterbium-precipitatable tannins ranging from 16.08 (±2.39)% of the DM in Gliricidia sepium to 30.31 (±2.42)% in Desmodium. intortum. The tannin content varied significantly (P<0.05) with species. The altitude did not have significant effect on tannin content, but tannins within species behaved differently with season. The protein content differed significantly (P<0.05) with species. Proteins were significantly (P<0.05) higher in wet than in the dry season. The altitude had no effect on protein content. The tannin : protein ratio also varied significantly (P<0.05) with species. Species also had a significant interaction with season. Tannins significantly (P<0.05) reduced the rumen effective degradability of both DM and crude protein in all species, resulting in large quantities of undegraded dietary nitrogen(N). Tannin also significantly (P<0.05) decreased the total tract digestion of the DM (DMD) and depressed the digestible crude protein (DCP) of leucaena, calliandra, cassava and aintortum, but had no effect on gliricidia, sesbania and velvet bean DCP. A large proportion of the rumen undegraded dietary protein that reached the intestines was degraded in most species except calliandra. Tannin significantly (P<0.05) altered the degradability constants a, b, and c for both DM and  CP by reducing fraction a and the rate of degradation c and increasing the b fraction. From the study it was concluded that the MPT and forage crop species, despite their high protein content, are not good protein supplements. Their tannins reduce both the CP digestibility and the DM degradability in the rumen. Lastly, potentially viable treatments that reduce the effect of tannin on digestibility are suggested.  Table of contents ABSTRACT ^ LIST OF TABLES ^  vii  LIST OF APPENDIX TABLES ^  viii  LIST OF FIGURES ^  ix  LIST OF APPENDIX FIGURES ^ ACKNOWLEDGEMENT ^  xi  GENERAL INTRODUCTION ^  1  CHAPTER 1. DETERMINATION OF TANNIN LEVELS AND THEIR VARIATION^  2  1.1 INTRODUCTION ^  2  1.2. LITERATURE REVIEW ^  2  1.2.1 DEFINITION AND SIGNIFICANCE OF TANNIN ^ 4 1.2.2 CHEMICAL CLASSIFICATION ^  6  1.2.2.1 Hydrolysable tannins ^  6  1.2.2.2. Condensed tannins ^  6  1.2.3 OCCURRENCE AND ECOLOGICAL SIGNIFICANCE OF TANNINS  ^  1.3 MATERIALS AND METHODS ^  6 8  1.3.1 SAMPLE COLLECTION ^  8  1.3.2 CHEMICAL ANALYSIS ^  9  1.3.3 STATISTICAL ANALYSIS ^ 1.4 RESULTS ^  11 12  1.4.1 TANNIN CONTENT ^  12  1.4.2 PROTEIN CONTENT ^  12  1.4.3 TANNIN: PROTEIN RATIO ^  16  1.4.4 DISCUSSION ^  18  iv  1.6 CONCLUSION ^  20  CHAPTER 2. DETERMINATION OF TANNIN EFFECT ON PROTEIN DEGRADATION IN THE RUMEN AND INTESTINAL DIGESTION21 2.1 INTRODUCTION ^  21  2.2 LITERATURE REVIEW^  22  2.2.1 FORMATION OF TANNIN-PROTEIN COMPLEX ^ 22 2.2.1.1 Mechanisms ^  22  2.2.1.2 Specificity of tannins ^  23  2 .2.1.3 Factors influencing tannin-protein interaction ^ 24 2.2.2 EFFECT OF TANNIN ON ANIMALS ^ 26  2.2.2.1 Intake ^  26  2.2.2.2 Digestibility and animal performance ^ 26 2.2.2.2.1 In the monogastric ^  27  2.2.2.2.2 Effect of tannins in ruminants ^ 28 2.3. MATERIALS AND METHODS ^  31  2.3.1 SAMPLE PREPARTION ^  31  2.3.2 IN VIVO INCUBATION ^  31  2.3.3 ANALYSIS AND CALCULATION ^ 33 2.4 RESULTS AND DISCUSSION ^  35  2.4.1 DM AND CP DISAPPEARANCE IN THE RUMEN ^ 35 2.4.2 INTESTINAL DM AND CP DIGESTIBILITY ^ 42 2.5 SUMMARY AND CONCLUSION ^  52  GENERAL CONCLUSION ^  54  BIBLIOGRAPHY ^  V  55  APPENDICES ^  68  A. The neutral detergent fiber and acid detergent fiber of the MPT forage species ^  68  B. The effect of tannins on rumen degradation and intestinal digestion of DM and CP in period one (40g.PEG per gram tannin) and period two PEG (100g PEG/ g tannin) ^  70  C. A bio-assay for the optimum level of polyethylene glycol (PEG) ,that would inhibit the effect of MPT and forage tannins on the DM and CP ^ 79  vi  List of Tables  Table 1 The sample collection and analysis summyary ^  10  Table 2 Mean variation of tannin and protein with species ^  13  Table 3 Variation of tannin:protein with species ^  16  Table 4 Effect of tannin on DM effective degradability in the rumen at assumed outflow 36 rate k=0.04 and 0.06 ^ Table 5 DM and CP effective degradability in the rumen values of different species 37 at k=0.04 and 0.06 ^ Table 6 Effect of tannin on degradability constants of DM and CP ^38 Table 7 Animal variation in the effective degradability of the DM at two rumen outflow 39 rates.^ Table 8 Effect of period on % DM and CP effective degradability at k =0.06 ^39 Table 9 The effect of tannin on effective degradability of CP at assumed outflow rate 40 k=0.04 and 0.06 ^ Table 10 The mean total tract DMD and DCP of different species ^43 Table 11 Mean proportion of the total tract digestion of the DM contributed by intestinal 44 digestibility (IDMD) of different species ^ Table 12 Mean intestinal DM digestibility (ID MD) of different species ^ 44 Table 13 Effect of period on mean total tract % digestibilty of untreated forage ^45 Table 14 The effect of tannin on IDMD of different species ^  vii  46  List of appendix tables  Page  Tables^ I. NDF % in MPT and forages based on DM ^ II. ADF % in the MPT and forages based on the DM^  68 69  III. Effect of tannins on ruminal degradation and intestinal digestibility of protein in different preiod ^  70  IV. Effect of tannin on effective degradability of the CP ^ 71 V. Effect of tannin on degradation constants of the DM ^ 72 VI. Effect of tannin on degradation constants of protein ^ 73 VII. Effect of tannin on total tract digetibility of the DM (DMD) ^ 74 VIII. The effect of tannin on the total tract digestibility of CP (DCP) ^ 75 IX. The effect of tannin on intestinal DM digetibility(IDMD) ^ 76 X. the effect of tannin on intestinal digestibility of CP (EDCP) ^ 77  viii  List of figures  1. Fodder species and season interaction ^  14  2. The interaction effects of species and seasons on protein levels ^ 15 3 The interaction effects of fodder species and seasons on tannin levels ^ 17 4 Proportional contribution of intestine to total tract DM digestibility^47 5. The interaction effects of species and treatment on total tract digestion ^ 48 6. The interaction effects of fodder species and treatments on intestinal protein digestibility^  50  7. Proportional contribution of intestine to total tract CP digestibility^ 51  ix  List of appendix Figures  Figure.^  Page  I-IV Percentage microbial activity change with increase in PEG concentration in the MPT species ( leucaena, sesbania, Calliandra and gliricidia) ^ 82 V-WI Percentage microbial activity change with increase in PEG concentration in forage crops (velvet bean, D. intortum and cassava) ^83  x  ACKNOWLEDGEMENT I would like to express my sincere gratitude to my research supervisor Dr. J. A. Shelford for his capable guidance and invaluable advice throughout the progress of my research and thesis preparation. I am most grateful to members of my graduate committee to DR. L. J. Fisher of Agriculture Canada Agassiz, Dr. R. M. Tait of Animal Science Dept. for reading and commenting on this thesis, and Dr. M. Pitt Associate Dean Faculty of Agriculture for invaluable advice during the preparation of this thesis. I am also grateful to my fellow students, the technical staff (both laboratory and farm), and members of staff Kenya Agricultural Research Institute (K.A.R.I) Embu, Muguga and Mtwapa for their support and assistant at different stages of my research. My gratitude also goes to K.A.R.I and Canadian International Development Agency (CIDA) for awarding the scholarship that made this study possible. Finally my special thanks goes to my dear wife Mary and children; Wanjiru, Kang'ara, Wanyoike and Mucina for their inspiration, patient and understanding during that long period of my absence. You are indeed a special family.  xi  GENERAL INTRODUCTION  The soil and water conservation technology involving the incorporation of MPT trees into arable lands is gaining acceptance in the farming community in Kenya (Scherr, 1992). This is because these trees not only provided fuel and conserve soil and water, but also are a source of high protein forage for livestock feeding. Using the current knowledge, the dairy cattle fed on these forages have not been able to exploit fully the potential of these crops. The milk yields of cows fed on these fodders have been lower than expected. Investigation on the level of feeding of these fodders to dairy cows have been going on although most of them are based on the protein content of the feed. Some concluded studies on cattle supplemented on tanniferous feed indicate a marginal increase in milk yield (Mwinga et al., 1992). Tannins, common to most MPT and some forages have been implicated as the cause for the low animal performance (Reed, 1986; Lefroy, 1992). There is therefore, a need to provide more information on the levels and distribution of these tannins in different altitudes and seasons. Farmers should be able to identify the species that are suitable to their area considering the elevation of their land, and when to harvest the MPTs considering that tannin levels are likely to change with the seasons. There is also a need to determine the effect of tannins in these MPT species and the conventional forages on digestion and utilization of nutrients by dairy cows so that the dairy farmers can be advised accordingly. This study therefore identified two areas requiring immediate attention. Mainly determination of levels of tannin and their distribution and the nutrient digestibility. The two were investigated in two phases and reported separately in two chapters. Phase one was mainly a survey into existing forages, which was partly based in Kenya for field work, and partly at UBC for laboratory work. Phase two was mainly digestibility trials, all conducted at UBC. 1  CHAPTER 1 DETERMINATION OF TANNIN LEVELS AND THEIR VARIATION 1.1 INTRODUCTION Energy and protein are the two major nutrients limiting dairy cattle production on small scale mixed farms in Kenya. A large proportion of these nutrients are provided by fodder grass, pastures or their combination. The level of supplementation with commercial concentrates currently being practiced is inadequate and provides only a small portion of nutrients required. Commercial dairy feed supplements are expensive. Consequently, farmers prefer feeding relatively cheap alternative sources to supply these nutrients. During the dry periods, growth of fodder and pastures after cutting is usually negligible. To counter these dry period shortages, cattle are fed with farm crop residues, namely: maize stovers, sorghum and other cereal straws. These residues have a low protein content ranging from 3-7% of the dry matter (DM), but have high of structural carbohydrates content whose digestibility partially depends on the level of crude protein (CP) intake (Kossila, 1984). Inexpensive sources of protein have been leguminous pasture species namely:Kenya white clover (Trifolium semipilosum.) silver leaf desmodium (Desmodium uncinatum), green leaf desmodium (Desmodium intortum) and alfalfa (Medicago sativa). These legumes were conventionally sown with grass as a mixed sward in a  grazing system of management up till the early 1970's. Since then dairy cattle management has been changing gradually to semi-zero and zero-grazing systems of management. In these systems, forage is cut and fed to the animals in stalls. The change from grazing was prompted by the gradual decrease in land available for pastures; a product of land subdivision due to high population pressure. 2  Under the zero-grazing system, Napier grass has been widely adopted due to its high dry matter yield per unit area. Alfalfa is sown in some areas where the soils are not very acidic. Dual purpose sweet potato (Ipomea batatas) varieties are grown in most areas to provide tubers for the family and vines for animal consumption. In the dry period both alfalfa and sweet potato vine yields are adversely affected by moisture stress. Since the majority of the farmers depend on rain water, and the land size limits availability of forage for conservation, protein deficiency in zero-grazed animals is rampant. There is a need for the appropriate protein sources compatible with zerograzing type of management, especially in areas where alfalfa can not thrive. The increase in population density has also resulted in increased tree felling for fuel, construction and cropping. As early as 1970 environmental degradation was evident in the deforested parts (Huggan and Westly, 1989). In 1978, some government ministries, local and international institutions, started working together in rural reafforestation and research in agroforestry in order to conserve the environment (Huggan and Westly, 1989). Since 1980, agroforestry practices were disseminated by both government and non-governmental agents, and are gaining wide acceptance (Scherr, 1992). These technologies involved integration of multipurpose trees with the conventional food and cash crops. The purpose of agroforestry is to conserve soil and water, provide wood for construction and fuel, and forage for livestock feeding. The introduction of multipurpose trees (MPT) in the dairy producing areas therefore provided an acceptable alternative source of protein. Most MPT are rich in protein (Jones, 1979), and their deep rooting system enables them to remain green even in dry period. This characteristic makes them the only source of green foliage when grasses and 3  herbaceous plants are dry, and probably the only inexpensive source of protein during this period (Lefroy et al., 1992). Feeding regimes which incorporate MPT and herbaceous forages like Desmodium sp. and cassava (Manihot esculenta) foliage are based mainly on their protein levels. (Leucaena leucocephala) is an exception to this, because in addition to protein content, mimosine levels limit its inclusion in dairy cattle rations to 25% of the DM (Jones, 1979). It has been established that the majority of these MPT have high levels of tannins (Lefroy et al., 1992). In East Africa, browse species were found to contain phenolics which include tannins to levels of up to 50% of organic matter (OM) (Reed, 1986). Tannins from different plant species have either adverse or beneficial effects on animal performance depending on their chemical structure (Waghorn et al., 1986; Kumar and Vaithiyanathan, 1990). Quantities of tannin in these MPT should also be taken into consideration when formulating cattle rations. Therefore, there is need for more information on the type and quantities of tannin in MPT and their role in the digestion of major nutrients. The objective of phase one in this study was:To determine the levels of tannin and their variation in multipurpose fodder trees and herbacious crops of Kenya. 1.2 LITERATURE REVIEW 1.2.1 Definition and significance of tannin  Tannins are secondary plant metabolites whose functions in plants are not well known (Waterman, 1992). Plants respond to attack by herbivores, fungi, insect and bacteria by increasing tannin levels (Bate-Smith 1973; Feeny 1976). Barry and 4  Duncan (1984) reported a decline in feed intake for sheep fed on high-tannin forage. Van Hoven (1984) also reported that greater kudus (browsers) died of starvation despite plenty of tanniferous browse. In birds, high selectivity for low-tannin sorghum was evident in a mixed field of low- and high-tannin cultivars (Butler, 1989). Such findings have led some workers to conclude that tannins serve as a chemical defense. These views however, are being challenged because some animals show some preference for tannic foliage (Robbins et al., 1987a). It has now been established that animals avoid excessive intake of high levels of tannins, but the tolerance of tannins varies from species to species (Mole and Waterman, 1987a). The significance of tannin in animal nutrition arises from the fact that it binds proteins to form a tanninprotein complex (Swain, 1979; Butler, 1989). The complex so formed is resistant or slows degradation by animal or microbial digestive enzymes (Swain, 1979; McLeod, 1974; Kumar and Singh, 1984) The ability of tannin to bind with protein has been exploited for centuries to convert hides and skin into leather. Tannin cross links with collagen chains of the hide to make durable, bacteria resistant leather (Mangan, 1988; Haslam, 1989). These tannins were extracted with water from macerated plant parts (Haslam, 1989). Tannins were then defined as water-soluble plant extracts with the ability to tan hides into leather. Since then it has been found that these extracts were composed of large molecules of phenolics. Tannin is therefore defined as water soluble plant phenolic metabolites with molecular weight ranging between 300 and 3000 Daltons, and ability to bind and/or precipitate proteins.  5  1.2.2 Chemical classification of tannins. Tannins are classified into two categories (Haslam, 1989) namely: hydrolysable and condensed tannins. 1.2.2.1 Hydrolysable tannins. Hydrolysable tannins consist of a carbohydrate moiety in which the hydroxyl groups are esterifial to gallic, digallic and hexahydroxydiphenic acids. The hydrolysable tannins are easily hydrolyzed by heating with weak acid or enzymes such as penicillin tannase (McLeod, 1974). The ability of hydrolysable tannin to bind and precipitate protein is controversial. Some workers state that hydrolysable tannins form a stronger precipitate with protein than condensed tannin (Cooper and Owen-Smith, 1985), while others maintain that hydrolysable tannin bonds are weak and have no effect on digestibility of protein in ruminants (Hagerman et al., 1992). The hydrolysable tannin protein complexes are hydrolyzed in the rumen to gallic acid which is absorbed and excreted in the urine.  1.2.2.2 Condensed tannins. These are polymeric compounds of flavan-3-ols or flavan-3-4 diol and related derivatives. About 50% of condensed tannin, when heated with mineral acid, produces a red color typical of anthocyanidins such as cyanidins and pelargonidin (Mangan, 1988). They are therefore generally referred to as proanthocyanidins. Unlike hydrolysable tannins, condensed tannins do not have a carbohydrate core. Two major examples of condensed tannins are procyanidins and leucoanthocyanins.  6  1.2.3 Occurrence and ecological distribution of tannins Tannins occur in almost all plant genera, but mainly in dicotyledons and particularly woody or tree legumes (McLeod 1974). Mangan (1988) reported that by 1954 over 500 species had been shown to have varying levels of condensed tannin. The levels of tannin vary with plant species (Ford, 1978). Although tannin can be found in all parts of the plant, some parts have higher tannin content than others. In Gramineae, such as sorghum and finger millet, tannins are found in grains only and none in the vegetation (Barney et al., 1989). Swain (1965) found that tanner's sumac tree (Rhus coraria) leaves, bark and wood had 27, 6, and 4% tannin in the DM respectively while quebracho tree (Schinopsis quebracho) had 7% in the bark 2.5% in the soft wood and 20% in the hard wood (Leinmuller et al., 1991). Within the same species tannin may vary with varieties (Akbar and Gupta, 1985). Usually the dark or brown coated seed of sorghum and finger millet have higher tannin content than the light coated (Bullard et al., 1981). The concentration of tannins or polyphenols changes in the same plant part with maturity. In sorghums, tannins increases sharply after pollination up till dough stage and decline as the grain dries (Bullard et al., 1981). In plum fruit, the tannin content is higher when green and declines as it ripens (Goldstein and Swain, 1963). Changes in tannin content with age or maturity is attributed to polymerization of phenolic monomers which are predominant in the immature stage. Under in vitro conditions, bovine serum albumin (BSA) precipitation increased with degree of polymerization (Horigome et al., 1988). This is probably because monomers are too small to cross link effectively with protein in young plants (Hagerman et al. 1992). After maturity and at ripening, tannins decline due to polymerization, becoming too  7  large to complex protein effectively (Goldstein and Swain, 1963; Bullard et al., 1981). Deficiency in major soil nutrients such as nitrogen (N), phosphorus (P) and potassium (K) results in higher levels of tannins in growing plants (Barry and Forss, 1983; Bryant et al., 1987). Soil pH affects the availability of some major nutrients such as P through fixation in acidic soil, thus affecting tannin distribution (Gartlan et al., 1980). Tannin levels in plants also vary with season due to changes in soil nutrient availability as a result of precipitation. This moisture change affects growth and alters the DM:tannin ratio (Coley, 1988). Increase in daylight, moisture, and temperature enhance plant growth in summer or the rainy season in tropical climates. Warmth and moisture also favor insect and microbial multiplication resulting in increased infestation and herbivory on plants (Coley, 1988). The plant responds to these attacks by further production of tannin, forming galls and callus in the affected parts (Feeny and Bostock, 1968; Haslam, 1989).  1.3 MATERIALS AND METHODS 1.3.1 Sample collection Foliage samples of eight Kenyan multi-purpose fodder trees and crops, were collected at Mombasa (low 300 m above sea level (ASL)) and Embu (high 1500m ASL) as summerised in Tablel. In Embu, samples were collected in both dry and wet season of February and June 1991 respectively, while in Mombasa only wet season samples were collected in early July 1991. The species collected were: Leucaena leucocephala, Sesbania sesban. velvet bean Mucuna pururiens, Calliandra calothyrsus, 8  Gliricidia sepium, cassava foliage Manihot esculenta, green leaf desimodium Desmodium intortum and silver leaf desimodium Desmodium uncinatum. Fresh  samples were oven dried at 60° C for 3 days weighed, ground and shipped for analysis at the University of British Columbia. 1.3.2 Chemical analysis The dry matter was determined by placing 1.2 to 2.0 g of the sample in a pre-heated  and weighed aluminum dish, and both were oven dried at 105° C for 24 hours and weighed. Two samples per species were used to calculate the percentage DM, using the following formula (W3-W1/ W2-W1) x 100 where W1 = dish weight W2 = original sample and dish weight W3 = cooled dry sample and dish The CP was determined through N determination using the wet oxidation procedure of Parkinson and Allen (1975). Tannin levels were determined using a gravimetric method described by Reed at al. (1985). This method exploits rare earth elements, affinity for phenolics at neutral pH. These elements are used to precipitate total soluble phenolics extracted from plant parts. The method uses ytterbium acetate to precipitate phenolics extracted from forage samples(100mg) using 70% acetone. The precipitates are refrigerated for 24 hours. After refrigeration, the ytterbium-tannin precipitates are then filtered, subjected to chlorophyll removal by washing with acetone, oven dried, weighed, ashed and reweighecl to determine the ytterbium-precipitable tannin. This method is inexpensive and avoids the problems associated with colorimetric methods of tannin determination. samples were analysed in triplicate and the mean of the two samples used for analysis. 9  Tablel .The sample collection and analysis summary Altitude  Dry season  Wet season  High  collected analyd/sam  collected analyd/sam  Leucaena  2^3  2  3  Sesbania  2^3  2  3  Gliricidia  nil^-  2  3  calliandra  2^3  2  3  Velvet bean  nil  2  3  D. intortum  2^3  2  3  D. uncinatum  2^3  2  3  Cassava  2^3  2  3  Leucaena  nil^-  2  3  Sesbania  nil  2  3.  Gliricidia  nil  2  3  Calliandra  nil^-  2  3  Velvet bean  nil^-  2  3  Low  1.3.4 Statistical analysis The analysis of variance and means comparisons (least significant difference) were carried out using the general linear model procedure from the SAS package version 6.04(SAS/STAT software, 1985), as an 8x2x2 factorial experiment. The model used was; Yijk =1-t ±0i+Ij+70-07ix+ Otij +Eiji( where YijK = measured variable = mean 13i = species effect ii = altitude effect YK  = seasonal effect  f3yhc= species x seasonal effect 13tii = species x altitude effect cjj = error effect  11  1.4 RESULTS 1.4.1 Tannin content The tannin content is presented as ytterbium precipitatable (Y-ppt) phenolics. The Y-ppt phenolics estimate the percentage total phenolics in the DM, which include hydrolysable tannins, condensed tannins and their monomers. The percentage Y-ppt phenolics varied significantly with species (P <0.05). Their least square means (LSM) ranged from 16.08 (± 2.39) % of the DM in Gliricidia sepium to 30.31 (±2.42) in Desmodium intortum (Table 2). The altitude  did not have significant effect on Y-ppt phenolics contents,nor did altitude interaction with species. Although seasons do not indicate significant differences on tannin levels, their interaction with species were significant (P< 0.05). Species behaved differently from each other in different seasons resulting in varying changes of tannin content (Figure 1). Calliandra tannin content showed a sharp increase (from 17% in dry season to 30% in wet season) in the wet season and decline in the dry season. Cassava tannins on the other hand were higher in dry season compared to the wet season decreased in wet season and increased in dry season. In D. uncinatum, changes in tannin were gradual but increased in wet season and declined in dry season. Leucaena, sesbania and D. intortum tannin remained almost unchanged regardless of season. 1.4.2 Protein content The protein content presented in Table 2 showed significant differences (P <0.05) between species and seasons. The altitude had no significant effect on protein content. There was a significant interaction between species and season (P < 0.05)(Figure 2). All the species showed a positive protein increase in the wet season with leucaena having the highest protein increase.  12  Table 2 Mean variation of tannin and protein with species Tannin(s.e. m)  Protein (s. e. m)  % of the DM  % of DM  Calliandra  25.96cd ±1.88  23.42cde +1.11  Cassava  26.24cd ±2.42  25.77de ±1.38  D. intortum  30•31d ±2.41  18.20a^±1.38  D.uncinatum  23.53bc ±2.41  19.06ab ±1.38  Gliricidia  16.08a ±1.39  20.69bc ±1.36  Leucaena  24.16c ±1.88  25.86e^±0.99  Sesbania  27.97cd ±1.88  23.49cd ±1.06  Velvet Bean  17.33ab ±2.39  24.90cde ±1.36  Values followed by similar superscript in a column are not significantly different(P <0.05).  13  35  -  .o 30  -  .---,  C/1  Z  > CD^25  ff.-4  r. ZS  E- 20  15  _ -  1  Dry  ^  1  Wet  Rainfall Season Figure 1. Fodder species and season interaction. • Cassava, 7 Desimodium, intortum • Sesba,nia, O Leuca,ena„ V Desmodiwm, uncinatu-m„ 0 Callia,ndra  14  15  32 30 28 26 24 2? 20 18 16 1 12  DRY  ^  WET  Rainfall Season Figure 2. The interaction effects of species and seasons on protein levels. • Cassava, 7 Dessmodium tin,tortum. • Sesbania, 0 Leucaena, V Desmodium uncinatum. 0 Calliandra  15  1.4.4 Tannin : protein ratio. The tannin:protein ratio Table 3. was obtained by dividing the percentage Y-ppt phenolics of a species by the percentage protein content of the same species. These ratios indicated the amount of tannin per unit of protein, and varied significantly (P <0.05) with species. Tannin:protein ratio also varied significantly with seasons. The interaction between species and season ffigure 3) was also significant (P <0.05).  Table 3. Variation of tannin : protein ratio with species Species  Tannin:protein (s.e.m)  Calliandra  1.081)  ±0.09  Cassava  1.15b  ±0.11  D. intortum  1.70d  ±0.11  D. uncinatum  1.21c  ±0.11  Gliricidia  0 . 88ab  ±0 . 11  Leucaena  1.02ab  ±0.08  Sesbania  1.28  ±0.08  Velvet bean  0.78a  ±0.11  Values with similar superscripts in the column are not significantly different (P<0.05)  16  Figure 3. The interaction effects of fodder species and seasons on tannin levels. • Cassava, 7 Desmoclium tintortum, • Sesbania 0 Leucaena, V Desmodium uncinatum, 0 Calltiandra  17  5 DISCUSSION In this study, tannins in the multi-purpose fodder trees and crops indicated significant (P < 0.05) differences between species which corroborates the observations made by Martin and Martin (1982), Reed (1986), and Burritt et al. (1987). However, even where similar species were studied, the methods of analyses were different from the one used in this study and therefore the values may not be comparable. In this study, tannin levels for gliricidia were lower than those reported by Vadiveloo and Fade! (1992), but those for leucaena were similar to theirs. The tannin levels did not vary with altitude. This contrasted with the findings of Gartlan et al. (1980) who reported significantly higher tannin content foliage number of species at lower altitude (<650 m above sea level (ASL)) compared to similar species at high altitudes (>1300 m ASL) in the tropics. The results from the current study agree with those obtained by Baldwin et al. (1987), who found no significant tannin variation with geographical location. Contrary to the findings of Feeny and Bostock (1968) in oaks, Shultz et al. (1982) and Baldwin et al. (1987) in sugar maple and birch, seasons had no significant effect on tannin content, which corroborated the findings of Cooper et al. (1988) in several South African savanna browse species. However, the significant interaction observed between species and season in this study, is an indication that MPT and crop species respond differently to moisture and temperature changes in tannin production. Cassava tannins were higher in the dry season and lower in the wet season, which was opposite to the other species. With the other species investigated, tannin levels were found to be higher in the immature stage of growth, which is often in the wet season, and then declined with maturity in the late wet and dry period (Gartlan et al., 1980; Schultz et al., 1982; Provenza and Malechek, 1984). The magnitude of decline in 18  tannin content varied in each species. Calliandra and D. uncinatum showed a remarkably sharp decline from wet to dry season, while Sesbania and D intortum had showed little change. Different species will therefore have altered levels of tannin depending on the season even in the same environment. If leaf life span expectation and light intensity result in increased production of tannin (Coley, 1988), then these tropical tree species would be expected to have high tannin content since they are evergreen, and light is not limiting in the tropics. In most studies involving tannin distribution (Feeny and Bostock, 1968; Gutlan et al., 1980; Baldwin et al., 1987; Cooper et al., 1988), trends of changes in tannin content  from bud to mature leaf were monitored in all the stages of development. In this study the samples were taken once in each season, and probably that is why seasonal changes in tannin content could not be detected, hence the need for more sampling times. Protein content differed significantly (P <0.05) with species and season. This is typical of most tropical forages, whose crude protein content is higher during the growing period, and declines with maturity. The CP levels are at the lowest in the dry period (Minson, 1988). The CP contents obtained in this study were similar to those reported in other studies (Jones, 1979; Reed et al., 1982; Akbar and Gupta, 1985; Van Eyes et al., 1986). The observed differences (P <0.05) in tannin: protein ratio between species and season in this study were similar to those reported by Tangendjaja et al. (1986). Elevating the protein content in tanniferous forages reduces the tannin : protein ratio, and results in increased acceptability of forages and performance of the animal (Provenza and Malechek, 1984; Cooper et al., 1988). Therefore the observed changes in tannin: protein ratios resulted from the significant seasonal changes in protein 19  content, while seasonal tannin changes were negligible. The animal performance attributable to tannin content would vary with the changing ratios. This may be true whether the CP changes are seasonal or there is a deliberate addition of protein into the animal ration. This is because tannins are competitive inhibitors of proteases (Mole and Waterman, 1987b). Therefore increasing the protein content results in more unoccupied binding sites on the protein for enzymes to attach. Proteolysis will then proceed though at a reduced rate relative to low tannin forages. 1.6 CONCLUSION In this study all the MPT and crops investigated had tannins characteristic of the specie. The average tannin levels did not vary much with the season, but the protein content varied with the season. This affected the tannin per unit protein (tannin : protein ratio), which may lead to seasonal change in animal performance. Frequent cutting of these fodders under zero-grazing type of dairy cattle management results in forage composed of young leaves and shoots which are rich in protein . This is likely to reduce the tannin: protein ratio. On the other hand, stress due to continuous disturbance through frequent defoliation, may lead to increased tannin content and the result would be an increase in tannin: protein ratio. There is need for further investigation to elucidate: (1) the effects of frequent cutting of MPT and crops on the tannin: protein ratio, (2) the effect of changing these ratios on animal performance (3) and the seasonal tannin distribution pattern.  20  CHAPTER 2 DETERMINATION OF TANNIN EFFECT ON PROTEIN DEGRADATION IN THE RUMEN AND INTESTINAL DIGESTION  2.1 INTRODUCTION The survival of ruminant animals on fibrous feeds is due to the symbiotic relationship with rumen micro-organisms. Of the rumen microbes, bacteria are the most abundant. The other micro-organisms include protozoa and fungi (Orskov, 1982). Once the animal is fed, a large portion of the dietary protein is hydrolyzed into short chain peptides or amino acids. These products are either incorporated directly by the microbes or deaminated to ammonia (NH3) by the bacterial deaminases. The resulting NH3 becomes the primary nitrogen (N) source for microbial protein synthesis (Armstrong and Weekes, 1983). About 75% of rumen microbial N is derived from NH3 (Oldham and Parker, 1981). Excess ammonia diffuses through the rumen wall into the blood stream, from where it finds its way to the liver, is converted into urea and excreted in urine, or recycled back into the rumen through saliva. The dietary protein and the recycled NH3 are not the only sources of N for microbial protein synthesis. The non-protein nitrogen (NPN) inherent in some feeds such as silage or urea are important sources of N. The NPN can provide sufficient N needs for maintenance and substantial milk production (NRC 1988). However, for high levels of production, some additional true protein would result in even better performance, because it not only maximizes microbial protein production but also increases the undegraded protein available for the host enzymatic digestion. 21  Some dietary protein escapes degradation in the rumen and together with microbes, are flushed into the lower gut (abomasum and intestines) where they are digested to provide the host animal with amino acids. The extent of protein degradation ranges from 35-80 % (Oldham, 1977), and depends on the chemical nature of the feed, mode of processing, and rumen outflow rate. The outflow rate is in turn affected by the frequency of feeding. The higher the frequency, the higher the outflow rate and the lower the extent of degradation, hence more protein bypass. The presence of some chemicals such as tannins and formaldehyde has been shown to reduce the extent of protein degradation and increase the dietary protein bypass. Digestion of the bypass proteins in the intestines is not 100% efficient as some, depending on their chemical composition, are voided with feces undigested. Overprotection of protein can lead to higher fecal nitrogen loss. In the previous chapter the levels of tannins and their variation with species, altitudes and seasons were reported. The objective of this study was; To determine the effect of these tannins on ruminal degradation and intestinal digestibility of protein.  2.2 LITERATURE REVIEW 2.2.1 Formation of tannin-protein complexes 2.2.1.1 Mechanism  The ability of tannin to bind and precipitate protein is inherent in its chemical structure. Hydrolysable tannins are polymers of seven to nine carbon ring (C7-C9) phenolic acids such as gallic and coumaric acids, while condensed tannins are polymers of flavanoid which is a C15 ring structure. Both hydrolysable and 22  condensed tannins have numerous phenolic hydroxyl groups exposed and unreacted on their surfaces. It is believed that the phenolic hydroxyl groups forms a hydrgen bond with the carboxyl group of the peptide to form a complex (Leinmuller et al. 1991). At one time this hydrogen bonding was claimed to be the main binding mechanism between tannins and proteins (Gustayson, 1954). Since then hydrophobic, electrostatic and covalent interactions have been suggested (Butler et al., 1984). Oh et al. (1980) following an intensive study involving cytochrome C tannins immobilized in a sepharose column and several eluents, provided strong evidence in favor of hydrophobic interaction over hydrogen bonding and the other interactions. Hydrophobic interaction is possible because both tannin and protein have hydrophobic regions, mainly the aromatic nuclei of tannin and the aliphatic and aromatic side chain of amino acids (Oh et al. 1980). Oh et al. (1980) also ruled out the possibility of electrostatic interaction for condensed tannin in acidic and neutral media. Butler et al. (1984) working with sorghum tannins found no evidence in support of covalent bonds. As for electrostatic interaction, Butler et al. (1984) found that binding was possible in a very high pH medium, and even then most proteins would be negatively charged, hence repulsion forces rather than attraction would be in effect. They however came out strongly in favor of both hydrogen and hydrophobic bonding. In this case then small phenolic molecules (monomers, dimers, trimers etc.) would be less effective in protein binding because of their small hydrophobic region. If present in large quantities, though not polymerized, small phenolic molecules can bind protein just like phenolic polymers such as tannins (McManus et al., 1981). 2.2.1.2 Specificity of Tannins  23  Tannins were assumed to be indiscriminate binders of all proteins (Goldstein and Swain, 1963). However studies by Butler et al. (1984) using affinity chromatography and a competitive binding assay, found that the proline rich proteins had higher affinity for tannins than the low proline proteins, large proteins with loose open structure tended to bind strongly with tannins, and presence of proline in the media tended to open the protein structures. An earlier report (Hagerman and Butler, 1981) showed that proanthocyanidins selectively precipitated out one protein in the presence of a large excess of another protein. Glycosylated proteins were reported to have low affinity for tannins (Butler et al., 1984; Strumeyer and Malin, 1970) although these findings are contrary to those of Asquith et al. (1987). It can then be concluded that tannins do not bind all protein equally. Some proteins are bound more strongly and faster than others. Gelatin for example, binds with tannin faster and more strongly than some proteins (Calderon et al., 1968). Condensed tannins from various plant species show structural differences. Some minor differences such as linkage isomerism at C3 and C4 may result in different affinity for proteins by relatively similar tannins (Clausen et al., 1990). This and the degree of polymerization could probably account for the differences in ability to bind protein, observed between condensed tannin from different plant species (Asquith and Butler, 1985). 2.2.1.3 Factors influencing tannin-protein interaction  The interaction of protein and tannin is influenced by metallic ions in the media. Divalent cations such as Ca2 + and Mg2 + enhances the precipitation of protein by tannin more than monovalent cations such as Na+ and K+ (Martin et al., 1985). The pH of the media also influences the ability of tannin to bind with protein. Jones and Mangan (1977) found that leaf protein fraction 1 formed stable complexes with 24  tannins between pH 3.5 and 7.0. Below pH 3.0 and above pH 8.0 the complex was unstable and easily dissociated. The interaction between pH and metallic ions has a complementary effect on precipitation of protein by tannin. Martin et al. (1985) found that at pH 6.15 and 6.90, the presence of sodium and potassium ions favored the precipitation of ribulose1,5 bisphosphate carboxylase (RUBPC) by tannic acid. At pH 6.15 in the presence of sodium and potassium salts, 49% of RUBPC in the media was precipitated and only 16% in the absence of salts. At pH 6.90 in the presence of salts, 84% of RUBPC was precipitated compared to 10% without salts. Higher pH above 7.55 resulted in little or no precipitate even in the presence of salt (Martin et al., 1985). Presence of detergents in the media result in the reduction of tannin-protein precipitation. Lysolecithin, a surfactant found in gut fluid of some insects, completely prevented precipitation of RUBPC by tannins at pH 6.15, 6.90, and 7.55, even in the presence of salts (Martin et al., 1985). Sodium dodecyl sulfate (SDS) is an effective solubilizer of the tannin-protein complex (Oh et al., 1980). Mammalian bile cholic acid dissolves precipitates of bovine serum albumin (BSA) and tannin when added at concentration similar to that found in the small intestine of humans (8mM) (Mole and Waterman, 1985). Polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) have been known to have very high affinity for tannin. They have therefore been used in purification of plant enzymes to prevent tannins from interfering with enzymes (Glen et al., 1972; Rayudu et al., 1970). PEG (mol. wt. 4000) effectively solubilized a complex of tannin and leaf protein fraction 1 under in vitro conditions (Jones and Mangan, 1977).Under in vivo conditions, spraying high tannin Lotus pedunculatus with PEG reduced the reactive tannin from 63g to 7g per kg DM and this resulted in enhanced intake and digestibility of DM, and total nitrogen digestibility (Mangan, 1988). Studies involving animals indicated that PEG applied through spraying or 25  drenching, reduced the effect of tannin on protein utilization (Barry and Duncan, 1984; Barry and Manley, 1986; Nunez-Hernandez et al., 1991). 2.2.2 Effect of tannin on animals 2.2.2.1 Intake  The ability of tannins to bind and precipitate protein is believed to be the cause of the decreased voluntary feed intake observed in animals fed on some tannin containing feeds (Barry and Duncan 1984, Van Hoven 1984, Cooper and Owen-Smith 1985, Kumar and Vaithiyanathan 1990). The intake reduction is attributed to binding of dietary protein, salivary mucoprotein and mucosal epithelial cells (Provenza and Malechek 1984). This causes a diffused feeling of extreme dryness and bitter taste in the mouth and throat of the animal commonly referred to as astringent (Mole and Waterman 1987a), prompting the animal to avoid tanniferous feeds (Haslam 1989). However, studies by Clausen et al.(1990) indicated that the snowshoe hare preferred bitterbrush foliage over blackbrush despite the fact that bitterbrush tannins have higher protein precipitating capacity than blackbrush tannin. Cooper et al. (1988) found that, despite the fact that Acacia nilotica has very high tannin content (about 300 mg/g) which taste highly astringent, it was highly favored by impalas over other less astringent species. Robbins et al. (1987a) could not find conclusive evidence that protein precipitating ability of tannin is an effective defense against herbivory. The three studies therefore question the validity of tannin as an inhibitor of voluntary intake. Jones and Mangan (1977) found that bovine sub-maxillary mucoprotein could bind with tannins only at temperatures below 250 C, and dissociated at temperatures above 250 C. This ruled out binding of salivary mucoprotein at normal body temperatures. Robbins et al. (1987a) suggested that some depolymerisal tannins are absorbed into the blood stream and most likely inhibit intake through toxicity in the 26  liver. A combination of factors appears to be involved in inhibition of voluntary feed intake and tannin is one of them (Cooper et al., 1988; Clausen et al., 1990). 2.2.2.2 Digestibility and animal performance 2.2.2.2.1 In the monogastric  Tannins ingested with food have been found to inhibit digestion of food both in monogastric and ruminant animals. In pigs, feed intake and digestibility values are lower in pigs fed on high than low tannin sorghums diets (Cousins et al., 1981, Mitaru et al., 1984). Digestibility and growth reduction have also been reported for rats fed on tannin containing feeds (Joslyn and Glick, 1969; Jambunathan and Mertz, 1973; Horigome et al., 1988) and in chickens fed on sorghum based diets (Armstrong et al., 1973). The reduction in digestibility was attributed to irreversible binding of dietary protein by tannins, forming a tannin-protein complex that resists the effect of digestive enzymes (Mole and Waterman, 1987a; Horigome et al., 1988) or to binding digestive enzymes and inactivating them (Goldstein and Swain, 1965) or to binding both enzyme and substrate (Hagerman and Butler, 1978; Horigome et al., 1988). The tannin-protein complex is then passed out in feces undigested, hence the increased fecal nitrogen observed in both humans and animals (Kumar and Singh, 1984). Prolonged feeding of animals with tanniferous feed leads to a decline in growth, weight and egg production in the case of chickens (Armstrong et al., 1973; Schaffert et al., 1974; Sell and Rogler, 1983; Mitaru et al., 1984; Myer et al., 1986). The binding of enzymes by tannin as an inhibitor of protein digestion has been disputed following the finding that most enzymes isolated from rats feeding on tanniniferous diet are fully active (Mitjavila et al., 1977). Mole and Waterman (1987b) also found that tannic acid does not bind with tryptic enzyme but rather competes with it for protein substrate. In some cases tryptic hydrolysis of BSA was increased in the presence of condensed tannins (Mole and Waterman, 1985; Mole and 27  Waterman, 1987b; Oh and Hoff, 1986). Therefore tannin inhibits protein digestibility by depriving enzymes of the substrate in a tannin-protein complex. The tannin-protein complex can form only under suitable pH and charge, in a detergent-free environment, thus showing some environmental selectivity and specificity to some proteins. The selectivity of tannin through a high affinity for proline rich proteins, the pH, and the surfactants have been exploited by animals to counter the effects of dietary tannin on the dietary protein. The raised midgut pH in some insect herbivores dissociates the tannin-protein complex, freeing protein for digestion in the gut (Berenbaum, 1980). Humans, some rodents, domestic goats and some wild browsers after a period of exposure to tannic diets show some degree of tolerance to tannin due to the production of salivary proline-rich protein (PRPs) (Mehansho et al., 1983; Robbins et al., 1987b). These PRPs bind with tannin leaving the dietary protein to digestive enzymes (Mehansho et al., 1983). The elevated fecal nitrogen observed in some animals feeding on tanniferous feed could have originated from the PRPs, and do not reflect the apparent digestibility of the dietary protein. The PRP production and other adaptative mechanisms enables animals to thrive on tanniferous feed. The effect of tannin therefore will vary with species, age and experience of the animal, the nutrient composition and other secondary metabolites in the feed. Tannin therefore should not be generally regarded as detrimental to animal nutrition. 2.2.2.2.2 Effect of tannins in the ruminant  In ruminants, tannins have been shown to have both positive and negative influence on protein digestibility and animal performance. The negative attributes are similar to those of monogastrics such as reduced apparent digestibility with tanniferous feeds. Robbins et al. (1987a) reported lower digestibility for the high-tannin containing foliage fed to mule deer compared to low tannin foliage. Increased fecal nitrogen and 28  reduced DM digestibility were reported in cattle fed on high tannin Sericea lespecleza (Donelly and Antony, 1969). In goats (Nastis and Malechek, 1981) and elk (Robbins, 1983; Provenza and Malechek, 1984), the growth rate was not affected, although fecal nitrogen was increased by feeding on tanniferous feed. This suggests that the increased N loss in the feces may be non-dietary in origin since the animal performance was not affected, but result from increased consumption of tannins. It probably originates from endogenous PRPs. There are several positive effects of tannin on protein digestion and animal performance that have been reported. These include 1) bloat control, 2) production of growth hormone (GH), and 3) protein protection. McArthur et al. (1964) implicated the fraction one (F1) leaf protein found in fresh alfalfa and clover as the foam causing agent in the bloat animals. Tannins control bloat through binding this highly soluble protein to form a stable complex in the rumen (Jones and Mangan, 1977). Plasma GH in sheep was found to increase linearly with varying levels of condensed tannin of Lotus pedunculatus (Barry, 1984). Growth hormone stimulates N retention and lipolysis, and results in a leaner meat carcass (Barry et al., 1986b). Essential amino acids are needed by dairy cattle to meet the daily maintenance, growth and lactation requirements. These essential amino acids are absorbed from the small intestine of the animal. Their sources are dietary protein that escapes rumen degradation, and the microbial protein. Usually microbial protein will meet maintenance and a substantial part of production needs. For a high milk producer and a fast growing heifer, the microbial source alone may not be adequate, and bypass protein is advocated (NRC 1989). Bypass protein is provided by supplementing the animal with low rumen degradable protein such as meat and bone meal or by protecting high quality highly degradable protein from microbial degradation in the rumen (Orskov, 1982). Heat, 29  formaldehyde and tannin treatments have been found to have a protective effect on protein (Nishimuta et al., 1974, Ferguson 1975; Chalupa, 1975). Ferguson (1975) reported increased wool production when sheep were fed on formaldehyde-protected protein. The problem with formaldehyde is that it affects the availability of three essential amino acids, namely, lysine, tyrosine and cystine (Ashes et al., 1984). Heat treatment was found to increase the quantity of amino acids reaching the abomasum (Nishimuta et al., 1984). The problem with heat treatment is timing and balancing of the heat to ensure protection of protein without affecting intestinal digestion through formation of maillard products (Knipfel, 1981). Dietary tannin binds with Fl leaf protein to form a stable tannin-protein complex at the rumen pH 5.5-6.8 (Jones and Mangan 1977). The complex so formed is not degradable by the rumen microorganism. Therefore the bound protein bypasses the rumen to the abomasum and anterior duodenum where the pH is about 2.5. In this very acidic medium, the complex dissociates and protein is released for digestion by the intestinal enzymes (Jones and Mangan, 1977). Subsequent studies, using Lotus species and PEG to reduce the effect of tannin, reported reduced ammonia concentration in the rumen, increased duodenal N flow and intestinal essential amino acid (EAA) flux (Egan and Ulyatt, 1980; Barry and Manley, 1984; Barry et al., 1986a; Waghorn et al., 1987; McNabb et al., 1993). Unlike formaldehyde, tannins do not affect the availability of EAAs tyrosine and lysine (Waghorn et al. 1987). This is one the advantages of using tannin over formaldehyde. In this study it was assumed that the multi-purpose fodder trees and forage crops have different types and quantities of tannins. It was hypothesized that : a) when these tannins are consumed by ruminant animals, they will bind to form a complex with dietary protein and protect it from microbial degradation in the rumen. 30  b) On reaching the duodenum the complex breaks down and proteins are digested in the small intestine. The effect of tannin on protein degradation in the rumen could therefore be determined through use of the mobile nylon bag technique (Arieli et al., 1988; Faldet et al., 1991). In this technique, feed materials are incubated in the rumen for a predetermined period in small bags, and the same bags are removed, washed and inserted into the intestines through the duodenal cannula to estimates the rumen degradation and intestinal digestibility and the total tract digestibilty.  2.3 MATERIALS AND METHODS 2.3.1 Sample preparation  Seven of the species samples obtained from the high altitude during the wet season were ground enough to pass through a 5 mm sieve in a Wiley mill, and each divided into two portions, to be used in periods one and two. The portions were further subdivided into two halves. To one of the halves PEG was added at the rate of 40 mg per g of tannin as used by Jones and Mangan (1977). Tannin content was determined in part one of this study. PEG was added to suppress the effect of tannin. Approximately 60 g of feed and PEG mixture were placed in 200 ml of water in a beaker and mixed thoroughly with a magnetic stirrer. The other half also was placed in water and thoroughly mixed. All the samples were individually freeze-dried. Approximately 1 g of the dried feed sample was weighed into a pre-weighed, labeled nylon bag and heat sealed. The bag dimensions were 3.5 x 5.5 cm. Pore size was 40 lam, averaged from six samples observed under Jenameci 2 microscope 400X magnification. 2.3.2 In vivo incubation 31  The mobile nylon bag technique as described by Arieli et al. (1988) was used in this experiment. Four Holstein cows fitted with both ruminal and duodenal cannulas, were fed in individual stalls on a mixture of orchard grass and alfalfa hay for 14 days prior to experimentation in June 1992. The PEG treated samples were placed in different animals from the untreated samples. The bags were introduced into large net bags suspended in the rumen, and connected with a nylon cord to the cap of the rumen cannula. Duplicate samples of each forage species were introduced at a time and remained in the rumen for 96, 48, 24, 12, 6, 0 hours, except in 24 and 12 hours where four bags at a time were placed. The placements into different cows were delayed for 20 minutes to give allowance for other activities to be done in between placements. The time 0 samples were incubated at 37° C in a borate-phosphate buffer (pH 6.8) with a gentle shake (in a shaker/incubator) for 30 minutes. All the samples were removed at once and washed in cold tap water for 15 minutes. Except for four samples of each species, from 12 and 24 hours (2 each), all the samples were oven-dried at 60° C for three days and weighed to determine the DM of the remains. The two wet bags per species from 12 and 24 hours were further incubated at 37° C for 2 hours in a pepsin solution composed of 2 g pepsin per liter in 0.1 N HC1. The samples bags were rinsed again and inserted into the intestine through duodenal cannula of the same cow they had been incubated in. The bags were recovered from the feces, washed, oven dried at 60° C for 3 days and weighed for DM determination. The experiment was repeated again in the second period with the same animals, but the treatments were switched so that those animals that received PEG added forages received forages without PEG and vice-versa. The level of PEG was also increased from 40 mg to 100 mg per g tannin (i.e. from 0.04% to 0.10%) . The decision to increase the PEG was due to little response observed in period one, when PEG was applied at the Jones and Mangan (1977) rate of 40 mg per g tannin, which 32  had proven effective with sainfoin (Onobrychis viciifolia Scop) tannins. An in vitro bioassay was conducted separately to determine the optimum PEG levels for each MPT species giving the highest rumen microbial degradation activity over the untreated samples. 100g PEG per g tannin was found to be effective and thus adopted for period two (Appendix C). 2.3.3 Analysis and calculation The DM of the residue was calculated by subtracting the bag weight from the weight  of the dried residues and bag. Residue N was determined using wet oxidation (Micro kjeldahl method) described by Parkinson and Allen (1975). The rumen DM disappearance was calculated by subtracting the residue weight from the initial DM of the sample (determined in part 1). The results of these calculations were fitted to the non linear regression equation P = a +b(1—e-') (Orskov and McDonald 1979). where p is the nutrient disappearance from the rumen over time t, a is the fraction of nutrient that rapidly disappears from the rumen, b is the potentially degradable amount of nutrients with time, and c is the fractional rate of degradation of the b fraction. The parameters a, b, and c, were estimated using a SAS program procedure developed by Agriculture Canada, Research Center, Lethbridge. The effective degradability was also estimated with the same Lethbridge SAS procedure using the bc^  equation p= a + ^ e (-(c+kr) c+k  where k is the assumed rumen flow rates (0.04 and 0.06) and to is the lag. This equation takes into consideration the lag phase. The intestinal DM and CP disappearance was taken as the difference between the whole tract digestion and the rumen disappearance. All the statistical comparisons for effective degradation of DM and CP in the rumen, and the digestible DM and CP reaching the intestines, were done using the general linear model (least square means) of SAS package release 6.04 (SAS/STAT 33  software, 1985). These were analyzed as 7x4x2x2 factorial experiments, with the following model = ,u +1-, + +  B k + Cm ± 131q +^+ TBik  TCnn etiking  where Y= Effective degradability or digestion A= Mean  = Treatment effect A= Animal effect B= Species effect  C= Time effect D= period effect TA= Animal x treatment interaction TB= Treatment x species interaction s = random error The period treatment effect was tested by comparing the controls in both periods separately from the treated because of confounding with treatment effect.  2.4 RESULTS AND DISCUSSION  2.4.1 Dry matter and Crude protein disappearance in the rumen  The effects of tannins on effective degradability of DM at two rumen outflow rates (k) are presented in Table 4. Tannin significantly (P <0.05) reduced the effective degradability of DM of all forages in the rumen. Differences in response to PEG treatment ranging from 41% to 200% in DM effective degradability, for gliricidia and calliandra, respectively were observed between PEG treated and untreated forages. The differences in effective degradability of the DM between species were also significant (P <0.05) (Table 5). Calliandra DM, with effective degradability of 13.8% , was found to be lowly degradable in the rumen followed by D. intortum (21.76%) at k= 0.04, while Gliricidia had the highest DM degradability (46.49%). These DM disappearance results for untreated samples were similar to those reported by Jones et al. (1992) for Leucaena and Calliandra. The DM disappearance values for Leucaena and Gliricidia (untreated) were higher than those obtained by Vadiveloo and Fade! (1992), which could probably be attributed to lower phenolic content in this study compared to theirs. Sesbania DM disappearance values were similar to those reported by Khalili and Varvikko (1992). Except for velvet bean and cassava whose in situ values were not available, the DM disappearance values compared well with the values obtained by Van Eyes et al.(1986). These results corroborate the findings of Barry and Manley (1986) and Waghorn et al. (1987), that PEG reduces the effect of tannin on the DM and enhances the degradation of DM in the rumen.  35  Table 4. Effect of tannin on mean DM effective degradability in the rumen at assumed rumen outflow rate k=0.04 and 0.06 Without PEG (s.e.m)  with PEG (s.e.m)  %increase after  % of initial DM  % of initial DM  PEG addition  Leucaena  38.51 ±1.53  63.71 ±1.53  65.54  Sesbania  44.79 ±1.53  71.58 ±1.53  59.81  Gliricidia  46.49 ±1.54  65.44 ±1.53  40.76  Calliandra  13.80 ±1.53  41.51 ±1.53  200.80  Velvet bean  38.62 ±1.53  57.92 ±1.53  49.97  D. intortum  21.76 ±2.26  55.55 ±1.53  155.28  Cassava  44.82 ±1.54  65.07 ±1.53  45.18  Mean  35.54a ±0.64  60.11b ±0.57  69.13  Leucaena  30.70^±1.53  58.97^±1.54  92.08  Sesbania  35.83^±1.53  66.50^±1.54  85.75  Gliricidia  39.76^±1.56  60.45^±1.54  52.01  Calliandra  9.93^±1.54  37.80^±1.54  281.05  velvet bean  32.59^±1.54  53.20^±1.54  63.24  D. intortum  15.65^±2.29  51.23^±1.54  227.56  Cassava  37.73^±1.56  59.61^±1.54  58.03  MEAN  28.88a ±0.64  55.40b ±0.58  91.82  k=0.04  k=0.06  Values with different supersripts on a row are significantly different (P <0.05)  36  Table 5. DM and CP effective degradability in the rumen mean values of different species at k=0.04 and 0.06 k=0.04 k=0.06 Species  % Effdgrd DM (s.e.m)  % Effdgrd DM (s.e.m)  Leucaena  51.11c  ±1.08  44.83c  ±1.09  Sesbania  58.18f  ±1.08  51.16f  ±1.09  Gliricidia  55.97e  ±1.09  50.10e  ±1.10  Calliandra  27.66a  ±1.08  23.86a  ±1.09  Velvet bean  48.27c  ±1.08  42.89c  ±1.09  D. intortum  38.65b  ±1.37  33.43b  ±1.38  Cassava  54.94d  ±1.09  48•67d  ±1.10  Effdgrd CP  Effdgrd CP  Leucaena  72.12c  ±1.49  77.18c  ±1.42  Sesbania  76.37c  ±1.49  80.97c  ±1.42  Gliricidia  76.30c  ±1.62  80.76c  ±1.55  Calliandra  53.05a  ±1.62  57.18a  ±1.54  Velvet bean  72.58c  ±1.62  77.21c  ±1.55  D.intortum  59.2213  ±1.98  64•34b  ± 1.89  Cassava  72.67c  ±1.62  78.23c  ±1.55  Values with similar superscripts in a column are not significantly different (P <0.05) Effdgrd= effective degradability  37  ^  Tannin affected the rumen DM degradability constants a, b and c differently as shown in Table 6. Tannin significantly (P <0.05) lowered the rapidly degradable fraction a and increased the time dependent degradable fraction b, and at the same time decreased the rate c at which b was degraded. These alteration resulted in the low DM effective degradability shown in (Table 4). Table 6. Effect of tannins on the degradation constants of DM and CP Fraction^Without PEG (s.e.m)^With PEG (s.e.m) DM Lag^4.6a^±0.50^3.20a ±0.47 a^8.51a ±1.41^37.65b ±1.28 63•59b ±2.44^42.37a ±2.22 4.66a ±0.53^6.41b ±0.48 CP Lag^3.07a ±0.75^4.46a ±0.69 a^39.99 ±1.19^60.29b ±1.10 53.37b ±1.57^34.77a ±1.45 5•90a ±0.63^8.71b ±0.58 Values with similar superscripts in a row are not significantly different (P<0.05) Similar low rumen DM disappearances has been reported by other workers on different tanniferous feeds (Chiquette et al., 1988; Mangan, 1988; Kumar and Vaithiyanathan, 1990). Tannin inhibits the carbohydrate degradation and activity of cellulolytic bacteria by binding with structural carbohydrates (Jung, 1985; Barry et al., 1986a). Hence the lower DM degradation values observed this study. In his 38  ^  studies, Jung (1985) found that the cellulose digestibility potential was not affected by cinammic acid (phenolic), but the rate of degradation was significantly affected.  Table 7. Animal variation in effective degradability of the DM at two rumen out flow rates COW  No.  % of initial DM  % of initial DM  k=0.04 (s.e.m)  k=0.06 (s.e.m)  1  58.22c  ±0.82  53.47c  ±0.83  2  36.44a  ±0.88  29.59a  ±0.88  3  39•65b  ±0.88  33.10b  ±0.88  4  56.99c  ±0.82  52.38c  ±0.83  Values with similar superscripts in a column are not significantly different (P<0.05). Table 8. Effect of period on % DM and CP effective degradability at k=0.06 period 1 (s.e.m.)^Period 2 (s.e.m.) DM^36.81a ± 0.57^47.46b ± 0.67 ^ CP 66•97a ± 0.877083b ± 0.90 Values in a row bearing similar superscripts are not significantly different (P <0.05). The effective DM degradabilities were significantly different (P <0.05) between animals (Table 7). Significant animal variation in degradability has been documented (Orskov et al., 1980; Lindberg, 1985; Nocek, 1988). Effective DM degradability differed significantly (P< 0.05) with the period (Table 8). Although variation with period of incubation is known to occur (Orskov et al., 1980), the 39  variation in this study was attributed to the increase in PEG from 0.04 to 0.1 . This was likely the case because there was no significant difference between controls in period one and two (P <0.05) on both DM and CP degradability in the rumen and the total tract digestibility. Table 9. The effect of tannin on effective degradability of the CP at assumed rumen outflow rate k=0.04 and 0.06 k=0.04  Without PEG (s.e.m)  with PEG (s.e.m)  Species  %increase after addition of PEG  Leucaena  71.75 ±2.00  82.63 ±2.00  15.16  Sesbania  74.91 ±2.00  87.04 ±2.00  16.19  Gliricidia  75.12 ±2.36  86.40 ±2.35  15.00  Calliandra  50.01 ±2.00  64.34 ±2.35  28.63  Velvet bean  72.65 ±2.36  81.77 ±2.00  12.54  D. intortum  53.12 ±2.98  75.57 ±2.35  22.44  Cassava  72.41 ±2.36  84.06 ±2.00  16.09  MEAN  67.35a ±0.88  80.04b ±0.82  19.34  Leucaena  65.75 ±2.10  78.50 ±2.10  12.75  Sesbania  69.18 ±2.10  83.57 ±2.10  20.80  Gliricidia  69.76 ±2.47  82.83 ±2.10  18.75  Calliandra  45.54 ±2.10  60.56 ±2.47  32.98  Velvet bean  67.62 ±2.47  77.53 ±2.10  14.66  D.intortum  46.49 ±3.12  71.96 ±2.47  54.79  Cassava  65.67 ±2.476  79.65 ±2.10  21.23  MEAN  61.43a ±0.91  76.37b ±0.84  24.32  k=0.06  Values with similar superscripts in a row are not significantly different (P < 0.05) 40  Tannin significantly (P <0.05) reduced the effective degradability of the CP (table 9). The forages treated with PEG had 19.3% and 24.3% higher effective degradability than those without PEG, at k=0.04 and 0.06 respectively. The differences between treated and untreated values were likely to be the contribution of tannin. Desmodium intortum had the largest mean effective degradability reduction of 54.8 %, followed by calliandra with 33.0% reduction. The effective degradability of CP in different MPT species at different rumen outflow rates are presented in Table 5 Only calliandra and D. intortum showed significant differences (P <0.005) from each other and from the other five species, which had no significant differences among them. Even with the slower outflow rates, calliandra and D. intortum effective degradabilities were less than 65% of the CP. The CP degradability values obtained in this study for Calliandra (45.5% and 50.0%) were higher than those of Jones et al.(1992), who reported only 9% N disappearance after 48 hours of incubation. However, Leucaena degradability values compared well with their findings.The values of Khalili and Varvikko (1992) for Sesbania were close but lower than the ones obtained in this experiment which could be attributed to the similar phenolic levels in both studies. Tannin reduced the effective degradability of CP in the rumen by binding with protein, thus significantly (P <0.05) reducing the rapidly degradable soluble fraction a (Table 6), and turns it into the slowly degradable time dependent fraction b. Tannin also significantly reduced the rate at which fraction b was degraded i.e. the degradation rate constant c. This means that, if the ruminant animal maintains or increases the feeding frequency and quantities, then higher quantities of protein would be expected to leave the rumen undegraded. This therefore, results in the low ruminal degradation reported by other workers on other tanniferous forages (Barry and Manley, 1984; Barry et al., 1986a; Robbins et al., 1987b). 41  Effective degradability of CP varied (p <0.05) significantly with animal and period. Such variations have been documented in different feeds (Orskov et al., 1980; Nocek, 1988). However, the periods (Table 8) variation in this experiment was to a great extent due to increase in PEG content in period two. The decrease in effective degradability of the DM observed in this experiment and others (Chiquette et al., 1988; Muellar- Harvey et al., 1988) could also be attributed to a reduction in rumen degradable nitrogen (RDN) for optimal microbial activity in tannin containing feeds. Adding urea to tanniferous feeds has been found to cater to microbes and improve in vitro DM digestibility (Schaffert et al., 1974). This supports the RDN deficiency hypothesis.  2.4.2 Intestinal DM and CP digestibility The whole gastro-intestinal tract digestibilities of DM and CP are presented in  Table 10. Both digestible dry matter (DMD) and digestible crude protein (DCP) differed significantly (P <0.05) with species. Table 11 presents the intestinal dry matter digestibility (IDMD) of different MPT species and forage crops. The IDMD was obtained by subtracting the percentage rumen dry matter degradation values from whole-tract percentage DM digestion and reflects the intestinal flux and digestibility of the DM. It also represents the portion of the total tract percentage digestibility contributed by intestinal digestion. Three out of seven species namely; calliandra, cassava foliage and velvet bean had significantly lower IDMD than the rest of the species. The effect of tannins on the whole tract DMD, simply referred to as DMD are presented in Table 12. Tannin significantly (P <0.05) reduced the DMD in all the species as evidenced by addition of PEG. The highest differences were in calliandra followed by D. intortum . These results are consistent to those reported by Nastis and 42  Malechek (1981), who found decreased DM, cell wall and protein digestibility when tanniferous feeds were added to goat feeds. Period of incubation had a significant (P <0.05) effect on DMD, which was attributed to the increase of PEG in period 2, because the controls in both periods were not significantly different from each other. The effect of tannins on the IDMD are presented in Table 14. Tannins significantly (P <0.05) increased the post rumen dry matter digestibility. Cassava foliage, velvet bean and Sesbania had over 100% increase in IDMD.  Table 10. The mean total tract DMD and DCP of different species DMD^(s.e.m)  DCP^(s.e.m)  % of the initial DM  % of initial CP  Leucaena  64.65cd  ±1.25  92.48cd  ±0.85  Sesbania  77.23e  ±1.23  97.83d  ±0.83  Gliricidia  73.88e  ±1.23  97.70d  ±0.83  Calliandra  38.43a  ±1.25  73.67a  ±0.85  Velvet bean  61.33c  ±1.23  93.31d  ±0.83  D.intortum  52.14b  ±1.25  87.11b  ±0.85  Cassava  67•38d  ±1.23  92.22c  ±0.83  Species  Values with similar superscripts in a column are not significantly different (P <0.05).  43  Table 11. Mean propotion of the total tract digestion of the DM contributed by the intestinal DM digestibility(IDMD) of different species Species  IDMD  s.e.m  Leucaena  14.71b  ± 1.41  Sesbania  19•86b  ± 1.38  Gliricidia  17.55b  ± 1.38  Calliandra  12.17a  ± 1.41  Velvet bean  13.48a  ± 1.38  D. intortum  16•82b  ± 1.41  Cassava  11.81a  ± 1.38  Values with similar superscripts in a column are not significantly different (P <0.05).  Table 12. The effect of tannin on total tract DMD in different species Species  Without PEG  With PEG(s.e.m)  %change  (s.e.m) Leucaena  54.30a ±1.70  75 . 00b ±1.82  38.12  Sesbania  68.57a ±1.76  85•67b ±1.70  24.93  Gliricidia  67.04a ±1.70  80•71b ±1.76  20.39  Calliandra  25•40a ±1.70  51•83b ±1.82  104.05  Velvet bean  55•17a ±1.70  67•49b ±1.76  22.33  D. intortum  38.69a ±1.70  65•60b ±1.83  69.55  Cassava  59•53a ±1.70  75•23b ±1.76  26.37  MEAN  52.62a ±0.65  71•68b ±0.67  36.22  Values with similar superscripts in a row are not significantly different (P <0.05).  44  ^  Table 13 . Effect of period on mean total tract % digestibility of untreated forage Period 1^Period 2 DMD  56•25a^±2.02^60.55 a ±2.09  DCP  88.84a  ±2.09^g5 77a ±2.07  Values with similar superscripts on the same row are not significantly different (P <0.05) Calliandra IDMD was not significantly different between PEG treated (control) and untreated, implying either, PEG was not sufficient or could not reverse the effect of tannin on dry matter that has escaped rumen degradation. It could as well be that another unknown factor may be contributing to the low IDMD. Jones et al. (1992) found DMD of Calliandra to be about 23% and suspected the low DMD to have been caused by the large alummina and silica content in calliandra. Similar DMD values for calliandra were reported by Bamualin et al. (1980). Figure 4 presents the proportional contribution of IDMD to the DMD. The IDMD increased significantly (P <0.05) as tannin level increased except in calliandra while the DMD in all the species decreased. The whole digestion tract digestibility values for CP (DCP) are presented in Table 9. There were significant differences between species in DCP. Except for calliandra and D. intortum which had 73.7% and 87.1%, most species had over 90% DCP. There was a significant interaction between species and treatment, suggesting that each species behaved differently to different levels of tannin. Alternatively, tannins from different plant species respond differently to PEG. Figure 5 depicts this interaction. A look at the figure indicates that calliandra DCP declines sharply as 45  leucaena, D. intortum, and cassava without PEG had significantly (P <0.05) lower DCP than with PEG, while in velvet bean, sesbania and gliricidia did not respond to PEG treatment. Period did not have any effect on DCP probably due to compensation by the intestinal digestion of bypass protein. These results are consistent to those reported by other workers (Jones and Mangan, 1977; Barry and Manley, 1984; Barry et al., 1986a; Waghorn et al., 1987), who found high tannin to result in a decline of total tract apparent digestibility, but increased intestinal N. Table 14. The effect of tannins on EDMD of different species Species  without PEG  with PEG (s.e.m)  (s.e.m)  % increase on PEG addition  Leucaena  18•90b^±1.92  10.52a^±2.00  79.65  Sesbania  27.20b^±1.99  12.53a ±1.92  117.15  Gliricidia  22•59b^±1.92  12.51a ±1.99  80.58  Calliandra  13.57a^±1.92  10.77a ±2.05  26.00  Velvet bean  18•81b^±1.92  8.14a^±1.99  131.08  D.intortum  19•31b^±1.92  14.32a ±2.07  34.84  Cassava  16•38b^±1.92  7.23a^±1.99  126.56  MEAN  19.5410^±1.92  10.86a ±0.76  79.92  Values with similar superscripts in a row are not significantly different (P <0.05).  46  A  ^  B  Leu Ses Gil Cal Vel Desi Cas  Leu Ses Gli Cal Vel Desi Cas  Without PEG  With PEG  MN DMD M IDMI) Figure 4 Proportional contribution of intestine to total tract DM digestibility.  MI  DMD M IDMD  100 95  -  90  -  85  _  80  -  75  -  70  -  65  -  60  I  Without PEG  ^  1  With PEG  Figure 5. The interaction effects of species and treatment on total tract digestion. 0 Leucaena, • Sesbania, 7 Gliricidia, V Calliandra, 0 Velvet bean, • Desmodium intortum, LI, Cassava  The intestinal digestible protein (IDP) values, which were obtained from subtracting the rumen-degradable CP, indicate the portion of the total tract digestibility contributed by the intestinal digestion. The IDP reflects the intestinal dietary N flux and its availability. IDP varied significantly (P <0.05) with species, treatment and their interactions (Figure 6). All species behaved differently with increasing tannin levels. Calliandra IDP decreased at higher tannin levels, while in all other species, higher levels of tannin resulted in varying increases in IDP. This means that Calliandra proteins are bound irreversibly by tannins and are resistant to rumen microbial degradation as well as intestinal enzymes. In such a case very high fecal nitrogen and declining animal performance would be expected (Lohan et al., 1983). This means that calliandra protein must be treated in order to enhance its digestibility. In the other species, the results indicated that undegraded dietary proteins that entered the intestine were digested. Therefore the results of this study agree with Jones and Mangan (1977) and Barry and Manley (1984) that presence of tannin increased the post ruminal protein digestion compared to low or no tannin. Figure 7 indicates the proportional contribution of IDCP to the total tract digestibility of CP. The IDCP contribution significantly (P <0.05) increases with increase in tannin content but lowers The DCP although not to the same extent as in the dry matter.  49  2 '7 20 0  18 biJ  16  C-1 a)^1 4 0  12 10 oJ  8 6  Without PEG  ^  With PEG  Figure 6. The interaction effects of fodder species and treatments on intestinal protein digestibility. 0 Leucaena, • Sesbania, 7 Gliricidtia, V Calliandra, 0 Velvet bean, • Desmodium intortum,^Calliandra  50  B  A 100 --/ 90 P 00  p 80 -  -  e r  e I  c 70  -  t 60  -  e H a  c 70  -  t 60  -  e H a  g C  g C U  f  Vel^Desi^Cas  Vel^Desi^Cas  Without PEG  ^  DCP M IDCP  ^  Figure 7. Proportion of contribution of intestine to total tract CP digestibility.  With PEG 11111 DCP M 1DCP  2.5 SUMMARY AND CONCLUSION This study investigated the effect of tannins in multipurpose fodder trees and forage crops of Kenya, on the rumen degradation and the whole GI tract digestibility of DM and CP. The results indicated varying but significant reduction in DM and CP degradation in the rumen, with large and varying quantities of the dietary DM and protein escaping the rumen undegraded depending on the plant species. These therefore increased the dietary DM and N flow into the intestine. Except in the case of calliandra, a significantly larger proportion of the escaped DM and CP was digested in the intestines. Generally both DM and CP digestibilities in the total tract were lower in forages without PEG than with PEG treatment. This implied that tannins reduced the apparent digestibility of both DM and CP. These results support Barry et al. 1986a findings that increasing the dietary tannin concentration linearly increases the duodenal N. flow, but linearly decreases the apparent digestibilty of energy and organic matter, and rumen digestion of hemicelluose,but not of cellulose. At the same time this also confirms Jones and Mangan (1977), finding that PEG is an effective suppresser of tannin effects, and is useful for future animal feeding experiments involving tannins. However, there is need to assay the optimum levels of PEG to be applied to each fodder species. The results also identified two categories of forages in the multipurpose fodder trees and crops. Category one composed of Calliandra calothyrsus and Desmodium intortum with less than 55% DMD and less than 90% DCP, which must be treated  before feeding, otherwise it may not be beneficial, or even may be deleterious to the animal. Category two: composed of forages with more than 55% DMD and 90% DCP which may be fed untreated or treated. These include leucaena, gliricidia, sesbania,  52  velvet bean and cassava, whose treatment may be beneficial to animals, especially in the periods when the tannin:protein ratios are likely to have an adverse effect. Most of these MPT and forage crops are fed as protein supplements to dairy cattle especially in periods of feeds shortage, when the bulk of the ration is mainly composed of fibrous farm crop residues and poor quality grasses. In most cases these may be deleterious to the cattle in that tannin decreases the DM degradation and the energy available in addition to a protein reduction in digestibility. There is therefore a need to treat some of these MPT species in order to achieve better animal performances. In cases where bypass protein is advocated, tannin may increase the bypass protein. The polyphagous tanniferous feeders have developed mechanisms to counter the effects of tannins in order to thrive. The mechanism, such as use of surfactants which dissolves the protein-tannin complex as used by herbivorous insects (Martin et al., 1985; Mole and Waterman, 1985), may be used as a pre-feeding treatment. This area needs further investigation. Tannins have high affinity for proline-rich proteins and PEG, because of their strong hydrophobic and hydrogen bonding characters. These characteristicss have been exploited by tanniferous browsers to suppress the effect of tannins. The PRPs (with low protein value) are produced in salivary glands of animals adapted to high tannin feeds. The PRPs strongly and preferentially bind with tannin, allowing the high quality protein in the diet to be digested (Hagerman and Butler, 1981; Mehansho et al., 1983). The PRPs and other cheap protein of low nutritive value and with similar characters needs further investigation as treatment agents. There is need to study the cultural practices relating to the fodder trees and crops that may modify the tannin:protein ratio. These may involve the fertilizing and harvesting regimes. 53  GENERAL CONCLUS ON  54  BIBLIOGRAPHY Akbar, M.A. and P.C. Gupta, 1985. Proximate composition, and tannin mineral  contents of different cultivars and of various plant parts of subabul (Leucaena leucocephala). Indian J. Anim. Sci. 55:808-812. Arlen, A., A. Ben-Moshe, S. Zamwel, and H. Tagari, 1988. In situ evaluation of the  ruminal digestibility of heat-treated whole cottonseeds. J Dairy Sci. 72:1228-1233. Armstrong, W.D, W.R. Featherston, and Rogler, 1973. 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Table I NDF % in MPT and forages based on DM High altitude  Species  ^  Dry season  ^  Wet season  Leucaena^34.68^39.86 Sesbania^41.68^35.48 35.78  Gliricidia^  Calliandra^47.19^40.87 49.05  Velvet bean^  Desmodium intortum^42.46^40.62 D. uncinatum^51.88^47.34 cassava foliage^36.62^44.34  Low altitude  28.38  Leucaena^  S esbania^-^37.61 Gliricidia^  38.62  Calliandra^  27.93  Velvet bean^  42.63  Table II. ADF % in the MPT and forages based on the DM 68  High altitude  Species^Dry season^Wet season Leucaena^20.26^20.1 Sesbania^26.62^18.96 21.09  Gliricidia^  Calliandra^29.79^23.88 Velvet bean^  33.85  D. intortum^30.55^32.82 D. uncinatum^43.01^24.99 Cassava foliage^22.81^27.13  Low altitude  Leucaena^  25.85  Sesbania^  26.13  Gliricidia^  24.83  Calliandra^  21.91  Velvet bean^  29.34  69  APPENDIX B  Effect of tannins on ruminal degradation and intestinal digestibility of protein in different periods  Table III. Effect of tannin on effective digestibility degradability of DM in the rumen Species^ % of DM with PEG % increase with % of DM without tannin PEG suppression Period 1 (40 mg PEG/ lg tannin) 43.78a. ±3.64  Leucaena Sesbania Gliricidia Calliandra  51.26a 45.37a 16.86a  Velvet bean D. intortum Cassava  45.05a 27.10a 44.93 a  Leucaena Sesbania Gliricidia Calliandra Velvet bean Cassava  ±3.64 ±2.97 ±3.64  46.45a 58.08a 52.08a 25•67b 46.80a  ±3.64 ±3.64 +3.64 +3.64 ±3.64  3.90  38.08b 51.98a  ±3.64 ±3.64  40.0 15.70  +3.64 ±3.64 ±2.97 Period 2 100g PEG/ g tannin 33.24a +1.52 38.30a +1.52 40.95a ±2.16  80.96b 85.08b  +1.52 +1.52  78.56b  +1.52  10.72a 32.18a 35.58a  57.35b 69.05b 78.161  +1.52 +1.52 +1.52  ±1.52 +1.52 ±2.15  6.00 13.30 15.30 52.20  143.60 122.10 91.80 435.00 114.60 119.70  Values with similar superscripts in a row do not differ significantly (P<0.05)  70  Table IV Effect of tannin on effective degradability of the CP Period 1  Without PEG  With PEG  % Change  Leucaena  73.65a  ±1.96  78.27a  ±1.96  6.30  Sesbania  81.28a  ±1.96  84.11a  +1.96  3.50  Gliricidia  80.02a  ±2.77  82.21a  ±1.96  2.70  Calliandra  46.59a  ±1.96  59.5013  +1.96  27.70  Velvet bean  80.21a  ±2.77  78.74a  ±1.96  -1.80  D. intortum  55.40a  ±1.96  67.30b  ±1.96  21.50  Cassava  74.60a  ±2.77  78.67a  ±1.96  5.50  Period 2 Leucaena  69.83a  ±2.61  86.98b  ±2.61  24.60  Sesbania  68.54a  ±2.61  89.96b  ±2.61  31.30  Gliricidia  72.18a  ±2.61  90.58b  ±2.61  25.50  Calliandra  53.42a  ±2.61  70.57b  ±2.61  32.10  Velvet bean  68.37a  ±2.61  84.79b  ±2.61  24.00  Cassava  70.80a  ±2.61  89.45b  ±2.61  26.30  Values with similar superscripts in a row do not differ significantly (P<0.05)  Table V. Effect of Tannin on degradation constants of DM Fraction  Without PEG  With PEG  Period 1 Lag  3.12 a^±0.80  3•35a  ±0.84  a  7.45 a^±0.79  11.62b  ±0.83  60.57a^±1.69  59.60a  ±1.77  5.78a^±0.90  8.63b  +0.95  Period 2 Lag  5•67b^±0.53  3.21a  +0.45  a  6.38a^±0.28  64.18a  ±0.24  70.00b^+4.95  24.32a  +4.29  4.17a^±0.51  4.30a  +0.44  Values with similar superscripts in a row do not differ significantly (P<0.05)  Table VI. Effect of tannin on degradation constants of protein Fraction  Without PEG  With PEG  Period 1 Lag  1.33a  ±0.72  4.04b  ±0.60  a  38.30a  +1.78  45.05b  ±1.49  b  53.30a  ±2.64  49.02a  ±2.21  c  7.05a  ±0.72  11.20b  ±0.60  Period 2 Lag  4.67a  +1.14  5.43a  ±1.23  a  44.89a  ±0.52  74.6913  ±0.56  b  52•49b  +1.42  21.66a  +1.58  c  5.14a  +0.98  7.36a  ±1.06  Values with similar superscripts in a row do not differ significantly (P<0.05)  TableVII . Effect of tannin on total tract digestibility of the DM (DMD)  73  Without PEG  With PEG  Species Period 1 Leucaena  60.15a  ±2.59  63.74a  ±2.78  Sesbania  71.60a  ±2.59  79.26b  ±2.78  Gliricidia  71.68a  ±2.59  73.22a  Calliandra  27.63a  ±2.59  36.10b  ±2.78  Velvet bean  59.16a  ±2.59  57.73a  4_2.78  D. intortum  40.16a  ±2.59  47.78b  ±2.78  Cassava  65.57a  ±2.59  63.07a  ±2.78  ±2.78  Period 2 Leucaena  48.45a  ±1.92  87.12b  ±2.07  Sesbania  65.37a  ±1.92  92.50b  ±1.92  Gliricidia  62.40a  88.93b  ±1.92  Calliandra  22.44a  ±1.92  67.60b  -±2.07  Velvet bean  51.18a  ±1.92  76•81b  ±1.92  D. intortum  37.21a  83•87b  ±2.07  Cassava  53.48a  86.72b  ±1.92  ±1.92  ±1.92 ±1.92  Values with similar superscripts in a row do not differ significantly (P <0.05)  Table VIII. The effect of tannin on the total tract digestibility of CP (DCP).  74  Species  Without PEG  With PEG  Period 1 Leucaena  92.8a  Sesbania  97.56a  Gliricidia  ±1.53  94•70a  ±1.64  ±1.64  98.80a  ±1.53  97.91a  ±1.53  98.20a  ±1.64  Calliandra  67.501  ±1.53  79. 89b  ±164  Velvet bean  94.28a  ±1.53  94.70a  ±1.53  D. intortum  78.33a  ±1.53  86.39b  ±1.64  Cassava  94.15a  ±1.53  92.62a  ±1.64  95. 81b  ±1.54  Period 2  Leucaena  86.65a  ±1.44  Sesbania  95.74a  ±1.44  99.12a  ±1.54  Gliricidia  95.86a  ±1.44  98.84a  ±1.54  Calliandra  60.37a  ±1.44  86.82b  ±1.54  Velvet bean  89.48a  ±1.44  94.52b  ±1.54  D. intortum  85.86a  ±1.44  97.94b  ±1.54  Cassava  86.90a  ±1.44  95.20b  ±1.54  Values with similar superscripts in a row do not differ significantly (P <0.05) Table IX. The effect of tannin on intestinal DM digestibility (IDMD) 75  Species  Without PEG  With PEG  Period 1  Leucaena  17.88 a  Sesbania  17.96a  Gliricidia  18.98a  ±2.09  14.94a^±2.25  Calliandra  11.76a  ±2.09  9.93a^±2.25  Velvet bean  14.71b  ±2.09  7.55a^±2.09  D. intortum  14.46a  ±2.09  8.86a^±2.25  Cassava  13.13b  ±2.09  6.19a^±2.25  ±2.09 ±2.25  12.33a^±2.25 16.34a^±2.09  Period 2 Leucaena  19.92b  ±3.08  8.30a ±3.31  Sesbania  35.40b  ±3.08  8.72a^±3.08  Gliricidia  26.20b  ±3.08  10.20a^±3.08  Calliandra  15.39a  ±3.08  11.46a^±3.31  Velvet bean  22.91b  ±3.08  8.9a^±3.31  D. intortum  24.1V  ±3.08  19.63a^±3.31  Cassava  19.6313  ±3.08  8.77a^±3.08  Values with similar superscripts in a row do not differ significantly (P <0.05)  76  Table X. The effect of tannin on the intestinal digestibility of crude protein (IDCP)  Without PEG Species  With PEG  Period 1 Leucaena  17.49b  ±1.47  11.58a^±1.58  Sesbania  10.73a  ±1.58  9•59a^±1.47  Gliricidia  15.92b  ±1.47  9.90a^±1.58  Calliandra  20.59a  ±1.47  21.86a^±1.58  Velvet bean  13.43a  ±1.47  10.68a^±1.47  D. intortum  22.54b  ±1.47  15.17a^±1.58  Cassava  13.46b  ±1.47  8.87a^±1.58  Period 2  Leucaena  18.20b  ±1.98  10.90a^±2.12  Sesbania  30.45b  ±1.98  9.58a^±1.98  Gliricidia  23.53b  ±1.98  7.59a^±1.98  Calliandra  7.64a  ±1.98  19.54b^±2.12  Velvet bean  23.90b  ±1.98  8.75a^±2.12  D. intortum  16.53a  ±1.98  12.48a^±2.12  Cassava  16.77b  ±1.98  4.89a^±1.98  Values with similar superscripts in a row do not differ significantly (P <0.05)  77  The results of individual periods, indicate that; at 40 mg PEG per 1 g tannin (low PEG) used in period 1 the tannin effect on the DM disappearance in the rumen was not clearly manifested in most MPT and forage species. Calliandra calothyrsus and Desmodium intortum are the only species that responded to the addition of 40 mg PEG per 1 g tannin The degradation constants in low PEG behaved differently with those of high PEG (100 mg PEG/1 g tannin). Generally the high PEG enhanced the DM and the, CP disappearance in the rumen and the total tract digestibility , but reduced the intestinal contribution of the total DMD and DCP.  78  APPENDIX C A bio-assay for the optimum level of polyethylene glycol (PEG): that would inhibit the effect of MPT tannins on protein  When protein feeds enter into the rumen or are incubated insacco or in vitro, the rumen microbes actively hydrolyse the protein into short chain peptides and amino acids which they utilize directly for microbial protein synthesis or deaminate them into ammonia which is then used as nitrogen (N) source for microbial protein synthesis (Armstrong and Weekes 1983). Tannin in feed binds with the dietary protein and forms a tannin protein complex which is resistant to microbial enzymes hydrolysis. PEG has been found to suppress the effect of tannin and in some cases dissociating the formed complex (reversing the effect of tannin) (Jones and Mangan, 1977). The rate and mode of PEG application has been variable (Jones & Mangan, 1977; Barry and Duncan, 1984; Waghorn et al., 1987). In period one of this study, the rate of 40 mg/g tannin as used by Jones and Mangan (1977) on Lotus pedunculatus was adopted. This rate was found to be less effective on MPT species. Therefore, there was a need to change this level to most appropriate one for these tropical forages. When PEG is added to tanninferous feeds, and incubated, the microbial activity would be higher and more amino acids released than untreated feed indicating a low microbial activity. The released amino acids can be quantified through ninhydrin method described by Rosen (1957). Objective of this assay was to determine the PEG levels that would result in high microbial activity. 79  MATERIALS AND METHODS  Sample Preparation: A 0.1 g samples from each species were drawn and diluted with phosphate sulphur (pH 6.8) to make up a suspension of 100 g/l. Preparation of rumen fluid:  Rumen fluid obtained from two rumen cannulated cows maintained on orchard grass and alfalfa hay mixture was strained on a 4 layer cheese cloth to fill a 2 1 thermos. The strained fluid was then prepared as described by Forsberg (1978), which involved centrifuging the rumen fluid at 600 xg for 5 minutes at 30°C to remove the debris and protozoa. The resulting supernatant was further centrifuged for another 15 minutes at 1600 xg at 30°C under CO2 atmosphere. The pellet was reconstituted with phosphate buffer to 600 nil. An aliquot of lml feed suspension was drawn and placed in 10 test tubes. The test tubes were added 1 ml PEG solution with different concentration of PEG viz. 0, 50, 100, 150, 200, 250, 300, 400, 500 mg/g tannin. One ml of rumen fluid was added to all test tubes. Two more test tubes were added to this set. One of the two had fluid only and the other feed suspension only. All twelve were incubated in anaerobic environment at 39° C for 4 hours. these were replicated 3 times. Immediately after incubation, fermentation was stopped using 1 ml icy cold 10% trichloroacetic acid (TCA) and let to stand on ice for 30 minutes. Tubes were then centrifuged at maximum (27000 xg) and 1 ml of supernatant drawn and treated as described by Rosen (1957) for absorbance reading. 80  The absorbance was read using the Shimantzu uv-spectrophotometer at 570 mu  and at 440 mu for proline and hydroxyproline. Calculations: True amino acids absorbance was obtained by subtracting from individual feed hydrolysate absorbance, the absorbance value for rumen fluid alone and substrate suspension alone (i.e without rumen fluid).  Results The microbial activity versus PEG level graphs were plotted (figure I -VII). The optimum PEG level was taken as that activity where additional PEG resulted in little or no increase the microbial activity (or was at asymptote). However, a lower value was considered where PEG levels were exceptionally high (or may not be reasonable). The 100 mg PEG was selected because in most species the microbial activity was high.  ^ ^  Figures I — VII. Percentage microbial activity change with increase of PEG concentration in the MPT species. 0 Run 1, • Run2  T 100 7 1 80 L Loucaena 60 -  •  4.0  E-7C....) <  -20 -40  II  '70 -  • 100  300  200  400  500  Sesbania 10 -^•  /C^  -10 -20 C.7  Ez^-40 ^  E^  40 30 [  r;  10 ^0*^  100^200^300^400^500  • &  ,„  -10 .4.-y0 -Callia,ndra -.30 -40 100^200  0 300  400  500  Concentration of PEG (mg,/g tannin) 32  ^  iv  20 -  ON  ^•  - 7 0  10 ^ ^ >100 200^300^400^500 E> V120 -Velvet Bean E--^100 80 7 -  60 <^:to 7  o  20 0•  100  T 600 - Cassava  ^  200  ^  300  ^  400  ^  500  C.,^ I 500 400 7Cz;  300  -  200 100 -100  100^200^300^100^500  VII20C D e s-rno &um, int ortu-m, 5k: -  10c.5C rC 100^200^300^400^500  Concentration of PEG(mg/g tannin) S3  

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