Detannification of Calliandra calothyrsus: the effect on digestibility and performance of rabbits

E Wina, I W R Susana and B Tangendjaja

Indonesian Research Institute for Animal Production, P.O Box 221 Bogor 16002
winabudi@yahoo.com

Abstract

Although the protein level is high, Calliandra calothyrsus also contains high level of tannin. This experiment aimed to study the effect of reducing tannin content in Calliandra calothyrsus on its digestibility and performance of rabbit. Polyethylene glycol (PEG) and Ca(OH)₂ were used to reduce tannin content in Calliandra calothyrsus. The first experiment was a digestibility trial, using 9 Rex rabbits which were raised in the metabolism cage individually. Rabbits were randomly allocated and each was fed  with either of 3 dietary treatments  which were 1) fresh Calliandra calothyrsus , 2) PEG treated Calliandra calothyrsus   and 3) Ca(OH)₂  treated Calliandra calothyrsus  as a sole diet. After 7 days of feeding, faeces were collected for 7 days. Then, a plastic neck’s collar was put on each rabbit to prevent coprophagy. The same feed was fed to rabbits for 7 days and then, faeces were again collected for 7 days. At the end of experiment, all rabbits were slaughtered and the contents of digestive tract compartment were collected and analysed.

Detannification treatments by PEG and Ca(OH)₂   increased dry matter, protein, NDF (neutral detergent fibre) and NDF-bound protein digestibilities (P<0.05). PEG not Ca(OH)₂  treatment reduced protein and NDF-bound protein level in the digestive tract compartments of rabbit. The second experiment was a growth trial, used 27 New Zealand White rabbits which raised individually in wire cage.   Rabbits were randomly allocated and each was fed with either of 3 dietary treatments  contained 30% of : 1) Calliandra calothyrsus  leaf meal (untreated), 2) PEG treated Calliandra calothyrsus  leaf meal and 3) Ca (OH) ₂  treated Calliandra calothyrsus  leaf meal. Feed consumption and body weight were recorded for 6 weeks. There was no statistical difference on daily consumption among treatments, however, a significant improvement (P<0.05) on daily gain was obtained by PEG and Ca(OH)₂   treatments compared to  the untreated treatment (26.62 and 26.35 g/day vs 24.04g/day, respectively).

In conclusion, detannification of Calliandra calothyrsus by PEG or Ca(OH)₂ improved Calliandra calothyrsus digestibility and performance of rabbit.

Keywords: detannification, Ca(OH)₂, forages, PEG

Introduction

Raising rabbit is getting popular in Indonesia especially in high altitude areas in Java and Sumatra since it can be handled by small land owners. Raising rabbit can give several benefits to the farmers and people in the surrounding areas since rabbit’s meat can be sold as grill meat or is processed as sausages or nuggets. The rabbit’s skin can be used for hat, bag or wallet etc. The rabbit’s urine and faeces are known as good organic fertilizer for growing vegetables and the young rabbit can be sold as pet animal.

To establish rabbit’s farm, forage is the most important feed as rabbit requires high fibre content in their diet. Calliandra calothyrsus is a well known multipurpose shrub legume that can fix nitrogen. It has been planted in Indonesia for reforestation, soil erosion control especially for high slope area, source of fuel wood, production of honey and source of forages for animal.

Calliandra calothyrsus is a good source of protein since it contains high protein level. However, Calliandra calothyrsus  also contains high level of tannin which is the highest among other legumes such as Leucaena leucocephala, Gliricidia sepium, Sesbania grandiflora, etc. Direct toxicity of tannin in Calliandra calothyrsus  has never been reported. In vitro work showed that tannin in Calliandra calothyrsus could be useful to protect protein of tofu waste and soybean meal from the rumen degradation (Wina et al 2008). However, some tannins depending on the type and level reduced the nutritive value of the forages by reducing intake or protein digestibility or affecting enzyme activities (Villalba and Provenza 2001, Al-Mamary et al 2001). Addition of  tannin containing sorghum grains to rabbit’s diet significantly changed the growth rate, food consumption and feed conversion ratio when the tannin level was high (3.5% catechin equivalent) but not when the tannin level was low (1.5% catechin equivalent) (Al-Mamary et al 2001).

Many attempts to reduce tannin effect in different forages have been conducted.  Physical treatments such as drying, wilting or aerobic and anaerobic storage  (Ben Salem et al 2005a, Vitti et al 2005) or chemical treatments such as urea, calcium hydroxide, hydrochloric acid, wood ash, polyethylene glycol (PEG)  (Vitti et al 2005; Wina et al 2005; Ben Salem  et al 2005 a, b) have been done to reduce tannin in different forages.  Very limited studies have been reported on the effect of detannified forages on rabbit performance. Therefore, this experiment aimed to study the effect of reducing the tannin content in Calliandra calothyrsus  by soaking in PEG or Ca(OH)₂  solution on digestibility of Calliandra calothyrsus and performance of rabbit.

Materials and methods 

The experiments were carried out at the Indonesian Research Institute for Animal Production which located at 400 m above sea level with average daily temperature 27^oC and rainfall 3000 mm/year. Calliandra  calothyrsus trees were grown at the site and were routinely cut at 12-14 weeks interval.

Preparation of :

1.                        untreated Calliandra calothyrsus  : The leaves were plucked from the branches.

2.                         PEG treated Calliandra calothyrsus  : One hundred grams of  leaves were sprayed with 4% polyethylene glycol in 100 ml of water.

3.                        Ca(OH)₂ treated Calliandra calothyrsus  : The leaves were soaked in the saturated Ca (OH) ₂  solution  in the plastic pail for 24 hours and then, were taken out from the pile and put on the bamboo sieves to let the solution drip off.

For experiment 1. All leaves were fed directly to rabbit as a sole diet.

For experiment 2. All leaves were, then, dried in the oven at 60^oC and  milled using hammer-mill and Calliandra calothyrsus  leaf meal was incorporated at the level of 30% in the rabbit ration, which then made into a pellet form.

Experiment 1

Nine growing Rex rabbits were placed individually in metabolism cage. Rabbits were originally fed a standard pelleted ration and then gradually fed Calliandra calothyrsus  forages until they were fed Calliandra calothyrsus  forages as a sole diet. There were 3 dietary treatments, i.e: 1) fresh Calliandra calothyrsus , 2) PEG treated Calliandra calothyrsus  and 3) Ca(OH)₂  treated Calliandra calothyrsus  and each was fed to three rabbits. After adaptation for one week, faeces were collected for 7 days period. Faeces, offer forages and forage residues were freeze dried and milled for further analysis. After that, a collar made from  rigid plastic sheet was put around rabbit’s neck to prevent coprophagy. Coprophagy is a habit of rabbit to eat their own soft faeces. Rabbits with neck’s collar were adapted to eat Calliandra calothyrsus  for a week and followed by faeces collection for 7 days. Nutrient digestibilities with and without coprophagy were calculated. At the end of feeding, all rabbits were slaughtered and the content of every section of digestive tract (stomach, small intestine, caecum, large intestine) was collected, freeze dried and milled. The content of digestive tract compartment was analysed for its protein, NDF and NDF-bound protein and tannin.

Experiment 2

This experiment was designed to evaluate detannification process of Calliandra calothyrsus  leaves when detannified C.calothyrsus leaf meal was used as the feed ingredient for rabbits.  The detannification process on Calliandra calothyrsus  leaves was done with polyethylene glycol and Ca (OH)₂  solution. Twenty seven New Zealand white rabbits were allocated individually in wire cages. They were fed with either  of 3 different complete rations. The rations were 1) feed contained untreated Calliandra calothyrsus  leaf meal (untreated), 2) feed contained PEG treated Calliandra calothyrsus  leaf meal and 3) feed contained Ca(OH)₂ treated Calliandra calothyrsus  leaf meal. The diet composition is presented in Table 1.

Each dietary treatment was given to 9 rabbits and the experiment was carried out for 6 weeks. Feed consumption and body weight were recorded.

Statistical analysis

All data were subjected to statistical analysis.  Nutrient digestibilities (Experiment 1) were calculated and subjected to statistical analysis by factorial design with 2 factors. The first factor was the effect of detannification process and the second factor was the effect of coprophagy. Randomized Completely Design was used in the feeding trial (Experiment 2) and any significant treatment was further analysed using a Least Significant Difference.

Results  

Table 2 shows nutrient digestibilities Calliandra calothyrsus in rabbit without coprophagy were relatively low.

The highest value was dry matter digestibility (39.7%) and the lowest was NDF digestibility (9.2%). Allowing rabbits to eat their own faeces (coprophagy) resulted in higher digestibilities of  all nutrients. The highest effect of coprophagy was obtained when rabbit was fed with fresh calliandra. If  it is calculated in percent increase of digestibility (Table 3), coprophagy improved significantly the NDF digestibility of fresh Calliandra calothyrsus (162% increase), followed by that of Ca (OH) ₂ treated Calliandra calothyrsus (75.2% increase).

However, the effect of detannification process was more significant  (P<0.01) than the effect of coprophagy on all nutrient digestibilities (Table 2). 

The result shows that detannification using PEG increased very significantly protein digestibility of Calliandra calothyrsus from 44.2 to 74.5% and the NDF bound protein digestibility from 45.1 to 74.9%, whereas Ca (OH) ₂ treatment increased protein digestibility from 44.2 to 66.4% and NDF bound protein digestibility from 45.1 to 55.1% (Table 2). PEG and Ca (OH) ₂ treatments  gave higher effects on nutrient digestibility when the rabbit’s condition was with no coprophagy than with coprophagy. The percent increase of NDF digestibility of fresh Calliandra calothyrsus due to PEG  and  Ca (OH) ₂ treatments were 232% and 43.6% higher than control, respectively (Table 4).

Figure 1A. shows that crude protein (CP) level in every compartment of rabbit’s digestive tract varied and reached the highest in the small intestine.

After passing the small intestine, CP level in the digestive content decreased slowly and reached the lowest in the faeces. Detannification using PEG caused lower of CP level in all digestive contents. In average, the CP level in all digestive contents due to Ca (OH) ₂ treatment is in between control and PEG treatment.

The curve of NDF content (Figure 1B)  in every digestive tract compartment was different from that of CP content. The lowest NDF content occurred in the small intestine and increased slowly and reached the highest  in the faeces. The NDF level of digestive contents from rabbit fed PEG was higher than other treatments. Ca (OH) ₂ treatment did not change the NDF level of the stomach, small intestine contents  and faeces (same as control), but it caused higher NDF level of  the caecum and large intestine contents.

Figure 1C. The NDF-bound protein level of all digestive contents from rabbit fed PEG treated Calliandra calothyrsus were lower than those from rabbit fed untreated  or  Ca (OH) ₂ treated Calliandra calothyrsus.       

The performance of rabbit fed untreated and treated Calliandra calothyrsus  (Experiment 2) is presented in Table 5.

The daily feed consumption was not significantly different among treatments (P>0.05). However, the consumption slightly increased when the rabbits were fed with Ca (OH) ₂  or PEG-treated Calliandra calothyrsus. The average daily gain of rabbit significantly increased when rabbits were fed with either Ca (OH) ₂  treated (26.4g/day) or PEG treated Calliandra calothyrsus  (26.6 g/day)  compared to those fed with untreated Calliandra calothyrsus   (24.0 g/day) (P<0.05). Although detannification improved average daily gain but it did not give any effect on Feed Conversion Ratio.

Discussion 

Effect of tannin

Tannin indeed showed a negative effect by reducing the nutrient digestibility of Calliandra calothyrsus. Tannin bound feed protein and  this tannin-protein complex made protein unavailable for digestion and, hence, reduced protein digestibility. Tannin also reduced the activity of some enzymes. Al-Mamary et al (2001) reported that tannin in sorghum grains reduced the activity of α-amylase, trypsin and lipase in the digestive system of rabbit and the higher the level of tannin, the bigger the reduction.  

NDF digestibility of Calliandra calothyrsus was very low in rabbit having no coprophagy. There are two possible explanations. First, when tannin bound the protein, the tannin-protein might not be degraded in the digestive tract and came out as part of the NDF fraction of faeces and increased the NDF content of faeces. This phenomenon has been previously observed in feeding wilted Calliandra calothyrsus to rabbit (Tangendjaja et al 2007) and also was reported by Makkar et al (1995). Makkar et al (1995) mentioned that there was a problem with detergent extraction technique for tannin-rich materials especially faeces material. The NDF fraction of faeces might contain tannin bound protein and was much higher than feed NDF, hence, the NDF digestibility value seemed extremely low or sometimes the value became negative. Therefore, caution should be taken when interpreting results obtained by the detergent methods. Second,  rabbit could not digest the fibre well when it was not allowed to eat its soft faeces The benefit of allowing coprophagy could be seen clearly (Table 3) that it improved drastically the fibre (NDF) and also NDF-bound protein digestion. Allowing coprophagy on rabbit was beneficial in improving fibre digestibility (Tangendjaja et al 2007; Carabano et al 1989) and increase ingestion of vitamin K and all B vitamins, amino acids and volatile fatty acids (Kent and Carr 2001).

Effect of detannification process

Detannification process using PEG  and Ca (OH)₂ significantly reduced the  tannin content of Calliandra calothyrsus from 15.58% to 1.00 and 3.79%, respectively (measured by protein precipitation method). Although both chemicals reduced tannin content,  the chemical reaction between PEG and  Ca (OH)₂ to tannin is different. PEG (polyethylene glycol) will bind tannin strongly by hydrogen bond between oxygen of PEG chain and phenolic hydroxyl group of tannin but the bonding can be released at low pH.  Ca (OH)₂ which is a basic solution allowed oxidation of the phenolic groups of tannin so that the structure of tannin changed (Makkar and Singh 1992).

As a result of reducing tannin content, detannification process using PEG and Ca (OH)₂ significantly improved nutrient digestibility. The increase of crude protein digestibility was also reported by Ayers et al (1996) when they fed rabbit with black locust (Robinia pseudoacacia) together with PEG. Protein, which previously bound to tannin, was now released and could easily be digested by gut enzymes, hence, increased the protein digestibility. It has also been proved that PEG promoted microbial attachment and activity in the presence of tannin (Bento et al 2005). PEG treatment gave a higher effect on nutrient digestibility than Ca (OH)₂ treatment. Similar results have been reported with other species such as sheep (Wina et al 1994) and goat (Alam et al 2005).

Although rabbit has the ability to digest fibre, it is shown that fibre was more difficult to be digested by rabbit especially if the rabbit could not eat their soft faeces. The habit of eating soft faeces improved significantly fibre (NDF) digestion (Table 3). The NDF digestion was more pronounced when the rabbit without coprophagy consumed PEG or Ca (OH)₂  treated Calliandra calothyrsus compared to the fresh forage (Table 4). So, PEG or Ca (OH)₂ treatments seemed to improve the digestion rate not only protein but also fibre fractions of Calliandra calothyrsus. In PEG treatment, as the degradation of feed was higher, the absorption of nutrients might be higher as it was indicated by lower of protein and NDF bound protein levels and higher NDF fraction in the contents of digestive compartment (Figure 1 A, B and C).

Beside higher nutrient digestibility or absorption, higher consumption of feed contained Ca (OH)₂ or PEG treated Calliandra calothyrsus might contribute to the higher daily gain of rabbit. Villalba and Provenza (2001) have reported that PEG increased the intake of tannin-containing plants. Since tannin has been bound with PEG, other nutrients especially protein is available to be degraded and its amino acids absorbed by the body. Beside rabbit, PEG treatment has also improved the performance of other livestock such as sheep (Ben Salem et al 2005b), goat (Alam et al 2005). Only a few reports showing the beneficial effect of Ca (OH)₂  treatment  on animal performance and this experiment could show that Ca (OH)₂  treatment improved daily weight gain of rabbit and would be easily applied in villages as it is much cheaper technology than PEG treatment  

Beside  improving digestibility and rabbit’s performance, feeding low level of tannin gave another beneficial effect. Maerten and Struklee (2006) reported that feeding only 0.5% chestnut tannin extract to does and litters reduced the mortality rate of rabbit which caused by diarrhea’s problem. They hypothesized that tannin-protein form avoided a protein to overload in the gut and tannins could partially cover the mucus membrane and became a barrier against toxins. After Ca (OH)₂ or PEG treatment, treated Calliandra calothyrsus leaves contained only small amount of tannin which may give beneficial effect as above.

Conclusion 

• Tannin is an important factor affecting calliandra digestibility in rabbit.

  
  

• The detannification process improved  nutrient digestibilities higher than coprophagy.  

  
  

• Polyethylene glycol (PEG)  treatment gave  higher increases than Ca(OH)₂ treatrment on dry matter,  protein, NDF and NDF-bound protein digestibilities.

  
  

• As a result of increasing nutrient digestibility, both PEG and Ca(OH)₂ treated Calliandra calothyrsus improved body weight gain of rabbit, however Ca(OH)₂ treatment would be more easily applied and cheaper than PEG treatment.

References 

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Received 8 December 2008; Accepted 19 February 2009; Published 1 June 2009

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