Relationship between tannin contents and short-term biological responses in male rabbits supplemented with leaves of different acacia tree species grown in Limpopo province of South Africa

L Mashamaite, J W Ng’ambi, D Norris, L R Ndlovu and C A Mbajiorgu

Deprtment of Animal Production, University of Limpopo, Private Bag X 1106, Sovenga 0727, South Africa
anayochukwumbajiorgu@gmail.com

Abstract

This study related the amounts and types of tannins in some acacia tree species grown in Limpopo province to effects on short-term biological responses in rabbits. Eight New Zealand White male rabbits with a mean live weight of 0.68 ± 0.05 kg were randomly assigned to metabolic crates in a two 4 x 4 Latin Square design. The four dietary treatments were a control diet (ground rabbit feed), control diet mixed with Acacia karroo leaf meal (4 % of diet), control diet mixed with Acacia nilotica leaf meal (4 % of diet) or control diet mixed with Acacia tortilis leaf meal (4 % of diet).

There were no differences (P > 0.05) in diet intakes and digestibilities between the treatments. However, rabbits on Acacia tortilis diet drank more (P < 0.05) water than those on the other treatments. Faecal DM and nitrogen outputs, urine output, urine nitrogen output and nitrogen retention in rabbits were not affected (P > 0.05) by supplementation. Nitrogen digestibility and retention were most accurately predicted by contents of total phenolics, radial diffusion, precipitable phenolics by filter paper and polyethylene glycol in the leaves.

Extracted condensed tannin contents provided no reliable indication of diet dry matter and nitrogen digestibilities, and faecal nitrogen output in rabbits.

Keywords: Acacia tree species, digestibility, intake, trees

Introduction

Acacia trees are important components of the fodder resources for ruminant livestock and wildlife in Limpopo province of South Africa. There is also interest in the use of leaves of acacia trees as sources of protein for non-ruminant animals (Abdulrazak et al 2000; Al-Mamary et al 2001; Skarpe and Bergstrom 1986). The main features of these browse plants are their high protein and mineral contents, and their ability to survive in unfavorable soil and climatic conditions (D’Mello et al 1987; Tangendjaja et al 1990). However, Acacia tree species contain tannins which either have positive (Al-Mamary et al 2001) or negative (D’Mello et al 1987) effects on animal performance. The amounts and type of tannins synthesized by plants vary considerably depending on the plant species, cultivars, tissues, stage of development and environmental conditions (Giner-Chavez et al 1997). Therefore, the study of the nutritional effects of tannins on animals requires quantification of the tannins present in a particular diet. Due to the complexity of tannins, several methods have been developed for their quantification. These include colorimetric (Schofield et al 2001; Singleton et al 1999; Waterman and Mole 1994), gravimetric (Makkar 1993) and protein precipitation (Schofield et al 2001; Silanikove et al 2001) assays. Studies done, mostly, are about determining the tannin content of a particular plant or observing the biological responses of animals. Few studies have been done correlating tannin contents and biological responses (Makkar 2003). The scarcity of information relating the amounts and types of tannins that they contain to effect on animal performance has tended to delay development of management strategies that minimize the negative effects of tannins whilst optimizing the positive ones. The objective of this study was to determine the relationship between tannin contents by different chemical analytical techniques and short-term biological responses in rabbits supplemented with tanniniferous leaves from different acacia tree species found in the Capricorn region of Limpopo province in South Africa.

Materials and methods 

Study site

This study was carried out at the University of Limpopo Experimental farm, South Africa, between July and November 2007. The ambient temperatures around this area are above 25 ⁰C during summer and around 25 ⁰C or lower during the winter season. Mean annual rainfall is 446.8 mm with the dry season occurring between April and October and the rainy season occurring between November and March.

Collection, drying and storage of plant material

Acacia leaves were harvested in July 2007. Branches of each shrub were removed manually and placed in a shed. Leaves were allowed to air-dry on the branches and then removed by carefully beating the branches with sticks. The leaves were then ground through a 1 mm sieve and kept in a cool and dry-room until feeding times.

Animals, management, diets and experimental design

Eight New Zealand White male rabbits with a mean live weight of 0.68 ± 0.05 kg were used in this study. The rabbits were allocated at random to four treatments in a two 4 x 4 Latin Square design. The rabbits were housed individually in metabolic cages measuring 40.6 x 45.7 cm² and given the experimental diets for an adaptation period of 10 days and a collection period of 5 days. Prior to this, the rabbits had been kept in the cages for one month to get used to the metabolic cages and new environment. The four dietary treatments were a basal diet, a basal diet mixed with 4 % Acacia Karroo leaf meal, a basal diet mixed with 4 % Acacia nilotica leaf meal and a basal diet mixed with 4 % Acacia tortilis leaf meal. Feed intake was recorded on daily basis during the collection period. Water intakes were monitored by use of graduated water bottles, which indicated the amount of water consumed. The rabbits were weighed prior to being assigned to a treatment and body weight changes (gain/loss) were recorded during the collection period, daily output of faeces for each rabbit were collected, mixed and recorded. Ten percent of the faeces were sub-sampled and kept. All the sub-sampled faeces were pooled on animal basis, dried and kept until required for chemical analysis. The urine was collected in graduated bottles containing a solution of 50 ml of 10% H₂S0₄ to prevent ammonia-nitrogen loss and maintain pH below 3.0 (Garrido et al 1991). A 10 % aliquot of urine was retained daily and bulked for each rabbit within the collection period.

Chemical analyses

Dry matter, crude protein, ash, calcium and phosphorus were determined using the methods described by AOAC (2000). Total phenolics (TP) were determined using Folin Ciocalteau method and expressed as tannic acid equivalent (% DM) (Waterman and Mole 1994). Condensed tannins were (both extracted and unextracted) determined using Butanol- HCL method and expressed as leucocyanidin equivalent (% DM) (Porter et al 1986). Protein-binding capacity by filter paper (PPFP) was determined using the methods described by Dawra et al (1988). Radial diffusion (RD) was determined using the methods described by Hagerman (1987). Reaction of polyethylene glycol 4000 with tannins (PEG) was determined according to the methods described by Silanikove et al (1994).

Statistical analyses

The effects of tannin level and type on feed and water intakes, digestibility and nitrogen retention in rabbits were analyzed using GLM procedures of SAS (2000) as in a two replicates of a balanced 4 x 4 Latin Square design. Means separation was attained through the procedure of least significant difference (P < 0.05). Stepwise regression was used to relate tannin amounts determined by different assays to animal performance indices (intake, digestibility and nitrogen retention).

Results 

The control diet contained 160 g crude protein per kg DM and 12.86 MJ metabolisable energy per kg DM. However it contained no traces of phenolic compounds. Data on tannin contents of acacia leaves are presented in Table 1.

This control diet is not included in  table 1 because the analyses did not indicate any presence of tannins. Acacia nilotica had higher (P<0.05) total phenolics (2.04 % DM) than both Acacia karroo (1.51 % DM) and Acacia tortilis (1.25 % DM). Similar differences were observed when tannins were measured by radial diffusion method. The analysis by polyvinylpolypyrrolidone method showed that Acacia nilotica (1.22 % DM), Acacia karoo (0.57 % DM) and Acacia tortilis (0.4 % DM) wee different (P< 0.05). Similar differences were observed when butanol-HCL, polyethylene glycol and precipitable phenolics by filter paper were used. Results of diet intakes and digestibility are presented in Table 2.

There were no differences (P>0.05) between treatments in diet DM intake, nitrogen intake and DM and nitrogen digestibilities. Rabbits on Acacia tortilis diet drank more (P<0.05) water than those on the other treatments. However, there were no differences (P>0.05) in water intake between rabbits on control, Acacia nilotica and Acacia karoo diets. There were no differences (P>0.05) between treatments in faecal DM and nitrogen outputs, urine output, urine nitrogen output and nitrogen retention (Table 3).

A series of linear regressions that were used to predict intake in rabbits from total phenolics are presented in Table 4.

Dry matter intake was moderately predicted from total phenolics (r = -0.64) and precipitable phenolics by filter paper (r = -0.64) contents, but correlations with extracted condensed tannins (r = -0.13), polyethylene glycol (r = -0.37) and radial diffusion (r = -0.57) contents were less accurate. Water intake by rabbits was poorly and positively correlated with total phenolics, extracted condensed tannins, polyethylene glycol and radial diffusion values. However, water intake by rabbits was poorly and negatively correlated with precipitation phenolics by filter paper. Water intake was positively correlated with extracted condensed tannins (r = 0.68). Nitrogen intake was poorly and positively correlated with extracted condensed tannins (r = 0.32) and polyethylene glycol (r = 0.07). However, negative and poor correlation coefficients were observed between nitrogen intake and total phenolics (r = -0.42) and radial diffusion (r = -0.22) contents of acacia browses.

A series of linear regression equations that predict faecal nitrogen output, digestibility and nitrogen retention in rabbits from total phenolics, extracted condensed tannins, polyethylene glycol precipitable phenolics by filter paper and radial diffusion contents of acacia browse species are presented in Table 5.

Faecal nitrogen output in rabbits was poorly and negatively correlated with extracted condensed tannin contents (r = -0.01). However, positive correlation co-efficients were observed with total phenolics (r = 0.59), polyethylene glycol (r = 0.71), precipitable phenolics by filter paper (r = 0.61) and radial diffusion (r = 0.66) tannin contents. Nitrogen digestibility was poorly and negatively correlated with extracted condensed tannin values of the browses (r = 0.13). However, nitrogen digestibility was highly and negatively correlated with total phenolics (r = -0.93) and radial diffusion (r = -0.96) tannin contents of the browses. Diet dry matter digestibility was positively and poorly correlated with different tannin assays. Nitrogen retention values were highly and negatively correlated with browse contents of total phenolics (r = -0.99), polyethylene glycol (r = -0.91), precipitable phenolics by filter paper (r = -0.97) and radial diffusion (r = -0.99). Poor correlation coefficients were observed between nitrogen retention (percentage of total nitrogen intake) in rabbits and extracted condensed tannins (r = -0.23) in the browses. The correlation coefficients between nitrogen retention (g/rabbit/day) and different tannin assays were good except with extracted condensed tannins (r = -0.24), which was less accurate.

Discussion 

The acacia species used in this study contained different types of tannins in varying levels. Although Acacia nilotica had highest concentrations of total phenolics, simple phenolics, polyethylene glycol, precipitable phenolics by filter paper and radial diffusion, it had the lowest amounts of condensed tannins, both extracted and unextracted. Thus, few of the phenolics in Acacia nilotica were condensed tannins. Most of the phenolics in Acacia nilotica are catechin gallates (Muller – Harvey 1987), which may be absorbed by the gut, and may have toxic effects on the animal (Silanikove et al 1997; Makkar 2003). Acacia tortilis had the lowest contents of total phenolics, simple phenolics, radial diffusion and protein precipitation by filter paper. Acacia karroo was highest in condensed tannins. These tannins bind to proteins and hence render them unavailable for use by the animals. The condensed tannins are not absorbed into the blood streams, therefore, under normal physiological conditions, they are not likely to damage organs such as the liver, kidney, spleen, etc, as has been the case for hydrolysable tannins (McSweeney et al 1988). The present analyses are consistent with the findings of Dube and Ndlovu (1995), Dube et al (2001), Kahiya et al (2003) and Motubatse et al (2008).

Results with rabbits reported here indicate that there were no significant (P>0.05) differences in intake and digestibility between the control diet and those supplemented with tanniniferous browses of Acacia karroo, Acacia nilotica and Acacia tortilis. These results are similar to those of Al-Mamary et al (2001) who reported that addition of low-tannin (1.4 % catechin equivalent) sorghum grains in the diets of rabbits did not significantly change growth rate, feed intake or feed conversion ratio. However, they reported that the addition of high-tannin (3.5 % catechin equivalent) sorghum grains significantly reduced their live weight gain, feed conversion ratio and slightly increased fed intake with respect to the control diet. The authors suggested that lack of toxicity in rabbits fed low-tannin sorghum grain may indicate the existence of a threshold limit. Similarly, McNabb et al (1998) and Barry and Duncan (1984) showed that the effect of tannins depends on the type and level of tannins, and dietary nutrients involved. Therefore, in the present study, a possible explanation for the lack of significant differences may be that rabbits did not take in sufficient amounts of tannins from the 4 % browse supplementation to cause intake and digestibility decreases. Rabbits on a diet supplemented with leaves from Acacia tortilis drank more water than those on the control, Acacia karroo and Acacia nilotica. It has been postulated that astringency, caused by tannins, may cause animals to drink more water than they would on low tannin diets (Hove et al 2001). However, the mechanism of this is not clear. There were no differences between treatments in urine output, possibly indicating that substantial amount of water in rabbits on Acacia tortilis diet was excreted in the faeces. There were no significant differences between treatments in rabbit faecal nitrogen output, urine nitrogen output and nitrogen retention. However, the faecal nitrogen output of rabbits supplemented with Acacia browses tended to be higher than those on the control diet. It is likely that the amounts of tannins from the browses (4 % of the feed mixture) used in the present study were not large enough to significantly bind with proteins (dietary and enzymic) and consequently affect the faecal and urine nitrogen outputs (Al-Mamary et al 2001). The nutritional effects of tannins are associated with their ability to bind proteins, structural carbohydrate polymers and minerals with an overall effect of lowering the bioavailabilty of the nutrients at specific sites in the gastro-intestinal tract (Chang et al 1998; Al-Mamary et al 2001). Al-Mamary et al (2001) found that addition of low-tannin sorghum grains to the rabbit diet did not significantly have any effect on faecal nitrogen excretion and nitrogen retention with respect to the control diet. However, in their experiment, they also observed that the addition of high tannin sorghum grains significantly increased faecal nitrogen output and reduced nitrogen retention.

Rabbits eat their faeces and hence drive substantial amounts of microbial proteins from such diets (Al-Mamary et al 2001). However, digestibility of the re-ingested tannin-complexes formed along the digestive tract is not known and may require further investigation.

A major constraint to proper management of tanniniferous feeds has been that the assays are not well proven to correlate with the performance of the animals that eat such feeds (Makkar 2003). Such correlations would help in formulating management strategies that enhance the positive effects and reduce the negative effects that tanniniferous feeds cause to the productivity of animals that eat such feeds. In the present study diet dry matter intake in rabbits was negatively correlated with tannin contents in leaves. The negative correlation coefficients were expected since tannins tend to decrease diet digestibility through their ability to bind with proteins and other materials, resulting in decreased diet intake (Chang et al 1998; Al-Mamary et al 2001). The correlation coefficients between dry matter intake in rabbits and tannin assays ranged from low to moderate values. Such relationships have been reported elsewhere in ruminant animals (Dube et al 2001; Al Mamary et el 2001). The correlations between dry matter intake in rabbits and tannin contents in leaves by different assays were negatively and well correlated but extracted condensed tannins and polyethylene glycol contents were less accurate. These negative correlation coefficients were expected since tannins have been reported to be responsible for decreases in feed intake (Chang et al 1998; Ben Salem et al 2000; Al- Mamary et al 2001). These coefficients indicate that the higher tannin content in a plant the less is its dry matter intake.

Water intake in rabbits was moderately and positively correlated with extracted condensed tannins; but total phenolics, polyethylene glycol and radial diffusion contents were less accurate in predicting water intake. Water intake was negatively correlated with precipitable phenolics by filter paper. Positive correlation coefficients were expected since tannins dehydrate animals (Dube and Ndlovu 1995), thus increasing water intake by the animal. The correlation coefficients between nitrogen intake and browse contents of total phenolics, polyethylene glycol, precipitable phenolics by filter paper and radial diffusion indicated that these different tannin assays provided no reliable indication of diet nitrogen intake in rabbits.

The correlation coefficients between faecal nitrogen output in rabbits and tannin contents in leaves by different assays were moderate and positive except with that of extracted condensed tannins, which was low and negative. The positive correlation coefficients were unexpected since tannins are expected to bind with proteins (dietary and enzymic) and consequently increase faecal nitrogen output in rabbits (Al- Mamary et al 2001). Diet nitrogen digestibility in rabbits was highly and negatively correlated with total phenolics, polyethylene glycol, precipitable phenolics by filter paper and radial diffusion contents in browse supplements. These negative correlations were expected since tannins decrease nitrogen digestibility in animals through their ability to aggregate and precipitate proteins and various digestive enzymes, thus rendering them unabsorbable (Chang et al 1998; Al-Mamary et al 2001; Makkar 2003). However, the predictions of diet dry matter digestibility from different assays for measuring tannin contents were less accurate. Extracted condensed tannins in leaves of acacia species provided no reliable indication of diet dry matter and nitrogen digestibilities.  There were no reports found on the relationships between diet digestibility in rabbits and tannin contents of leaves of acacia species. High and negative correlations were found between nitrogen retention in rabbits (percentage of total nitrogen intake) and total phenolics, polyethylene glycol, precipitable phenolics by filter paper and radial diffusion contents in leaves of acacia species. Similar trends were observed when total phenolics, polyethylene glycol, precipitable phenolics by filter paper and radial diffusion contents in leaves of Acacia species were used to predict nitrogen retention. Prediction of nitrogen retentions (both in g/rabbit/day and nitrogen retention as a percentage of total nitrogen intake) by extracted condensed tannin contents in leaves of Acacia species was less accurate. The negative relationships can be explained by the detrimental effect of tannins on diet nitrogen digestibility, which eventually impacts negatively on nitrogen retention (Ben Salem et al 2000).

Diet dry matter and nitrogen digestibilities, faecal nitrogen output and nitrogen retention in rabbits were accurately predicted by total phenolics, polyethylene glycol, precipitable phenolics by filter paper and radial diffusion contents in leaves of Acacia browse supplements. These different assays are worth further study with the aim of developing an accurate prediction equation for use with tanniniferous feeds. However, extracted condensed tannin contents in leaves of acacia browse supplements provided no reliable indication of diet dry matter and nitrogen digestibilities, faecal nitrogen output and nitrogen retention in rabbits.

Conclusions  

• Acacia nilotica had the highest total phenolics, simple phenolics and phenolics that bind to proteins. However, it had the lowest amount of condensed tannins. Acacia Karroo had the highest amount of condensed tannins, both extracted and unextracted. Results indicated that there were no significant differences in diet intakes and digestibilities between the control diet and those supplemented with 4 % leaf meal of Acacia karoo, Acacia nilotica or Acacia tortilis. However, rabbits on a diet supplemented with leaf meal from Acacia tortilis drank more water than those on the other treatments. Faecal dry matter and nitrogen output, urine nitrogen outputs and nitrogen retention in rabbits were not significantly affected by supplementation with 4 % leaf meal of Acacia karoo, Acacia nilotica or Acacia tortilis. The amount of tannins from the browses (4 % of the feed mixture) used in the present study were ideal for supplementation without having negative effects. Negative correlation coefficients were observed between nitrogen digestibility and nitrogen retention in rabbits and total phenolics, polyvinylpolyprrolidone, precipitable phenolics by filter paper, radial diffusion, extracted condensed tannin and polyethylene glycol contents in leaves of acacia species. Nitrogen digestibility and retention were most accurately predicted by contents of total phenolics, precipitable phenolics by filter paper, radial diffusion, and polyethylene glycol in the leaves. However, extracted condensed tannin contents in leaves of acacia browse supplements provided no reliable indication of diet dry matter and nitrogen digestibilities, faecal nitrogen output and nitrogen retention in rabbits. Poor to moderate relationships were observed between diet dry matter, nitrogen and water intakes in rabbits and tannin contents in acacia leaves determined by different tannin assays. It is concluded that for the prediction of nitrogen digestibility and retention in rabbits, RD, TP, PPFP and PEG contents in Acacia leaves were most accurate. These are worth further study with the aim of confirming present results and developing accurate prediction equations for use with tanniniferous browses.
   

References 

Abdulrazak S A, Fujihara T, Ondriek J K and Orskov E R 2000 Nutritive evaluation of some Acacia tree leaves from Kenya. Animal Feed Science and Technology 85: 89-98

Al Mamary  M,  Molham A, Abdulwali A and Al-Obeide A  2001 In vivo effect of dietary sorghum tannins on rabbit digestive enzyme and mineral absorption. Nutritional Research  21: 1393-1401

AOAC  2000 Official Methods of Analysis, (17^th Edition) Association of Official Analytical Chemists. Washington DC

Barry T N and Duncan S J 1984 The role of condensed tannins in nutritional value of lotus pendunculotus for sheep I. Voluntary intake. British Journal of Nutrition 51: 485–491

Ben Salem H, Nefzaoui A, Ben Salem C and Tisserand J L 2000 Deactivation of condensed tannins in Acacia cyanophylla Lindl. Foliage by polyethylene glycol in feedblocks. Effect on feed intake, diet digestibility, nitrogen balance, microbial synthesis and growth by sheep. Livestock Production Science 64: 51-60

Chang K T, Wong T Y, Wei C L, Huang Y W and Lin Y 1998 Tannins in human health. A review, critical revision. Food Science Nutrition 38: 421-464

D’Mello J P F, Akamovic T and Walker A G 1987 Evaluation of Leucaena leaf meal for broiler growth and pigmentation. Tropical Agriculture (Trinidad) 64: 33-35

Dawra R K, Makkar H P S and Singh B 1988 Determination of protein binding capacity by filter paper assay. Analytical Biochemistry 170: 50-53

Dube J S and Ndlovu L R 1995 Feed intake, chemical composition of faeces and nitrogen retention in goats consuming single browse species or browse mixture. Zimbabwe Journal of Agricultural Research 33: 133-141

Dube J S, Reed J D and Ndlovu L R 2001 Procanthocyanidins and other phenolics in Acacia leaves of Southern Africa. Animal Feed Science and Technology 91: 59-67

Garrido A, Gomez-Cabrera A, Guerrero J J and van der Moer J M 1991 Effects of treatment with polyvinylprrolidone and polyethylene glycol on Faba bean tannins. Animal Feed and Technology 35: 3599-3603.

Giner-Chavez  B I, Van Soest P J,  Robertson J B, Lascano C and Pell A N 1997 Comparison of precipitation of Alfalfa meal protein and bovine serum albumin by tannins in the radial diffusion method. Journal of Science Food and Agriculture 74: 573-523

Hagerman  A E 1987 Radial diffusion method for determining tannins in plant extracts. Journal Chemistry of Ecology 13: 437-449

Hove L, Topps J H, Sibanda S and Ndlovu L R 2001 Nutrient intake and utilization by goats fed dried leaves of the shrub legumes Acacia angustissima,  calliandra aglothyrsus and Leucaena leucocephala as supplements to native  pasture hay. Animal Feed Science and Technology 91: 95-106

Kahiya C, Mukaratirwa S and Thamsborg S M 2003 Effects of Acacia karoo and Acacia nilotica diets on Haemonchus contortus infection in goats. Veterinary Physiology 115: 265-274

Makker H P S 1993 Antinutritional factors in food for livestock. Animal production in developing countries. Occasional Publication No. 16,  British Society of Animal Production.

Makkar  H P S 2003 Effects and fate of tannins in ruminant animals, adaptation to tannins and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Ruminant Research 49: 241-256

McNabb W C, Peters J ,S Foo L Y, Waghorn G C and Jackson F S 1998 Effect of condensed tannins prepared from several forages on the in vitro precipitation of ribulose 1.5 biosphophate caboxylase (rubisco) protein and its digestion by trypsin (EC 2.4.21.4) and chymotrypsin (EC2.4.21.1) Journal of Science and Agriculture 77: 201-212

McSweeney C S, Kennedy P M and John A 1988 Effect of ingestion hydrolysable tannins in Terminalia oblongata on digestion in sheep feed Stylosanthes hamata. Australia Journal of Agricultural Research 39: 235-244

Motubatse  M R,  Ng’ambi J W, Norris D and Malatje M M  2008 Effect of polyethylene glycol 4000 supplementation on the performance of indigenous Pedi goats fed different levels of Acacia nilotica leaf meal and ad-libitum Buffalo grass hay. Tropical Animal Health and Production 40: 229-238

Mueller- Harvey I,  Reed J D and Hartley R D 1987 Characterization of phenolic compound, including flavonoids and tannins of ten Ethiopian browses species by high performance liquid chromatography. Journal of Science Food and Agriculture 39: 1-14

Porter L J, Fritsch L W and Chan B G 1986 The conversion of proanthocyanidin and prodelphinidins to cyanidin and delphinidin. Phytochemistry 25: 223-230

SAS 2000  SAS User’s Guide: Statistics. SAS Institute, Cary, North Carolina, USA

Schofield P, Mbugua D M and Pell A N 2001 Analysis of condensed tannins: A review. Animal Feed Science and Technology 91: 21-40

Silanikove N, Gilbou N and Nitsau Z 1997 Interaction among tannins supplementation and PEG in goats given Oak leaves, effect on digestive digestion and food intake. Animal Science 64: 479-483

Silanikove N, Niteau Z and Perevolotsky A 1994 Effect of polyethylene glycol supplementation on intake and digestion of tannins containing in leaves (Ceratonia siliqua) by sheep. Journal of Agriculture and Food Chemistry 42: 2844-2847

Silanikove  N, Perevolotsky A and Provenza F D 2001 Use of tannin-binding chemicals to assays for tannins and their negative post ingestive effects in ruminants. Animal Feed Science and Technology 91: 64-81

Singleton V L, Orthofer R and Lamuela-Roventos R M 1999 Analysis of total phenolics and other oxidation substance and antioxidants by means of Folin-ciocalteu reagents. Methods of Enzymology 299: 152-178

Skarpe and Bergstrom 1986 Nutrient content and digestibility of forage plants in relation to plant phenology and rainfall in the Kalahari, Botswana. Journal of Arid Environment 11: 147-164.

Tangendjaja et al 1990:  Tangendjaja B, Raharjo Y C and Lowry J B 1990 Leucaena leaf meal in the diet of growing rabbits, evaluation and effect of low mimosine treatment. Animal Fed Science and Technology 29: 63-72.

Waterman P U and Mole S 1994 Analysis of Phenolics Plant Metabolites. Blackwell Oxford

Received 10 April 2009; Accepted 19 April 2009; Published 1 July 2009

Go to top