18

II. REVIEW OF LITERATURE

2.1. Mechanism of fibre degradation 2.1.1. Microbes affecting fibre degradation 2.1.2. Factors affecting fibre utilization

2.2. Microbial biomass synthesis 2.2.1. Microbial efficiency 2.2.2. Dietary factors influencing microbial biomass yield

2.3. Techniques for assessing the nutritive value of straw 2.3.1. Rate of fermentation, yield of biomass and partitioning factor 2.3.2. Estimation of microbial mass by purines 2.3.3. Volatile fatty acid production

2.4. Manipulation of rumen fermentation 2.5. Effects of bioactive agents on rumen fermentation

2.5.1. Tannins 2.5.1.1. Chemistry of tannins and their effect on rumen fermentation 2.5.1.2. Effects of tannin on digestibility and partitioning of the fermentation

products 2.5.1.3. Effects on biomass production and the microbial efficiency 2.5.1.4. In vivo effects 2.5.2. Saponins 2.5.2.1. Chemistry of saponins and their effect on rumen fermentation 2.5.2.2. Effect of saponin on availability and partitioning of nutrients 2.5.2.3. Effect of saponin on biomass production and the microbial efficiency 2.5.2.4. In vivo effects 2.5.3. Tannin-saponin interactions 2.5.4. Mulberry leaf as supplement 2.5.5. Non ionic surfactant 2.5.5.1. Chemistry of surfactant and their effect on rumen fermentation 2.5.5.2. Effects of surfactant on availability and partitioning of the nutrients 2.5.5.3. Effects of surfactant on biomass production 2.5.5.4. In vivo effects 2.5.6. Surfactant-saponin interaction

19

2.5.7. Surfactant-tannin interaction

The feeding value of straw is generally poor and provides nutrients at sub-

maintenance level only for ruminants. The straws are characterized by low energy and

low protein content. Straw is also a poor source of minerals, high in lignin, which is the

major impediment in release of potential energy present in the form of polysaccharides

(Walli, 2004). Rice straw has minerals such as silica, Phosphorus, Calcium, Potassium

and Sulphur in insoluble forms, which encrust plant structures and impede the digestion

of cell wall material (Le.T. Trung, 1986).

2.1. Mechanism of fibre degradation

Ruminant have the ability to convert low quality feeds into quality protein and to

utilize feeds from land not suitable to grow crops for human consumption. This is made

possible by the ruminal microorganisms that synthesize and secrete β1-4 cellulase

enzyme complex, thereby allowing hydrolysis of plant cell walls. However, the actual

conversion of feeds, especially fibrous forages, to meat and milk is not very efficient.

Only 10–35 per cent of energy intake is captured as net energy, while 20–70 per cent of

cellulose may not be digested by the animal. If a greater percentage of the total dietary

energy from forages was available to ruminants, lower cost diets could be formulated and

environmental resources would be used more efficiently (Varga and Kolver, 1997).

2.1.1. Microbes affecting fibre degradation Rumen represents a complex ecosystem and an efficient biological fermentation

vessel where a variety of fermentation reactions essential for growth and productivity of

the ruminant are brought about with the help of microorganisms. The efficiency of

ruminant nutrient utilization is greatly dependent upon the balance of fermentation

products, which is regulated by types and activities of rumen microorganisms. The

microflora inhabiting the rumen include cellulolytic, hemicellulolytic, xylanolytic,

pectinolytic, proteolytic, lipolytic and methanogenic organisms (Hungate, 1966). Among

the rumen bacterial population, the most predominating types include R.albus,

R.flavefaciens, Butyrivibrio fibrisolvens, Fibrobactor succinogenes, Prevotella

ruminicola and Selenomonas ruminantium etc. which play vital role in feed utilization

20

(Pattnaik et al., 2004). Fibrolytic bacteria tend to degrade the more readily digestible

structures such as the mesophyll cells, although F. succinogenes digests parenchyma

bundle sheaths, epidermal cell walls and leaf sclerenchyma (Akin, 1989).

The major contributors to cell wall degradation are Bacteroides succinogenes,

acting through close adherence by a cell bound extracellular cellulase, which also

promotes cellulolysis (Akin et al., 1988, Latham et al., 1978). Fibrobactor succinogenes

s85 also adheres intimately with cellulose fibers (Bae et al., 1993). Another prevalent

colonizer of cell wall is Ruminococcus albus and it adheres to cellulose at a greater

distance (Cheng and Costerton, 1986).

The fungi have an important role in fiber digestion because they are able to

penetrate both cuticle and cell wall of lignified tissues (Akin, 1986) with their cutinase

activity (Kolattukudy, 1984). In addition, fungi can degrade more recalcitrant cell wall

materials, including the sclerenchyma and vascular tissue (Akin, 1989). This aids in

reducing the tensile strength of the tissue and provides additional sites for bacteria to

access and attach (Akin, 1986). Fungi degrade 37–50 per cent of barley straw, whereas

rumen bacteria digest only 14–25 per cent (Joblin et al., 1989). The fibrolytic activity of

fungi, which includes both cellulase and hemicellulase activities, is enhanced by

hydrogen-utilizing methanogens (Joblin et al., 1989), which decrease the repressive

effect of hydrogen (Orpin and Joblin, 1988). The anaerobic fungi also possess a broad

range of fibrolytic enzymes, including cellulases. Neocallimastix frontalis has the highest

cellulolytic activity of any organism ever reported in the literature. Borneman et al.

(1990) demonstrated the presence of both ferulic and coumaric acid esterase activities in

two monocentric and three polycentric fungi.

The protozoa in the rumen also possess cellulases, xylanases and a broad range of

glycosidases although none have been purified (Cheng et al., 1991). The microbial

fermentation products eventually become available as energy (volatile fatty acids) and

protein (microbial cells) for animal tissue metabolism. When straws are fermented, the

products are basically acetic (65%), propionic (20%), butyric (9%) acids and branched

chain acids (5%). These are further absorbed for fat and glucose production in the animal.

21

Hence, with low-quality feeds the maximum amounts of nutrients are to be

extracted by microbial digestion from the basal diet (crop residues and natural pasture),

and providing sufficient rumen degradable substrates to optimize the growth efficiency of

the rumen microbes (Teferedegne, 2000).

2.1.2. Factors affecting fiber utilization

Several components contribute to the nutritive value of forges namely chemical

composition, level of voluntary intake, digestibility and efficiency of metabolism at the

tissue or cell level. There are considerable variations within cereal straws especially in

readily fermentable components. Variation both between and within species could be

due to effects of environmental factors such as light, fertility, moisture, temperature, and

disease during growth of crops and or genotypic contributions. Devendra (1982)

indicated that exposure to weather decreased crude protein content of straw from 5.6 to

3.4 per cent and Calcium from 0.31 to 0.21 per cent and Phosphorus from 0.11 to 0.2 per

cent. Rice straw collected soon after the harvest of grain may be 55 per cent digestible

because of a high sugar level and will drop to 40-45 per cent during storage, as sugars are

lost. Sugars are fermented instantaneously on entering the rumen where as fermentation

rate of fiber may reach a peak at some 4-6 hours post ingestion of roughage (Annison and

Lewis, 1959).

The cellulolytic activity of the rumen microorganisms may be restricted when

poor quality forages are fed (Akin et al., 1988) and this is likely due to a variety of

factors such as presence of inhibitory compounds and steric hindrance (Varga and

Kolver, 1997).

Feed intake in ruminants consuming fibrous forage is primarily determined by the

level of rumen fill, which in turn is directly related to the rate of digestion and passage of

fibrous particles from the rumen (Van Soest, 1994). In vivo digestibility can vary from

20 to more than 50 per cent depending on degradation rate and outflow rate (Orskov,

1986). Low-quality diets (hay and crop residues) are characterized by low animal

productivity, resulting from the shortage of one or more essential nutrients required for

rumen microbial activity, typically N.

22

Addition of little amount of fermentable carbohydrate that provides energy for the

microbes would help proliferation of microbes and aid digestion of roughages. However

addition of concentrates ad lib to roughages increase the proliferation of amylolytic and

proteolytic bacteria producing high VFA and thus reducing the pH which would reduce

the proliferation of cellulolytics and affect fibre digestion. This will also cause

proliferation of protozoa that would be detrimental to the animal for two reasons.

Firstly it appears that a proportion of certain species of protozoa sequester on the

large plant particles within the rumen, eventually die and ferment rather than flow with

digesta and hence become unavailable for digestion in the small intestine. This reduces

the amount of amino acid N absorbed by the animal and changes the form of digestible

energy absorbed with less from digestion of microbes and more from absorbed VFA

(Dixon, 1985). Secondly there is evidence that large populations of protozoa are

associated with reduced populations of anaerobic fungi responsible for digestion of poor

quality fiber in the rumen, hence reduced cell wall fraction digestion (Dixon, 1985).

2.2. Microbial biomass synthesis Microbial biomass synthesis determines the microbial protein available to the

animal and the potential for the use of nonprotein nitrogen by rumen fermentation. The

net metabolizable protein received by the animal is the sum of these digestible microbial

protein and feed protein escaping the rumen (Van Soest, 1994).

2.2.1. Microbial efficiency

Microbial efficiency is expressed (i) in terms of Y glucose (yield per mole of

glucose) (ii) as grams of cells per mole of glucose or per 100 g of fermented feed (Y sub)

and (iii) yield of dry cells (or of microbial protein) per mole of ATP (YATP) or per 100 g

(or kg) of organic matter either apparently or truly fermented in the rumen (Yokoyama

and Johnson, 1979).

2.2.2. Dietary factors influencing microbial biomass yield Efficiency of microbial biomass yield increases as the dilution rate is increased.

Silages and high concentrate diets show lower yield than forages or mixed diets. Rymer

23

and Givens (1999) have shown that good quality feeds (grasses, silage, wheat, maize

molasses, sugarbeet feed and fish meal) which produces large amount of gas and SCFA

yield small amount of microbial biomass per unit feed truly digested. High rates of

passage promote on an average faster digestion rates in the rumen leaving slow digesting

substrates unavailable.

2.3. Techniques for assessing the nutritive value of straw

The digestibility of feeds can be estimated by biological methods, which stimulate

the digestion process. The three major digestion techniques currently available to

determine the nutritive value of ruminant feeds are (1) digestion with rumen

microorganisms as in the work of Tilley and Terry (1963) or gas method (Menke et al.,

1979) (2) cell free fungal cellulase and (3) in situ incubation of samples in nylon bag in

the rumen (Getachew et al., 1998).

2.3.1. Rate of fermentation, yield of biomass and partitioning factor

In vitro methods have the advantage not only of being less expensive and less

time consuming but they allow to maintain experimental conditions more precisely than

do in-vivo trials (Getachew et al.,1998). The in vitro methods are based on quantification

of substrate degraded or microbial protein produced using internal and external markers

and of gas or SCFA production in an in vitro rumen fermentation system based on

syringes (Menke et al., 1979). The method also facilitates to estimate not only DMD but

also efficiency of microbial protein synthesis (Makkar, 2000). By in vitro technique,

measurement of the gas produced also reflects SCFA production. But the microbial

biomass production that is inversely related to gas and SCFA production is measured in

relation to true degradable organic matter or TDOM.

Blummel et al. (1997a) have demonstrated how a combination of in vitro gas

production measurement with a concomitant quantification of TDOM provides important

information about partitioning of fermentation products. The in-vitro microbial biomass

production could be calculated as MBP = TDOM- (Gas volume*stoichiometric factor).

For roughages the stoichiometric factor is taken as 2.2 and for concentrates it is 2.34.

24

Blummel et al. (1998) examined the microbial growth kinetics for hays after 8,

12, 18 and 24 hours of incubation in the modified Hohenheim gas test and found

maximum microbial mass recovery is approximately coincide with the incubation time at

which half of the symptomatic gas volume was produced (halftime or t1/2). In fifteen

straws, microbial biomass production estimated at t1/2 (time at which half of the gas

produced) by purine bases and microbial nitrogen analysis were reasonably well related

to microbial mass estimated at t1/2 as MBP=TDOM- (GV*SF); (R2-0.65), in contrast to

gas volume alone amounted for 39 per cent variation in microbial biomass (Blummel et

al., 1999).

Partitioning factor (PF) is defined as ratio of substrate truly degraded (mg) to gas

produced (ml) is regarded as measurement of efficiency of microbial biomass synthesis

(Blummel et al., 1997b). Of late the estimation of PF, which in turn is a reflection of

potential of feedstuff to support microbial biomass synthesis, has evolved as criteria for

evaluation of microbial biomass synthesis potential and quality assessment of different

feedstuffs.

The production of microbial biomass per unit of ATP may vary from 10-32 mg.

At similar yATP, proportion of higher propionate leads to higher PF compared to higher

acetate production (3 vs 2.8 ands 3.6 vs 3.3 at yATP 10 and 20 respectively). For

conventional feeds (roughages) PF ranges from 2.74 to 4.65 mg/ml (Blummel et al.,

1997b).

PF cannot be determined by residue determination in case of tannin rich forages.

This is due to leaching of tannins from the feed during fermentation, contributing to (i)

drymatter loss but without contributing to gas production and (ii) inhibition of cell

solubles by tannins or combination of both (i) and (ii) (Getachew et al., 1998). In tannin

containing feedstuffs gas measurement should be combined with microbial mass

determination using either internal (e.g., purines, 2, 6-diaminopimelic acid) or external

markers (e.g.,15N or 32P incorporation) (Makkar and Becker, 1997b).

25

2.3.2. Estimation of microbial mass by purines

The rapid degradation of dietary nucleic acids in the rumen has led to the wider

use of nucleic acids or their constituent purine or pyrimidine bases as markers of

determination of microbial protein synthesis in the rumen. The method of Zinn and

Owens (1986) for quantification of purines has been used by several workers as it is

simple and inexpensive. Ushida and Jouny (1985) have shown good recovery of added

purine bases and of the purine bases in yeast RNA added to bacterial samples using the

method of Zinn and Owens (1982). The presence of undigested feed produces errors in

the determination of purines. Makkar and Becker (1999) suggested that additional

centrifugation in the procedure before purines are precipitated by silver nitrate would

help in removing the interfering substances. The use of 2M HClO4 instead of 12 M

HClO4 for hydrolysis would help in reducing the interference by undigested matrices.

2.3.3. Volatile fatty acid production

Blummel and Orskov (1993) have shown that the gas volumes in the bicarbonate

buffered Hohenheim in vitro gas production test for straw reflects SCFA production very

closely. Gas volumes were produced quantitatively and qualitatively according to the

stoichiometry of Wolin (1960) i.e. the amount of fermentative CO2 and CH4 could be

accurately calculated from the amount and proportion of acetate, propionate and butyrate

present in the incubation medium. The relationship between in vitro fermentation

products and substrate supply could be generalized as

Equation1: Substrate incubated-substrate truly undegraded = Microbial biomass +SCFAs

+ Gases

Equation2: Substrate incubated-substrate apparently undegraded = SCFAs+ Gases

Equation 3: Substrate truly degraded-substrate apparently degraded = Microbial biomass.

Fermentative CO2 and CH4 from carbohydrate fermentation were calculated according to

the equation

Mmol CO2 = a/2+p/4+1.5b

M mol of CH4= a+2b-CO2

26

Where a and b are proportions of sum of a, p, and b where CO2 is inserted from equation

(1)

Isobutyrate is produced from valine and H2O and one mmol of CO2 is produced from

generation of one mmol of isobutyrate

CO2 produced from buffering (CO2buty) is: mmol CO2buty= mmol total SCFA (a+p+b+bi)

Gas volumes calculated as: ml of gas = mmol of gas*gas constant*temperature where gas

constant is 0.0821 and temperature is 312 Kelvin (273 K+ incubation temperature of

390C. Hence the total gas volume is calculated as

ML gas = mmol (CO2ferm +CH4ferm+CO2isobuty+CO2but) x 0.0821 x 312.

2.4. Manipulation of rumen fermentation

The presence of protozoa in the rumen significantly decreases the amount of

bacterial protein leaving the rumen and becoming available for digestion in the intestine.

In terms of ratio of protein from microbes to VFA energy in the nutrients absorbed, this

ratio may vary from 25g microbial protein/MJ VFA in fauna free to below 12-14 g /MJ

VFA in faunated ruminants (Leng, 1990). There is increased microbial protein for

digestion in fauna free as compared with faunated ruminants (Ushida et al., 1989).

Increased digestibility of fibrous feeds and increase in live weight gain and wool

growth have been observed when protozoa are absent from the rumen of sheep on straw

based diet (Soetanto, 1986; Romulo et al., 1989) including that those supplemented with

substantial quantities of green and/or bypass protein (Foster and Leng, 1989; Habib et al.,

1989). These findings show the potential for improvement in ruminant productivity on

low quality forages by sustaining fauna free animals (Bird, 1989). The major advantage

of defaunated state is seen when Protein: Energy ratio in the nutrients absorbed is low

and when rumen microbial growth is not constrained by nutrient deficiencies.

2.5. Effects of Bioactive agents on rumen fermentation Many feed additives have been developed to improve the efficiency of nutrient

use by decreasing the total amount of methane or ammonia N produced. Many plants

produce secondary metabolites such as phenolic compounds, essential oils and saponins

27

that affect the microbial activity. Although many plant extracts affect the microbial

activity, their effect on rumen fermentation and improvement in the nutritive value of

feedstuff are to be studied in detail.

2.5.1. Tannins

Tannins are naturally occurring water-soluble plant polyphenols that precipitate

protein (Haslam, 1989). Horvath (1981) defined tannins as phenolic compounds of

sufficiently high molecular weight containing sufficient phenolic hydroxyls and other

soluble groups (i.e. carboxyls) to form effectively strong complexes with protein and

other macromolecules under the particular environmental conditions being studied.

Ability to complex with minerals could also be added to the definition (Reed, 1995).

2.5.1.1. Chemistry of tannins and their effect on rumen fermentation

Tannins are usually subdivided into two groups hydrolysable and condensed

(proanthocyanidins) tannins. Hydrolysable tannins are gallic acid and ellagic acid esters

of core molecules that consist of polyols such as sugars and phenolics such as catechin

and are susceptible to enzymatic and non-enzymatic hydrolysis yielding gallic acid or

ellagic acid and glucose.

Proanthocyanidins (PA) are polymers of flavan-3-ols linked through an

interflavan carbon bond that are not susceptible to hydrolysis. The ability of tannins to

form strong complexes depends on characteristics of tannin and protein molecular

weight, tertiary structure, isoelectric point and compatibility of binding sites. Tannins

have a large number of free phenolic hydroxyl groups and form strong hydrogen bonds

with protein and carbohydrates (Haslam, 1989). Tannins also complex with protein

through hydrophobic bonding (Oh et al., 1980). They also form covalent bonds with

protein through oxidative polymerization reactions as a result of heating, exposure to UV

radiation and by the action of polyphenol oxidase.

Makkar et al. (1990) have suggested that the protein precipitating capacity (PPC)

is more governed by the total phenolic content than the level of condensed tannin and

28

highly polymerized tannins have little protein precipitating capacity. The PPC activity of

any material is determined by their degree of polymerization, specific activity and the

material itself capable of binding proteins.

The interactions occur between PA and proteolytic enzymes or dietary protein and

inhibit the proteolysis by steric interference on the binding of the protease with

susceptible sites on the dietary protein (McManus et al., 1981) or by direct inhibition of

the proteolytic enzymes (Kumar and Singh, 1984). Tannins may decrease the enzyme

activity of closely associated microorganisms such as Butyrivibrio fibrisolvens and

Streptococcus bovis through binding directly with cell coat enzymes (Jones et al., 1994).

Tannins reduce the bacterial numbers by inhibiting growth (Brooker et al., 1999; Jones et

al., 1994; Min et al., 2002). Tannins may affect adhesion of rumen microorganisms to

substrate through binding to the substrate (Artz et al., 1987, Barry et al., 1986, Makkar et

al., 1995) or bacterial cell surface (Bae et al., 1993; Jones et al., 1994; Molan et al.,

2001). Tannins have shown to exert higher negative effect on the activity of

microorganisms that are surface active such as cellulolytic bacteria rather than

microorganisms present in liquid phase (Makkar et al., 1988). Addition of tannic acid

(1nM) to in vitro fermentation of cellulose by rumen fungus N. frontalis caused a

reduction in the degradation of cellulose due to depression of adhesion and inhibition of

enzymatic activity (Muhammed et al., 1994). The ruminal fungus Neocallimastix

patriciarum was able to degrade cellulose in presence of 100 ug/ml of condensed tannin

from L.corniculatus (McAllister et al., 1994). Addition of PEG to cellulose and Mimosa

Tannin treatment produced higher number of microbes attached to cellulose but these

were less efficient in the incorporation of 15 N (Bento et al., 2005).

Experiments done by Tanner et al. (1994) have shown that the inhibitory effect on

proteolysis occurs for PA: protein ratios above 1:5. Forage legumes containing between

20 and 50 g PA/kg DM are encountered growing under field conditions, and having PA:

soluble protein ratios in excess of those required to inhibit proteolysis. It is likely that

ruminants grazing such forages benefit from increased protein absorption due to reduced

rumen hydrolysis of leaf protein.

29

Molan et al. (2000) showed condensed tannin to bind both plant protein and

bacterial cells, and both interactions lead to decreased degradation rate of plant protein.

They further observed that the interaction between condensed tannin and plant protein

was reversible with PEG, whereas CT-bacterial interaction was stronger or the interaction

was of different type. Higher concentrations of CT may act by inhibiting the bacterial

proteinase enzyme directly by blocking the active site, or indirectly via inhibition of

general bacterial metabolism.

It is suggested that insoluble condensed tannin functions through reducing plant

protein degradation in the rumen by forming reversible complexes with protein (Barry et

al., 2001, Makkar, 2003a) while free condensed tannin can react with and inactive

microbial enzymes explaining why high levels of free CT reduce rumen carbohydrate

digestion. In the rumen, tannin protein complexes are stable. However, this dissociates

post-ruminally in response to extremes of pH (McNabb et al., 1996). The low pH in the

abomasum as well as high pH in small Intestine can stimulate the dissociation. This

condensed tannin could be exploited to improve total protein availability of protein in

forages by reducing wasteful deamination of protein in the rumen in diets with high

rumen degradable protein in excess of microbial requirement (Barry et al., 1986, Parez-

Maldonado et al., 1995).

2.5.1.2. Effects of tannin on digestibility and partitioning of the fermentation

products

Waghorn et al. (1994) have reported higher levels of rumen ammonia (μg N/ml of

rumen liquor) when PEG was infused (20010.5) ml as against 134.856.6 ml in

Romney marsh wethers fed on a tannin rich legume forage Lotus peduculatus, as sole

feed. The rise in rumen ammonia concentration in PEG treated animals are better

explained by increased degradation of plant proteins resulting from binding between CT

&PEG. In fibre and DM degradation, they found that the total DM digestibility and

proportion of DM digested in the rumen were lower in tannin fed sheep than those

receiving PEG but the greater loss of DM to digestion was observed in the large intestine.

They have concluded that condensed tannins did not affect the digestion of cellulose,

hemicellulose and ash but there were wide variations in the apparent digestibility of

30

hemicellulose and lignin but not cellulose as 97 per cent of cellulose was degraded in the

rumen. They also found that the intake of tannin rich browse is 12 per cent lower than

those receiving PEG.

Bae et al. (1993) showed the digestion of filter paper by Fibrobactor

succinogenes declined drastically to nil value when the level of condensed tannin from

Lotus corniculatus was increased from 300 ug/ml to 400 ug/ml. It was also demonstrated

that endoglucanase activity was inhibited at concentrations above 400 ug/ml.

Waghorn et al. (1994) suggested that tannin reduced the rate of fermentation

indicated by significantly lower levels of VFA, larger rumen pool size and lower levels of

intake but the longer residence time of fiber would enable a more complete digestion

even at a slower rate rather than inhibited or changed by CT which is expressed by

similar molar ratios of VFA after 21 days in tannin fed and tannin + PEG fed sheep.

Makkar (2003a) has suggested that the level of condensed tannin should not be high

enough to affect the true digestibility of the substrate to have the beneficial effect.

Negative correlation between condensed tannin and gas production (r=-.564) and

degradability parameters (r=-0.42) was reported by Tolera et al. (1997). Makkar (2003a)

suggested that the measurement of gas alone in in vitro systems could lead to misleading

conclusions on effect of tannins as potential rumen modulators in the extent of digestion

of tanniferous feeds and microbial protein synthesis. Makkar has also concluded that for

tannin rich feeds the determination of true and apparent digestibility by the use of Van

Soest’s detergent system of fiber analysis proved to be meaningless due to the appearance

of tannin-protein complexes in the true as well as the apparent residues (Makkar and

Becker, 1997b). They have suggested that for tannin rich feeds the gas measurements

should be combined with the determination of microbial mass using internal or external

markers.

2.5.1.3. Effects on biomass production and the microbial efficiency

Tannins at low levels have the potential to modulate rumen fermentation towards

maximizing microbial protein synthesis. The decreased rate of digestion of feedstuff by

tannins could help in synchronized release of various nutrients that in turn might be

31

responsible for increased microbial efficiency (Makkar et al., 1998). This may also bring

about reduction in the spillage or uncoupling of released energy. The higher molar

proportion of propionate in the in vitro fermentation system and lower protozoal counts

produced by tannins (Makkar et al., 1995) are consistent with the higher efficiency of

microbial protein synthesis observed in presence of tannins (Makkar, 2003a). Increase in

the efficiency of microbial protein synthesis and decrease in the protein degradability of

feed protein in the rumen are beneficial for ruminants as they increase the supply of non

ammonia nitrogen to the lower intestine for production purposes resulting in higher milk,

meat and wool production.

But effects may vary with plant material. It could be speculated that these plant

secondary metabolites increase the efficiency of microbial protein synthesis and hence

through these, higher proportions of degraded substrate could be channeled towards

microbial protein synthesis and lower proportion towards SCFA and gas.

Microbial protein synthesis in vitro expressed as 15 N incorporation into microbes

per unit of short chain fatty acid production was more efficient in the presence of tannin

(Makkar et al., 1995).

Bento et al. (2005) summarized on effect of mimosa tannin with pectin and /or

PEG on cellulose showed at 24 hour incubation, MT+C produced 113.7μg 15N/G residual

pellet as against 145.3 μg 15N/G residual pellet with control and with PEG 147 μg 15N /G

residual pellet showing a reduction of 32 μg15N/G (p<.001). Mimosa tannin reduced

(p<0.05) TN content and increased (p<0.05) 15N content of residual pellet.

2.5.1.4. In vivo effects

Typical effect of condensed tannin in forages includes low DM intake, poor DM

and N digestibility and poor livestock performance. At low levels of tannin in diets higher

animal performance has been attributed to protection of food proteins from degradation in

the rumen (Waghorn et al., 1994).

Forages containing up to 110 g PA/kg dry matter (DM) increase the duodenal

absorption of non-ammonia N in ruminants, resulting in faster lean weight gain (Barry

32

and Manley, 1984; Purchas and Keogh, 1984; Barry et. al., 1986). However, diets of

forages with high levels of PA (> 110 g/kg DM) may result in decreased voluntary intake,

digestibility, adipose deposition (Barry and Duncan, 1984; Barry and Manley, 1986;

Chiquette et al., 1988; Van Hoven and Furstenburg, 1992). The effect of tannins in forage

legumes and leguminous tree supplements on intake of cereal crop residues showed,

legume trees with high content of PA are associated with low straw intake whereas

legume trees that contain moderate levels of PA are associated with high straw intake

(Woodward and Reed, 1989).

Barry et al. (1986) reported that less than 4 per cent of tannin in the ration was

beneficial to ruminants in increasing duodenal NAN flow and the Nitrogen.

Sheep fed with dried sainfoin had a greater flow of essential amino acids to the

duodenum compared with sheep fed dried alfalfa (Harrison et al., 1973). PA in Lotus

pedunculatus are associated with an increased flow of essential amino acids through the

abomasum and increased net apparent absorption of threonin, valine, isoleucine, tyrosine,

phenylalanine, histidine and lysine (Waghorn et al., 1987). McNabb et al. (1993)

observed increased utilization of cystine in sheep fed L.pedunculatus in comparison with

sheep fed L.pedunculatus in which the tannins had been deactivated by the addition of

PEG.

Reddy et al. (1979) fed tamarind seed hauls containing 13.52 per cent tannin at 0,

10 and 15 per cent levels to calves and reported DMI of 3, 2.93 and 3.12 percent body wt,

the difference being not statistically significant.

Feeding Quebracho tannin at 5 per cent DM with pelleted dried grass reduced

apparent digestibility of NDF from 63.3 per cent to 58.8 per cent after 2 weeks of feeding

the diet to sheep (Dawson et al., 1999).

Bhatta et al. (2000) in his study on feeding tamarind seed husk to diet of lactating

cows at 2.5% and 7.5% levels showed no difference in digestibility of DM, OM, EE,

NFE, NDF, and ADF and significant reduction in CP digestibility. In vitro microbial

protein synthesis observed in presence of TSH with starch and cellulose showed

33

significant reduction at 7.5% level. However addition of TSH at 7.5% level to

compounded feed mixture showed beneficial effect with respect to body weight gain,

milk protein and nitrogen balance (Bhatta et al., 2001).

2.5.2. Saponins

Saponins are natural detergents found in many plants. Because of their surfactant

action, saponins are known for their bloat production, photosensitization, reduction in

palatability and feed intake and hemolytic, piscicidal, insecticidal, molluscicidal.

membranolytic, ammonia binding, hypo-cholesterolemic, anti-carcinogenic, immune

stimulating, antiprotozoal, antifungal, antibacterial and antiviral properties (Makkar and

Becker, 2000).

Selective suppression of the rumen protozoa has been suggested to be a promising

approach to reduce methane release (Moss et al., 2000) as shown by the results of

defaunation (Whitelaw et al.,1984; Dohme et al., 1999). However the complete

suppression is difficult on practical conditions hence a reduction rather than total

elimination could be achieved with tropical diets to improve productivity (Dominguez

Bello and Escobar, 1997).

Foliage (Rosales et al., 1989; Navas-Camacho et al., 1994; Newbold et al., 1997)

as well as fruits (Diaz et al., 1993; Navas-Camacho et al., 1994) of several tropical and

sub-tropical multipurpose shrubs and trees have been reported to suppress rumen

protozoa.

Eadie et al. (1956) showed that certain terpenes and other substances present in

plant material had marked toxic properties towards rumen protozoa. Warner (1962)

found that minor plant constituents such as terpenes or alkaloids might have specific

effects on individual species of rumen microorganisms. The compounds assumed to be

responsible for these anti-protozoal effects are saponins or saponin like substances.

(Newbold et al., 1996).

34

2.5.2.1. Chemistry of saponins and their effect on rumen fermentation

Saponins have detergent or surfactant properties because they contain both water-

soluble and fat-soluble components. They consist of a fat-soluble nucleus, having either a

steroid or triterpenoid structure, with one or more side chains of water-soluble

carbohydrates. Saponins have pronounced antiprotozoal activity. The mechanism of the

antiprotozoal effects is that saponins form irreversible complexes with cholesterol.

Cholesterol and other sterols are components of the cell membranes of all organisms

except prokaryotes (bacteria). Thus, significant reductions in ruminal protozoa numbers

observed when saponins are fed (Lu and Jorgensen, 1987; Wallace et al., 1994; Klita et

al., 1996) and within in vitro ruminal fermentation systems (Makkar et al., 1998; Wang et

al., 1998) are caused by reaction of saponins with cholesterol in the protozoal cell

membrane, causing breakdown of the membrane, cell lysis, and death. The antiprotozoal

activity requires the intact saponin structure with both the nucleus and side chain(s)

present.

Saponins may have potential as natural ruminal defaunating agents. The

incorporation of pure saponins or saponin rich feeds such as Sapindus saponaria into

diets reduced rumen ciliate population where as bacterial and fungal biomass was

increased (Diaz et al., 1993; Navas-Camacho et al., 1993; Makkar and Becker, 1997a).

Valdez et al. (1986) observed that sarsaponin from Yucca schidigera, decreased protozoal

numbers but not bacterial numbers in a 22 d semi-continuous system.

Thalib et al. (1996) reported that methanol extract of Sapindus rarak fruit fed at

0.07 per cent of body weight, every three days resulted in 57 per cent reduction in

protozoal population, 69 per cent increase in bacterial numbers, significantly reduced

ammonia nitrogen, greater daily body weight gain and improved feed conversion

efficiency.

Inclusion of E. cyclocarpum increased the rate of bodyweight gain by 24 per cent

(Leng et al., 1992) and 44 per cent (Navas-Camacho et al., 1993) and wool growth by 27

per cent (Leng et al., 1992), which was attributed to a decrease in protozoal numbers

caused by the saponin present in it (Teferedegne et al., 1999).

35

Newbold et al. (1996) fed rumen cannulated sheep with diet containing 25 per

cent Sesbania sesban, showed that the diet caused 60 per cent decline in protozoal

number after 2 days supplementation and 75 per cent reduction in the breakdown of

S.ruminantium and 70-80 per cent increase in total and cellulolytic bacterial population,

associated with reduced protozoal engulfment of bacteria. However, the protozoal

population recovered quickly and returned to pre-supplemental level by day 9, and the

cellulolytic bacterial population declined. Thus, complicating factor on supplementation

of saponin is the hydrolysis of saponin by ruminal bacteria that remove the carbohydrate

side chains (Makkar and Becker, 1997a; Wang et al., 1998). Because there may be an

adaptation of ruminal bacteria for metabolism of saponins, one approach for retaining

antiprotozoal activity would be to feed saponins intermittently. Such a regimen might

suppress protozoa and without the continuous presence of saponins, bacterial adaptation

might also be suppressed.

Thalib et al. (1995) found that administering saponins to sheep every 3 days was

effective in suppressing protozoa and reducing ruminal ammonia concentrations.

Primarily as a result of suppression of ruminal protozoa, dietary saponins increase the

outflow of bacterial protein from the rumen (Wallace et al., 1994; Makkar and Becker,

1996). Makkar and Becker (1997a) observed that Quillaja saponins were stable in the

rumen for up to 6 h after administration. It is possible that this time period may be

adequate for the saponins to have antiprotozoal activity. Thus, the fact that saponins are

rapidly degraded in the rumen may not necessarily eliminate their capacity to suppress

ruminal protozoa.

A remarkable observation was made by Odenyo et al. (1997) that S.sesban which

was introduced directly into the rumen remained toxic to protozoa, but dietary S.sesban

was ineffective.

2.5.2.2. Effects of saponin on availability and partitioning of nutrients

On investigation of saponins for their effects on whole fermentation and their

nutritional implications different results were obtained in different studies. Van Navel

and Demeyer’s (1990) study of sarsaponin in vitro showed no indication of toxic effects

36

or effects on microbial growth or protein breakdown. Goetsch and Owens (1985)

concluded that the benefits of sarsaponin would be diet dependent, increasing the

digestion of sorghum silage and other fibrous feeds but apparently decreasing digestion

of cereal and protein meals.

Makkar et al. (1998) studied the rate and potential extent of gas production when

hay was used as substrate with saponins found, Yucca and Acacia saponins decreased the

potential extent of gas production significantly at 0.6 and 1.2 mg/ml where as Quillaja

saponins had no effect at 0.6 mg/ml but increased significantly at a level of 1.2 mg/ml.

The rate of gas production decreased at a level of 0.6 mg/ml by Quillaja and

Acacia saponins. At higher levels of 1.2mg/ml, Yucca and Quillaja saponins decreased

but Acacia saponins increased the rate of gas production. The following table showed the

effect of saponins on the rate and potential gas production on hay based ration and total

mixed ration.

Table 2.1 : Table showing rate and potential gas production with saponins in hay

based and hay + concentrate rations

Hay based ration c b(ml) Control .0685.0032a 47.3 0.29a Yucca saponins (0.6mg/ml) .0639.0013abd 45.50.15b Yucca saponins (1.2mg/ml) .0599.0007bc 42.0 0.27c Quillaja saponins(0.6mg/ml) .0589 .0025bc 46.05.49ab Quillaja saponins(1.2mg/ml) .0576+.0007c 47.0.42a Acacia saponins(0.6mg/ml) .0609.0019cd 39.1.54d Acacia saponins(1.2mg/ml) .0682.0019a 37.7.55e Hay+Conc(70:30) Control .0713.0006a 51.9.089a Yucca saponins (0.6mg/ml) .0676.0012a 47.82.43b Yucca saponins (1.2mg/ml) .0662.0012a 46.2.96b Quillaja saponins(0.6mg/ml) .0631.0011b 53.3.97a Quillaja saponins(1.2mg/ml) .0662.0005b 52.91.18a Acacia saponins(0.6mg/ml) .0778.0026c 45.7.81b Acacia saponins(1.2mg/ml) .0901.0028d 44.4.56b

Using the hay and concentrate mixture as the substrate the potential extent of gas

production decreased significantly at both levels of Yucca and Acacia saponins. Yucca

37

saponins had no effect on the rate of gas production at either both level although they

decreased the potential extent of gas production significantly. On the other hand, Acacia

saponins increased the rate of gas production. Only Quillaja saponins decreased the rate

at both levels.

Mishra et al. (2004) studied the effect of Sapindus saponaria fruit on in vitro

ruminal fermentation on N deficit diet like wheat straw and N rich tropical forage diet,

Leucena leucocephala, found that there was marked decrease in methane (19.2 per cent)

and ammonia N levels (35.4 per cent) at 25 per cent inclusion level with wheat straw and

significant (p<.01) decrease in ciliate protozoal population by 28.2 and 49.6 per cent in

wheat straw and Leucena substrate. The acetate propionate ratio decreased from 3.81 to

2.98 in wheat straw and 3.5 to 3.24 in L. leucocephala.

Lu et al. (1987) discovered that alfalfa saponins appeared to suppress

fermentation and changed the bacterial population in continuous culture. Subsequent in

vivo investigation (Lu and Jorgensen, 1987) confirmed a general decrease in fermentative

activity when alfalfa saponins were supplied to the sheep rumen resulting in decreased

cellulose digestion, TVFA concentration and reduced acetate propionate ratio from 1.93

to 1.37 in presence of one per cent saponin in the medium.

Feeding S. saponaria fruits (containing 120 g of saponin per kg) at 10 per cent

level, Abreu et al. (2004) have reported no effect on apparent and total rumen

degradation of organic matter (OM) and NDF but decreased ADF digestibility (p<0.01).

Molar proportion of acetate and isobutyrate were decreased (p<0.001) and acetate:

propionate and increased the proportions of propionate and butyrate. The reduced

isobutyrate level and ammonia concentration, which are essential for the growth of

fibrolytic ruminal bacteria, could be the reason for reduced fiber digestibility. Total

ciliate protozoa counts were increased (P<0.01) by 67 per cent when fruit was

supplemented.

Hu et al. (2005) studied the effect of different levels of tea saponin (TS) in rumen

fermentation parameters showed, significant reduction in ammonia nitrogen

concentration (p<0.01) and protozoal count. The protozoal counts reduced by 19, 25, 45

38

and 79 per cent for 2, 4, 6 and 8 mg of TS. Addition of 6-8 mg of TS tended to increase

the propionate with little effect on acetate and butyrate levels. The microbial protein

yield was 1.92, 2.36 and 2.61 mg/ml with addition of 4, 6 and 8 mg TS, respectively

which was 28, 57 and 74 per cent higher (p<0.01) than the control feed consisting of

grass and corn meal. Methane concentration was reduced by 13, 22, 25 and 26 per cent

respectively with addition of 2, 4, 6 and 8 mg TS at 24 hours of incubation.

Neeta et al. (2004) have reported ethanolic extract of soapnut produced 96 per

cent inhibition of methane and reduced acetate to propionate ratio and reduced in vitro

drymatter degradability in rumen liquor inoculum.

The removal of protozoa from the rumen is normally associated with a decrease in

volatile fatty acid concentrations (Williams and Coleman, 1992) although there are higher

concentrations expressed by some studies after defaunation (Stern and Hinkson, 1974;

Grummer et al., 1983).

Newbold (1996) had reported prolonged increase in the molar proportion of

acetate and decrease in the proportion of branched chain acids and a transient increase in

propionate and reduction in butyrate, on supplementation of S.sesban. Bonsi et al. (1995)

have reported S.Sesban stimulated propionate production at the expense of acetate when

fed to sheep receiving teff straw.

Effects on rumen fermentation pattern of saponin containing plants such as

Sapindus saponaria, (crude saponins 120mg/g), Enterolobium cyclocarpum (crude

saponins 19mg/g) or Pithecellus saman (crude saponins, 17mg/g) supplemented to forage

based diets on RUSITEC showed, P.saman decreased ammonia concentration of rumen

fluid and E.cyclocarpum and P.saman increased n-butyrate proportion of total volatile

fatty acids. OM degradation in the S.saponaria diet did not differ from the control diet

but was higher in P. saman and E. cyclocarpum containing diets. Sapindus saponaria

decreased the protozoal count by 54 per cent and daily methane release by 20 per cent

relative to control without affecting the methanogen count (Hess et al., 2003).

39

Table 2.2 : Apparent anti-protozoal properties of some saponin containing tropical

plants measured by their ability to inhibit the breakdown of [14C] leucine-labelled

Selenomonas ruminantium (10 g/l) in strained rumen fluid in vitro

Species Percentage inhibition of bacteriolytic activity of rumen protozoa

Reference

Yucca shidigera 98 Wallace et al., (1994) Enterolobium cyclocarpum 91 ’’ Sesbania sesban 98 Odenyo (1997) Samanea saman 85 Teferedegne (1999) Acacia angustissima 95 ’’ Phytolacca dodecandra 85 ’’

Makkar et al. (1998) reported that addition of saponins caused higher microbial

mass for saponins of Yucca and Acacia, for which the gas production was lower than the

control (91.2 and 83.5 against 95.3). For saponins of Yucca and Quillaja gas production

was similar to that of the control but higher microbial mass production was observed.

With Acacia, there was shift in partitioning of nutrients as the same amount of truly

degraded substrate was partitioned to lesser SCFA and more of microbial mass

production from 90 to 121 mg but with a depression in truly degraded substrate from 300

mg to 297.6 mg. This result suggest that with Acacia saponin the same amount of truly

degraded substrate but different microbial mass and gas production was observed as

against the saponin of Quillaja which produced same volume of gas but different

microbial mass production depending upon the extent of truly degraded substrate. The

addition of Quillaja saponin increased the purine content and increased truly degraded

substrate by approximately 7 per cent (Makkar et al., 1998).

Addition of sarsaponin at 33, 55 or 77 ppm to an in vitro fermentation system

using rumen fluid from mature heifers had increased bacterial numbers and decreased

those of protozoa in the fluid and increased the apparent digestibilities of organic matter

and starch. Rates of disappearance of N, OM, and ADF from a diet containing

concentrate and sorghum silage DM (3:2) given to two rumen cannulated animals were

decreased when sarsaponin was added at 77 ppm (Valdez et al., 1986).

40

Wina et al. (2005) in a study with Sapindus rarak on rumen fermentation showed

that the saponin treatment did not decrease isoacids or ammonia concentration at earlier

hours of fermentation but depressed the isoacids and ammonia production (P<0.05) at 48

hours of incubation suggesting there was no inhibition of deaminase activity by saponins

but, the difference in microbial constitution as a result of defaunation. Higher levels of

supplementation (2mg/ml and 4mg/ml) showed a reduction in NDF digestibility.

Sapindus extract (SE) significantly depressed rumen xylanase activity in both trials and

carboxymethylcellulase activity in the long-term trial (p<0.01). CMCase activity

significantly increased in short term study. The SE in both trials did not affect

Fibrobacter sp., while Ruminococci and the anaerobic fungi showed a short-term

response to the application of saponins. Protozoal counts were decreased only in the long-

term trial with sheep.

2.5.2.3. Effect of saponins on biomass production and the microbial efficiency

In vitro studies suggest that the engulfment and subsequent digestion of bacteria

by ciliate protozoa is quantitatively the most important cause of bacterial protein

turnover in the rumen (Wallace and McPherson, 1987). Removing of ciliate protozoa

from the rumen avoids the cycle of bacterial protein breakdown and resynthesis in the

rumen and thereby increases the flow of protein to the animal (Lindsay and Hogan,

1972; Williams and Coleman, 1992).

The efficiency of microbial protein synthesis expressed as mg microbial mass-

produced per 100 ml of truly degraded substrate was 30 for the control and 38.3, 33, 36.9

and 40.7 for Yucca- butanol extract and aqueous extract and Quillaja and Acacia

saponins treatments, respectively. Makkar et al. (1998) have concluded that in the

presence of saponins, a higher proportion of the substrate degraded was canalized

towards the microbial mass production and a lower proportion towards the gas

production.

Saponins have the potential to increase the microbial protein production and

decrease the emission of environment-polluting gases such as methane and carbon

41

dioxide. Saponins can also shift the carbon from waste products of fermentation such as

carbon dioxide and methane to microbial mass.

Wina et al. (2005) in study with Sapindus rarak on rumen fermentation suggest

that there was increase in microbial biomass with increasing concentration of methanol

extract of the above, the membrane hybridization study showed that a higher bacterial

RNA concentration only occurred with 1 mg/ml of methanol extract of Sapindus (MS)

and no further increase in bacterial RNA concentration occurred at higher levels of MS.

Saponins have selective effects on ruminal microorganisms as it would reduce the

cellulose digestion by Ruminococcus spp, and Fibrobacter succinogenes species of

bacteria and Neocallimstix and Piromyces species of fungi, but is a safe and persistent

suppressor of ciliate protozoa, which may have wide application (Wallace et al., 2002).

Hence the effect on nitrogen retention as well as microbial protein synthesis and

biomass production has to be assessed in low protein diets primarily comprising the straw

based diets, as the major effect being suppression in rumen ammonia production and

higher propionate synthesis. There is limited information on the effect of natural plant

extracts on peptide metabolism in the rumen. The addition of 7.5 mg/Kg DM of Yucca

extract (containing 8 per cent of sarsaponin) increased the average peptide N

concentration by 26.2 per cent through the 8 h feeding interval indicating stimulation of

proteolysis or inhibition of peptidolysis (Cardozo et al., 2004). Newbold et al. (1997)

have reported an increase in proteolytic activity and transient peptidolytic activity and

increased deaminase activity on supplementation of S.sesban in sheep fed on mixed diet.

2.5.2.4. In vivo effects of saponins

The effect of in vivo experiments with lambs, the addition of Quillaja bark in the

diet corresponding to very low amount of saponin 40 and 60 ppm compared to those used

in the in vitro rumen fermentation system had positive effects on male lambs (higher

growth rate and carcass weight statistically significant only at 40 ppm) and adverse

effects in female lambs and no difference could be observed in rumen ammonia level or

protozoal counts between saponin free and saponins - containing groups (Makkar and

Becker, 2000).

42

Sheep consuming the seed pericarp of S.saponaria had a reduced rumen protozoal

count which was associated with an increased in live weight gain (Navas-Camacho et al.,

2001).

In sheep, ethanolic extract of Sapindus rarak (SE) depressed CMCase and

xylanase activities in a dose-dependent manner (p<0.001). The activities of CMCase and

xylanase were reduced by 50per cent at the highest inclusion level of SE (0·72 g kg 1

body mass) compared with that of the control. In contrast to goats, the protozoal counts

were reduced in a dose-dependent manner (p<0.01). At the highest level of SE, the

protozoal counts decreased by 80per cent compared with the control (Wina et al., 2005).

Goetsch and Owen, (1985) reported no significant effect on total and microbial

nitrogen entering the duodenum when sarsaponin of Yucca schidigera included in diet of

mature dairy cow at 44 mg/kg.

Lu and Jorgensen (1987) suggested, detrimental effect on rumen microorganisms

and reduced microbial protein synthesis and nitrogen flow to duodenum on

supplementation of 20 and 40 g per Kg dietary DM of alfalfa saponin to sheep.

Klita (1996) reported increased duodenal flow of nitrogen when sheep received

daily doses of 200, 400 and 800 mg/kg bodyweight of alfalfa root saponin. This increase

in microbial N flow was associated with a significantly decreased ruminal protozoa

count.

2.5.3.Tannin-saponin interaction

Studies on tannin- saponin interaction showed simultaneous presence of tannin

(Quebracho) and saponin (quillaja) produced additive effect on decrease in apparent and

true digestibilities and gas production. (Makkar et al., 1995). The biological effects of

both tannins and saponin depend upon their chemical properties.

Saponins bind with tannins in the gut (Freeland et al., 1985). The nullification of

the toxic effects of tannins was dependent on the relative proportions of tannin and

saponin (Freeland et al., 1985). The negative effect of A. angustissima (high tannins)

43

could be reversed if it is mixed with Sesbania spp. that have low tannin, low NDF and

high crude protein. This combination would decrease the binding of free tannins from A.

angustissima to rumen enzymes, and would increase the efficiency of protein utilization

of Sesbania spp. by reducing its rate of fermentation. The nullification of the toxic effects

of tannins was dependent on the relative proportions of tannin and saponin (Freeland et

al., 1985). Most saponin-containing plants also contained some tannin. Feeding a

combination of saponin-rich feeds could reduce the toxic effect of high-tannin-containing

plants. Conversely, the anti-protozoal activity of saponin containing plants might depend

on their tannin content (Teferedegne, 2000).

2.5.4. Mulberry leaf as supplement

The Mulberry plant in tropical belt is grown as low bush while as high bush in

temperate regions. In tropical conditions, individual leaf and branch harvest is done with

a yield of 10 to 30 ton/ha/yr, while it is shoot harvested in temperate regions with a leaf

yield of 25 to 30 ton/ha/yr. Mulberry is grown on an extensive scale in various parts of

India, particularly in Mysore, West Bengal and Jammu and Kashmir for its leaf, which

constitutes the food for the silkworm, and its cultivation is an integral part of the

sericulture industry. The contents of protein and soluble sugars in leaves decrease with

the maturity of leaves; fibre, fat and ash constituents increase.

Analysis of leaves collected from different silk producing localities in India gave

the following range of values (per cent in DM): crude protein 16.0-39.0, soluble sugars

7.6-26.0 and ash 8.0-17.0. On DM basis, the leaves contained 15.0 - 27.6 per cent crude

protein (CP), 2.3 - 8.0 per cent ether extract (EE), 9.1 - 15.3 per cent crude fibre (CF),

48.0 - 49.7 per cent nitrogen free extract (NFE), 63.3 per cent total carbohydrates, 14.3 -

22.9 per cent ash, 2.42 - 4.71 per cent Ca, 0.23 - 0.97 per cent P, 0.196 per cent S, 1.66 -

3.25 per cent K, 350 - 840 ppm Fe (Jayal and Kehar, 1962; Singh et al., 1984; Singh et.

al., 1989; Makkar et al., 1989). The cell wall constituents were: neutral detergent fibre

(NDF) 33 - 46 per cent, acid detergent fibre (ADF) 28 - 35 per cent, hemicellulose 5 - 10

per cent, cellulose 19 - 25 per cent, and lignin approximately 11 per cent (Lohan et al.,

1979; Makkar et al., 1989). The content of total phenols was very low (1.8 per cent as

44

tannic acid equivalent) and tannins by the protein precipitation capacity method were not

detectable (Makkar et al., 1989; Makkar and Becker, 1998).

Studies have been conducted at the Regional Station of Indian Veterinary

Research Institute, Palampur on characterization of Mulberry leaves for digestion kinetics

parameters and comparison of these values with other tree foliages (Devarajan, 1999).

Degradation kinetic parameters as studied by the in vitro gas production technique

(Menke et al., 1979) showed that the potential gas production in young leaves was

60ml/200 mg while the rate of degradation was 0.0703. The corresponding values for the

mature leaves were 35.4 ml and 0.0624 respectively, indicating the fall in fermentability

with maturity.

The rate, potential extent and effective degradability (at passage rate of 0.05/h) of

dry matter using the in sacco method of Orskov and McDonald (1979) were 0.0672, 85

per cent and 52 per cent respectively. The contents of NDIN and ADIN in Mulberry

leaves were 56.5 and 20.4 per cent of the total nitrogen, respectively and Rumen

Degradable Nitrogen to be 16.3, Undegradable Nitrogen 12.2.

Mulberry leaves being rich in nitrogen, sulphur and minerals, their

supplementation could increase the efficiency of utilization of crop residues by increasing

the efficiency of microbial protein synthesis in the rumen leading to higher microbial

protein supply to the intestine. Combination of trees and grassland would be a desirable

development and synergistic for cattle production. Strategic supplementation is justified

because of regular feed shortages that occur and the fact that ruminants subsist for most

of their life on fibrous crop residues on small farms. Henceforth, for high producing

animals, the feeding strategy should be aimed at supplementation with by-pass protein of

dietary origin in addition to creating an efficient ecosystem (Singh and Makkar,

(internet)).

2.5.5. Non-ionic surfactants

Non-ionic surfactants (NIS) have shown to improve rumen fermentation and

increase enzyme activity in batch fermenters as well as bacterial digestion of cellulose

(Akin, 1980 and Stutzenberger, 1987). Tween–80 (polyoxyethylenesorbitan monooleate,

45

IFN 8-08-031) is well known as an effective surfactant that stimulates the release of

enzymes from a range of aerobic fungi (Reese and Maguire, 1969; Hung et al.,1988). The

effect of surfactants have been attributed mainly to (i) action on the cell membrane

causing increased permeability, (ii) promotion of the release of bound enzymes (Reese

and Maguire, 1969) and (iii) decrease in growth rate due to reduced oxygen supply

(Hulme and Stranks, 1970). There have been studies on the effect of this surfactant on the

anaerobic growth of rumen microbes and fungi and rumen pure and mixed cultures and

enzyme distribution (Lee et al., 2003; Lee and Ha, 2003; Goto et al., 2003). The studies

have reported that, Tween-80, when used as feed additive in high grain diets have

stimulated succinate and lactate utilization by rumen microorganisms and induced

polysaccharide degrading enzyme activity (Lee et al., 2003).

2.5.5.1. Chemistry of surfactant and their effect on rumen fermentation

Surfactants produce loosening and/or partial breakdown of rigid cell wall

structure of forage resulting in greater accessibility of fibre degrading bacteria and their

associated enzymes to forage plants. Goto et al. (2003) have suggested that the

adsorption and orientation of surfactant molecules at the solid-liquid interface render the

substrate readily wettable by the enzymes thereby providing a highly localized substrate

concentration.

NIS has significantly increased the growth rate of M. elsdenii, P. ruminicola and

S.ruminantium at all concentrations and at all times and R. amylophilus after 12 and 16h

incubation. The growth rate of R.albus increased linearly up to 16h and the quickly

decreased and R. flavefaciens increased up to 30 hrs and subsequently decreased. In

general NIS influenced more gram-negative bacteria than gram-positive bacteria and non-

cellulolytic bacteria than cellulolytics.

Goto et al. (2003) studied the effect of surfactant Tween-80 on growth of bacterial

pure culture of Streptococcus bovis, Selenomonas ruminantum, Butyrivibrio fibrisolvens

Prevotella ruminicola, Megasphera elsdenni in the medium incubated with 1 per cent

barley grain and pure cultures of Fibrobactor succinogenes, Ruminococcus albus and

Ruminococcus flavefaciens in the medium containing 1 per cent orchard hay, showed the

46

growth of S.bovis, S.runinantum, B.fibrisolvens, P.ruminicola, M.elsdenni and

F.succinogenes were significantly higher than the control at 0.05 per cent concentration

of Tween-80 while R.albus and R.flavefaciens was not changed . The greater response of

the growth of noncellulolytic bacteria over cellulolytic may be due to higher degradation

of barley grain through relatively higher swelling capacity of starch granules.

2.5.5.2. Effects of Surfactants on availability and partitioning of nutrients

Experiments conducted by Lee et al. (2003) showed that Tween-80 stimulated the

release of polysaccharide degrading enzyme activities and increased the growth rate of

rumen bacteria and fungi and the rate of cereal grain digestion, succinate and lactate

dehydrogenase activities. Cellulase and Xylanase, the major polysaccharide degrading

enzymes and mostly cell bound and limited activities are witnessed in the free rumen

fluid. Hence release of these enzymes to the rumen fluid fraction or to the feed particle

fraction would beneficial to improve digestion of feedstuff (Lee and Ha, 2003).

Studies conducted by Lee et al. (2003) on the effects of Tween-80 addition to

rumen fluid, after 3 hours of incubation in lactating cows showed significantly (p<0.01)

increased production of the cell-free cellulase (CMCase) and total xylanase but activity of

cell bound cellulase and xylanase significantly decreased. The cell free enzyme activities

of protease, amylase and barley glucanase were also increased, above 200 per cent. NIS

has stimulated individual cell degrading and other enzyme activities as well as promoted

the release of microbial cell-bound enzymes in the rumen fluid or digesta. The effect may

be due to an increased permeability of anaerobic microbial cell membrane permitting

more enzyme release.

Surfactant Tween-80 increased DMD of plant fractions of young and matured

orchard grass by a cellulolytic commercial enzyme by 5-35 per cent units showing the

consistency of the improvement of enzymatic degradability with those of their water and

enzyme-holding capacities (Goto et al., 2002).

The effect of Tween –80 at 0.01 per cent and 0.02 per cent concentration in rumen

mixed culture in the medium containing barley grain and orchard grass hay showed

47

significant increase in DMD at 0.02 per cent and increase in total VFA. High propionate

acetate ratio was noticed showing increased activity of S.ruminantium, B.fibrisolvens and

M.elsdenni. On rumen fermentation characteristics, NIS increased the rumen ammonia

concentration by 14 per cent and total VFA concentration by 20 per cent after 3 hours

post feeding. The acetate and propionate concentration were elevated without appreciable

increase in isoacid concentration (Goto et al., 2003).

Lee and Ha (2003) in their study on the influence of surfactant Tween-80 on gas

production, cellulose digestion and enzyme activities by mixed rumen microorganisms

showed significant reduction in the cumulative gas production by 34-63 ml/g with barley

at 30 hrs and the cumulative gas production was maximum in orchard grass hay treated

with 0.05 per cent NIS. NIS supplementation to orchard grass hay showed increase in

cellulase (124.43 per cent), Xylanase (108.86 per cent) and amylase (271.22 per cent)

activities after 72 hours of incubation.

Wang et al. (2004) in their study on the effect of Tween-80 and monensin on

rumen fermentation of barley grain and barley silage based diets (58:42) for beef cattle

showed supplementation of Tween-80 at levels of 0.1 or 0.5 microlitre per ml of rumen

liquor showed Tween-80 applied at 0.5 μl/ml increased in vitro true dry matter

disappearance and reducing sugars at 4hrs and microbial nitrogen at 24 hrs with reduced

A:P ratio at 12 hr of incubation. At 0.1l/ml Tween-80 increased amylase activity at 4 hr

(p<0.001) and at 24 hr (P<0.05), but it decreased (p<0.01) CMCase activity at 12 hr. In

contrast, with Tween-80 at 0.5 μl/ml, amylase, CMCase and xylanase activities were

reduced (p<0.01) at 12 hr but amylase and beta glucanase activities were increased

(P<0.05) at 24 hr incubation of Tween-80- supplemented beef diets.

2.5.5.3. Effect of surfactant on microbial biomass synthesis

Lee and Ha (2003) in their experiment to find the influence of Tween-80 on

fermentation pattern and enzyme activities of mixed rumen microorganisms indicated

that Tween-80 did not influence the rate of growth of mixed rumen microorganisms. Lee

et al. (2003) showed the growth rates of rumen noncellulolytic bacteria were greatly

increased while growth of cellulolytic bacteria was not affected.

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Hristov et al. (2003), in their study on evaluating potential bioactive agents for

reducing protozoal activity in vitro showed the ruminal activity were unaffected by

Tween-80 other than marginal increase in ammonia concentration. There was an

inhibitory effect on 15 N incorporation into bacterial protein.

2.5.5.4. In vivo effects of surfactant

When lactating HF cows were fed with NIS, there was increase in the feed intake

and milk yield however concentrations of blood metabolites in gestating and lactating

Holstein cows showed blood NEFA (non-esterified fatty acid) concentration decreased on

addition of NIS, but the value quickly increased at the end of gestation and the value

slightly decreased after calving (Lee et al., 2003).

Hristov et al. (2000) fed 10 Jersey steers fed with basal diet containing 70 per cent

barley and 30 per cent alfalfa silage without and with additives including Tween-80

alone, salinomycin alone or Tween-80 and salinomycin with latter at two levels in a

replicated 5x5 Latin square experiment, showed highest levels of Carboxymethyl

cellulase and Xylanase enzymes with Tween-80 treated animals. Treatments did not

affect flow of microbial nitrogen with rumen solids. Drymatter intake by the steers and

total tract apparent digestibilities of DM, NDF, ADF and CP were not affected by dietary

treatments. Mc Allister et al. (2000) have discussed the in vitro response of Tween-80

which showed enhanced cellulase activity but failed to improve digestibilities of forage

or concentrate based diets by lambs.

Hristov et al. (2000) have pointed out that the concentrations determined to be

optimal in-vitro are not the same as those required to improve digestibility in vivo.

Ruminal protease might partially degrade Tween-80, an oleate ester of sorbitol or reduce

the effectiveness as a surfactant. He had concluded that the effective degradability is

depressed or not influenced by surfactant but improved the rate of disappearance.

Kim et al. (2006) observed that there was significant increase (3.9 per cent) in

milk fat and a transient increase in milk yield in cows fed diet containing NIS.

49

2.5.6. Surfactant-saponin interaction

There are no reports on the interaction of surfactant with saponins. It is expected

that non-ionic surfactant might behave like ionophores as there was a tendency for higher

propionate production, lower methane emission and reduced protozoa in both ionophores

and surfactants. Both saponins and ionophores suppress Gram-positive bacteria and

protozoa, so synergistic effects would not be surprising. In the antiprotozoal activity, they

act via different mechanisms: saponins cause cell lysis by interacting with cholesterol in

the protozoal cell membrane, whereas ionophores disrupt ion transport.

2.5.7. Surfactant-Tannin interaction

Miller et al. (1997) found that the addition of surfactants, SDS or alkanate 3SL3

to the diet of mulga-fed sheep did not improve N balance or digestion; however, apparent

digestibility of Phosphorus and Phosphorus and Sulphur balance were significantly

improved by SDS. There were no reports of addition of Tween –80 to alleviate the effect

of tannin in tropical forages. It is expected that the astringency caused by the tannins

would be alleviated and made more soluble by the surfactants.