Recent advances in ration balancing include manipulation of feed to increase the quantity and quality of protein and energy delivered to the small intestine. Selection of fibrous feeds based on high efficiency of microbial protein synthesis in the rumen along with high dry matter digestibility, and development of feeding strategies based on high efficiency as well as high microbial protein synthesis in the rumen will lead to higher supply of protein post-ruminally. This concept of feed evaluation has an extra element of the efficiency of microbial protein synthesis in addition to the more conventional one of the dry matter digestibility. The limited supply of protein post-ruminally under most feeding systems in developing countries is an important limiting factor which prevents an increase in animal productivity.
There are a number of methods used to determine net microbial protein synthesis in the rumen based on the use of microbial markers. They require the use of post-ruminally cannulated animals to determine flow of digesta. The cannulation approach is tedious and has several limitations (Chen et al., 1990) to its applicability under most research conditions in developing countries. A simpler technique for determination of microbial protein supply to the intestine is based on the determination of total urinary purine derivatives (IAEA, 1997). This approach is being thoroughly investigated under a Joint FAO/IAEA Coordinated Research Project (IAEA, 1998). Although the method is based on the collection of urine for determination of purine derivatives (allantoin and uric acid for cattle, and allantoin, uric acid, xanthine and hypoxanthine for sheep), the approach is being further simplified using spot urine samples. This technique does not require cannulated animals, but it involves feeding the diets under investigation to animals and therefore is not suitable for screening large numbers of samples or for developing feed supplementation strategies using various feed constituents.
In vitro methods for laboratory estimations of degraded feeds are important for ruminant nutritionists. An efficient laboratory method should be reproducible and should correlate well with actually measured in vivo parameters. 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. Three major biological digestion techniques are currently available to determine the nutritive value of ruminant feeds: 1) digestion with rumen microorganisms as in Tilley and Terry (1963) or using a gas method (Menke et al., 1979), 2) in situ incubation of samples in nylon bags in the rumen (Mehrez and Orskov, 1977), and 3) cell-free fungal cellulase De Boever et al., 1986). These biological methods are more meaningful since microorganisms and enzymes are more sensitive to factors influencing the rate and extent of digestion than are chemical methods (Van Soest, 1994). The nylon bag technique has been used for many years to provide estimates of both the rate and extent of disappearance of feed constituents. This technique provides a useful means to estimate rates of disappearance and potential ruminal degradability of feedstuffs and feed constituents whilst incorporating effects of particulate passage rate from the rumen. The disadvantage of the method is that only a small number of forage samples can be assessed at any one time, and it also requires at least three fistulated animals to account for variations due to animal. It is therefore of limited value in laboratories undertaking routine screening of a large numbers of samples. It is also laborious, and requires large amounts of samples. Substantial error could result in values obtained at early stages of digestion due to a low weight loss; and for poor quality roughages, adherence of microbes at early stages can even lead to higher weights and thus distortion of results if kinetic modelling does not incorporate the lag-phase (McDonald, 1981; Dhanoa, 1988).
The Tilley and Terry (1963) technique is used widely because of its convenience, particularly when large-scale testing of feedstuffs is required. This method is employed in many forage evaluation laboratories and involves two stages in which forages are subjected to 48 h fermentation in a buffer solution containing rumen fluid, followed by 48 h of digestion with pepsin in an acid solution. The method was modified by Goering and Van Soest (1970), in that the residue after 48 h incubation was treated with neutral detergent solution to estimate true dry matter digestibility. Although the method of Tilley and Terry (1963) has been extensively validated with in vivo values (Van Soest, 1994), the method appears to have several disadvantages. The method is an end-point measurement (gives only one observation) thus, unless lengthy and labour-intensive time-course studies are made, the technique does not provide information on the kinetics of forage digestion; the residue determination destroys the sample and therefore a large number of replicates are needed. The method is therefore difficult to apply to materials such as tissue culture samples or cell-wall fractions.
Both the Tilley and Terry and nylon bag methods are based on residue determinations and may result in overestimation of dry matter digestibilities for tannin-rich feeds, since tannins are solubilised in both these systems but might be indigestible and do not contribute to nutrient supply to animals (Makkar et al., 1993).
The gas measuring technique has been widely used for evaluation of nutritive value of feeds. More recently, the increased interest in the efficient utilisation of roughage diets has led to an increase in the use of this technique due to the advantage in studying fermentation kinetics. Gas measurement provides a useful data on digestion kinetics of both soluble and insoluble fractions of feedstuffs. Several gas measuring techniques and in vitro gas methods are in use by several groups. Advantages and disadvantages of these methods are discussed by Getachew et al. (1998a). The in vitro gas method based on syringes (Menke et al., 1979; Blümmel et al., 1997) appears to be the most suitable for use in developing countries. Other in vitro methods such as Tilley and Terry and nylon bag methods are based on gravimetric measurements which follow disappearance of the substrate (the components which may or may not necessarily contribute to fermentation), whereas gas measurement focuses on the appearances of fermentation products (soluble but not fermentable products do not contribute to gas production). In the gas method, kinetics of fermentation can be studied on a single sample and therefore a relatively small amount of sample is required or a larger number of samples can be evaluated at a time. The in vitro gas method is more efficient than the in sacco method in evaluating the effects of tannins or other anti-nutritive factors. In the in sacco method these factors are diluted in the rumen after getting released from the nylon bag and therefore do not affect rumen fermentation appreciably. In addition, the in vitro gas method can better monitor nutrient-antinutrient and antinutrient-antinutrient interactions (Makkar et al., 1995a).
A simple in vitro approach is described below which is convenient and fast, and allows a large number of samples to be handled at a time. It is based on the quantification of substrate degraded or microbial protein produced using internal or external markers, and of gas or short chain fatty acid (SCFA) production in an in vitro rumen fermentation system based on syringes (Menke et al., 1979). This method does not require sophisticated equipment or the use of a large number of animals (but one or preferably two fistulated animals are required) and helps selection of feeds or feed constituents based not only on the dry matter digestibility but also on the efficiency of microbial protein synthesis.
In the method of Menke et al. (1979), fermentations are conducted in 100 ml capacity calibrated glass syringes containing feedstuff and a buffered rumen fluid. The gas produced on incubation of 200 mg feed dry matter after 24 h of incubation together with the levels of other chemical constituents are used to predict digestibility of organic matter determined in vivo and metabolizable energy.
For roughages, the relationships are:
ME (MJ / Kg DM) = 2.20 + 0.136 Gp + 0.057 CP, R2= 0.94
OMD (%) = 14.88 + 0.889 Gp + 0.45 CP + 0.0651 XA, R2=0.92
Where ME is the metabolisable energy; OMD organic matter digestibility; CP, crude protein in %; XA, ash in %; and Gp, the net gas production in ml from 200 mg dry sample after 24 h of incubation and after correction for the day-to-day variation in the activity of rumen liquor using the Hohenheim standard
Aiple et al. (1996) compared three laboratory methods (enzymatic, crude nutrient and gas measuring technique) as predictors of net energy (as estimated by equations based on in vivo digestibility) content of feeds and found that for predicting net energy content of individual feeds, the gas method was superior to the other two methods.
The method of Menke et al. (1979) was modified by Blümmel and Ĝrskov (1993) in that feeds were incubated in a thermostatically controlled water bath instead of a rotor in an incubator. Makkar et al. (1995b) and Blümmel et al. (1997) modified the method further by increasing the amount of sample from 200 to 500 mg and increasing the amount of buffer two-fold as a result the incubation volume increase from 30 ml in the method of Menke et al. (1979) to 40 ml in the modified method. In the 30 ml system, the linearity between the amount of substrate incubated and the amount of gas produced is lost when the gas volume exceeds 90 ml because of the exhaustion of buffer of the medium; and in 40 ml system, the linearity is lost when the gas volume exceeds 130 ml (Getachew et al., 1998b). The exhaustion of the buffer decreases pH of the incubation medium; consequently the fermentation is inhibited. If the amount of gas production exceeds 90 ml using the 30 ml system and 130 ml using the 40 ml system, the amount of feed being incubated should be reduced.
The main advantages of the modified method (the 40 ml system and incubation in a water bath) are: i) there is only a minimum drop in temperature of the medium during the period of recording gas readings on incubation of syringes in a water bath. This is useful for studying the kinetics of fermentation where gas volume must be recorded at various time intervals, ii) because of large volume of water in the water bath and also its higher temperature holding capacity, drastic drop in the temperature of the incubation is prevented in case of power breakdown for a short duration, and iii) an increase in amount of sample from 200 to 500 mg reduces the inherent error associated with gravimetric determination needed to determine concomitant in vitro apparent and true digestibility (see below).
When a feedstuff is incubated with buffered rumen fluid in vitro, the carbohydrates are fermented to produce short chain fatty acids (SCFA), gases and microbial cells. Gas production is basically the result of fermentation of carbohydrates to acetate, propionate and butyrate. Gas production from protein fermentation is relatively small as compared to carbohydrate fermentation. The contribution of fat to gas production is negligible. When 200 mg of coconut oil, palm kernel oil and/or soybean oil were incubated, only 2.0 to 2.8 ml of gas were produced while a similar amount of casein and cellulose produced about 23.4 ml and 80 ml gas.
The gas produced in the gas technique is the direct gas produced as a result of fermentation and the indirect gas produced from the buffering of SCFA. For roughages, when bicarbonate buffer is used, about 50% of the total gas is generated from buffering of the SCFA and the rest is evolved directly from fermentation. At very high molar propionate the amount of CO2 generated from buffering of SCFA is about 60% of total gas production. Each mmol of SCFA produced from fermentation releases 0.8-1.0 mmol of CO2 from the buffered rumen fluid solution, depending on the amount of phosphate buffer present. Highly significant correlation has been observed between SCFA and gas production (see below).
Gas is produced mainly when substrate is fermented to acetate and butyrate. Substrate fermentation to propionate yields gas only from buffering of the acid and, therefore, relatively lower gas production is associated with propionate production. The gas that is released with the generation of propionate is only the indirect gas produced from buffering. The molar proportion of different SCFA produced is dependent on the type of substrate. Therefore, the molar ratio of acetate of propionate has been used to evaluate substrate related differences. Rapidly fermentable carbohydrates yield relatively higher propionate as compared to acetate, and the reverse takes place when slowly fermentable carbohydrates are incubated. Many workers reported more propionate and thus lower acetate to propionate ratio in the ruminal fluid of cows fed a high grain diet. If fermentation of feeds leads to a higher proportion of acetate, there will be a concomitant increase in gas production compared with a feed with a higher proportion of propionate. In other words, a shift in the proportion of SCFA will be reflected by changes in gas production.
The gas produced on incubation of cereal straws (Blümmel and Ĝrskov, 1993), a wide range of feeds including many dairy compound feeds and their individual feed components whose protein and fat contents vary greatly (Blümmel et al., 1999a), and tannin containing browses (Getachew et al., 2000a) in absence or presence of polyethylene glycol (a tannin complexing agent) in the buffered rumen fluid was closely related to the production of short chain fatty acids (SCFA) as per Wolin (1960) stoichiometry, which is as follows:
Fermentative CO2 = A/2+P/4+1.5B
where A, P and B are moles of acetate, propionate, and butyrate respectively.
Fermentative CH4 = (A+2B)-CO2
where A and B are moles of acetate and butyrate respectively and CO2 is moles of CO2 calculated from previous equation.
Assumption: one mole of SCFA releases one mole of CO2 from bicarbonate-based buffer described as buffering CO2 and therefore, mmol of buffering CO2 is equal to mmol of total SCFA generated during incubation.
Gas volume = mmol of gas x gas constant (R) x T
Where
R = the ratio between molar volume of gas to temperature (Kelvin zero; K) i.e.
(22.41l/273 = 0.082),
T = incubation temperature (Kelvin); 273 + 39o C = 312 K
Total volume of gas (ml) calculated from SCFA production = (BG + FG) x CF
BG = gas volume (ml) from buffering of SCFA,
FG = fermentative gas (ml) (CO2 + CH4),
CF = correction factor for altitude and pressure which is 0.953 for Hohenheim at altitude 400m above sea level (Blümmel et al., 1999).
(The volume of 1 mmol of gas at 39o C in Hohenheim would be;
1 x 0.082 x 312 x 0.953=24.4 ml).
The origin and stoichiometry of gas production have been described in details in Blümmel et al. (1997) and Getachew et al. (1998a).
The in vitro gas production measured after 24 h incubation of tannin containing browses in the presence or absence of PEG was strongly correlated with gas volume stoichiometrically calculated from SCFA. The relationship between SCFA production (mmol) and gas volume (ml) after 24 h of incubation of browse species with a wide range of crude protein (5.4-27 %) and phenolic compounds (1.8-25.3 % and 0.2-21-4 % total phenols and total tannins as tannic acid equivalent respectively) was (Getachew et al., 2000a):
In the absence of PEG
SCFA = 0.0239.Gas -0.0601; R2 = 0.953; N = 39; P<0.001 (I)
In the presence of PEG
SCFA = 0.0207.Gas + 0.0207; R2 = 0.925; N = 37; P<0.001 (II)
These relationships are similar to that obtained for wheat straw (Blümmel et al., 1993).
The SCFA production could be predicted from gas values using the above relationship. The level of SCFA is an indicator of energy availability to the animal. Since SCFA measurement is important for relating feed composition to production parameters and to net energy values of diets, prediction of SCFA from in vitro gas measurement will be increasingly important in developing countries where laboratories are seldom equipped with modern equipments to measure SCFA.
The stoichiometric balance also allows calculation of the CH4 and CO2 expected from the rumen fermentation if the molar proportions and amount of SCFA are known.
Kinetics of fermentation feedstuffs can be determined from fermentative gas and the gas released from buffering of SCFA. Kinetics of gas production is dependent on the relative proportion of soluble, insoluble but degradable, and undegradable particles of the feed. Mathematical descriptions of gas production profiles allows analysis of data, evaluation of substrate- and media-related differences, and fermentability of soluble and slowly fermentable components of feeds. Various models have been used to describe gas production profiles. France et al. (1993; 2000) combine modelling of the gas profiles with estimates of substrate loss and ruminal rate of passage and derive estimates of ruminal extent of degradation thus linking gas production to events in the rumen proper.
Determination of microbial mass. In vitro gas tests are attractive for ruminant nutritionists since it is very easy to measure the volume of gas production with time, but the measurement of gas only implies the measurement of nutritionally wasteful and environmentally hazardous products. In most studies the rate and extent of gas production has been wrongly considered to be equivalent to the rate and extent of substrate (feed) degradation. Current nutritional concepts aim at high microbial efficiency, which can not be achieved by measurement of gas only. In vitro gas measurements reflect only SCFA production. The relationship between SCFA and microbial mass production is not constant and the explanation for this resides in the variation of biomass production per unit ATP generated. This can impose an inverse relationship between gas volume (or SCFA production) and microbial mass production particularly when both are expressed per unit of substrate truly degraded. This implies that selecting roughages by measuring only gas using in vitro gas methods might result in a selection against the maximum microbial mass yield. Blümmel et al. (1997) have demonstrated how a combination of in vitro gas production measurements with a concomitant quantification of the truly degraded substrate provides important information about partitioning of fermentation products, and the in vitro microbial mass production can be calculated as:
Microbial mass (mg) = mg substrate truly degraded - (ml gas volume x stoichiometrical factor)
For roughages, the stoichiometrical factor was 2.20.
Partitioning factor. The parameters in the above equation also allow the calculation of a partitioning factor (PF). The PF is defined as the ratio of substrate truly degraded in vitro (mg) to the volume of gas (ml) produced by it (equivalent to the reciprocal of parameter Y in France et al., 1993). The above equation becomes
Microbial mass (units) = gas volume (PF - stoichiometrical factor)
A feed with higher PF means that proportionally more of the degraded matter is incorporated into microbial mass, i.e., the efficiency of microbial protein synthesis is higher. Roughages with higher PF have been shown to have higher dry matter intake. Different in vitro PF values are also reflected by in vivo microbial protein synthesis as estimated by purine derivatives (the higher the PF, the higher the excretion of urinary purine derivatives; (Blümmel et al., 1999b) and in methane production by ruminants (the higher the PF, the lower the methane output; (Blümmel et al., 1999b). These results show that the PF calculated in vitro provides meaningful information for predicting the dry matter intake, the microbial mass production in the rumen, and the methane emission of the whole ruminant animal.
The procedures for the determination of truly degraded substrate and the calculation of the stoichiometrical factor; stoichiometrical relationship between SCFAs and gas volume; and relationship between SCFA production, ATP production and microbial mass yield can be obtained from Blümmel et al. (1997) and Getachew et al. (1998a). It may be noted that these procedures and relationships are valid for substrates consisting predominantly of structural carbohydrates, and the findings might not extend to substrates such as those high in soluble carbohydrate, protein or fat. Rymer and Givens (1999) have shown that, as observed by Blümmel et al. (1997), good quality feeds (grass silage, wheat, maize, molasses sugarbeet feed and fishmeal) which produce large amounts of gas and SCFA yield small amounts of microbial mass per unit of feed truly degraded.
It seems therefore justified to suggest that feeds or feed ingredients should be selected that have a high in vitro true degradability but low gas production per unit of truly degraded substrate. Dijkstra et al. (2000) have described modelling of microbial protein synthesis in vivo from the in vitro gas parameters.
Incubation time and PF. Another study by Blümmel et al. (1998a), in addition to once again describing the importance of measuring microbial mass, has highlighted the importance of the fermentation time at which the microbial mass should be measured. In this study, substrate degradation and kinetics of in vitro partitioning of three hays, with similar in vivo digestibilities, into SCFA, microbial mass yield, and ammonia, carbon dioxide and methane production was examined at 8, 12, 18 and 24 h of incubation in the gas method under both low and adequate nitrogen levels. Microbial synthesis was quantified gravimetrically (Blümmel et al., 1997), by nitrogen balance (Getachew et al., 2000b) and by purine analysis (Makkar and Becker, 1999). SCFA and gas production were positively correlated (P < 0.0001) and cumulative at all times of incubation under both low and adequate nitrogen levels. On the other hand, microbial mass, microbial nitrogen and microbial purine yields declined after 12 h of incubation while ammonia production increased. Per unit of substrate degraded, gas and SCFA production were always inversely (P < 0.05) related to microbial mass yield regardless of incubation time and medium (low or adequate nitrogen). At later incubation times, continuously more SCFA or gas and less microbial mass were produced reflecting microbial lysis and probably increasing microbial energy spilling. All three hays differed (P < 0.05) consistently in how the degraded substrate was partitioned into SCFA and gas and into microbial mass in both the low and adequate nitrogen medium. Purine analysis indicated substantial differences in microbial composition across treatments, which might be one explanation for these different microbial efficiencies (Blümmel et al., 1998a).
The efficiency of microbial growth was higher for 16 h incubation than 24 h for tannin-rich feeds when these were incubated in presence or absence of PEG, a tannin-inactivating agent. Additional nitrogen in the medium also affected the efficiency of microbial protein synthesis from tannin-tannins feeds both at 16 h and 24 h (Getachew et al., 2000c). For proper characterisation of feed and feed ingredients, approaches need to be developed for measuring the PF at the incubation time at which the lysis of microbes is minimal. Some possible simple approaches worth investigating to identify this incubation time are: i) the time at which half of the maximum gas is produced, and ii) the inflection point at which the rate of gas production is maximum (the rate increases up to a certain incubation time and thereafter decreases as the incubation progresses), both these parameters can be mathematically calculated from the gas profiles. The effect of the nitrogen level in the medium on the PF and the significance in vivo of these PFs also need to be investigated.
Digestion kinetics of neutral detergent-soluble fraction. The gas measurement method has also been used to study digestion kinetics of the neutral detergent soluble fraction of forages, starch-rich feeds and other highly digestible carbohydrate components, which was obtained by subtracting the average gas production curve for the digestible neutral detergent fibre (NDF) from that of the unfractionated whole feed (Chen et al., 1999). The subtraction procedure might give some useful information relevant to low-NDF fibre feeds, e.g., corn grain Chen et al., 1999), but it is not suitable for forages rich in NDF (Blümmel et al., 1998b). Blümmel et al. (1998b) examined the rate and extent of fermentation of whole roughage and extracted NDF, dry matter degradability of extracted NDF and the PF for whole roughage and the extracted NDF of 54 roughages. The 24-h degradabilities of extracted NDF were higher than NDF degradabilities in whole roughages, and the PF values were lower for extracted NDF than for whole roughages (2.5 vs 3.1; i.e. the efficiency of microbial protein synthesis with extracted NDF was lower). Both the higher degradability and lower PF contributed to higher gas volumes obtained from extracted NDF compared with whole roughage. Supplementation of amino acids and sugars, which essentially constitute the solubles, may increase the efficiency of microbial synthesis from cell walls during fermentation (a situation similar to that in unfractionated forages) and the removal of solubles may result in lower microbial efficiencies. A considerable effect of cell solubles on partitioning of nutrients from the NDF raises doubts as to the significance in vivo of the kinetic parameters calculated using the subtraction procedure (Blümmel et al., 1998b).
Voluntary intake prediction. The main constraint to the utilisation of roughages by ruminants is voluntary feed intake so prediction of feed intake, particularly of fibrous roughage, is one of the important aspects of ruminant nutrition. In vitro gas production has been used to predict dry matter intake. Various workers have reported significant correlation between in vitro gas production and dry matter intake. Forage cell walls have considerable influence on voluntary feed intake through rumen fill mechanism (Van Soest, 1994). Gas production from extracted neutral detergent fibre was shown to be better correlated to voluntary feed intake than the values obtained from the incubation of whole roughage. The use of various models for intake prediction was investigated and it currently appears that combination of gas volume measurements (4-8 h) with concomitant determination of the amount of substrate degraded (> 24 h) is superior to the models based on kinetics of gas production only. The in vitro gas production from NDF explained more (82 % vs. 75 %) of the variation in dry matter intake than gas production from whole roughage (Getachew et al., 1998a).
Interaction between dietary constituents. Gas measurement was also employed for evaluation of the interaction between basal and supplementary diets by incubating basal diet and supplementary diet separately as well as in combination and monitoring gas production at different hours of incubation using the pressure transducer system (Sampath et al., 1995). This will indicate the availability of readily fermentable material as a ready energy source, which will stimulate the activity of the rumen micro-organisms which in turn would accelerate the digestion of roughages. These workers, by incubating the basal diet and the supplement, observed a positive interaction in gas production in the early hours of incubation, which according to the authors can be an approach to study the synergetic effects of supplementation. However, it must be pointed out that measurement of gas only, could lead to misleading results (see above) It is suggested to determine microbial mass production in addition to the gas measurement for such studies.
The need to determine microbial mass using internal or external markers. Unfortunately, the method based on the gas method and the detergent system of fibre analysis to calculate the microbial mass produced for fibrous feeds (the method outlined above) did not work for tannin-rich feeds. The PF for tannin-rich feeds (calculated as mg truly degraded substrate needed to produce one-ml gas) ranged from 3.1 to 16.1 (Getachew et al., 2000c), which is well above the theoretical range of PF (2.75 to 4.41) (Blümmel et al., 1997). The high PF could be due to: a) solubilization of tannins from the feed. These tannins would make no contribution to gas or energy in the system but would contribute to dry matter loss, b) the cell solubles contributing to dry matter loss but not to gas production because the gas production is inhibited by tannins or c) a combination of a) and b). In addition, the appearance of tannin-protein complexes as artefacts in the true residue also makes the gravimetric approach (Blümmel et al., 1997) of quantification of microbial mass redundant (Makkar et al., 1997a). The presence of tannin-protein complexes in faecal samples (the origin of proteins could be microbes, feed or endogenous secretion from the gastro-intestinal tract) from animals fed tannin-rich forage and their non-removal by the detergent system of fibre analysis (Van Soest et al., 1991) leads to misleading values of fibre and also causes problems in the in vivo evaluation of tannin-rich feeds (Degen et al., 1995; Makkar et al., 1995c). Therefore, caution is required in interpreting results obtained from in vivo or in vitro experiments on the evaluation of tannin-containing feeds using the detergent system of fibre analysis.
For the in vitro evaluation of tannin-rich feeds, the microbial mass should be quantified using diaminopimelic acid (DAPA) or purines as markers, or by 15N incorporation into the microbes (Makkar et al., 1998a), and the PF for tannin-rich feeds can be expressed as the microbial mass determined by these markers per ml of gas produced (or per mmol SCFA produced). The system developed for evaluation of tannin-containing feeds depends on incubation of the feed in the presence and absence of polyethylene glycol (PEG MW 4000 or 6000 preferably of 6000; (Makkar et al., 1995b)) and measurement of gas (or SCFA) and microbial mass using the above mentioned markers. PEG has a high affinity for tannins. Addition of PEG results in the formation of PEG-tannin complexes which inactivates tannins. The changes in gas (or SCFA) and microbial mass as a result of PEG addition represents ‘in totality’ the tannin effects (biological) as a function of the rumen fermentation parameters. This bioassay based on an in vitro rumen fermentation system coupled with the use of a tannin-complexing agent, polyethylene glycol (PEG) could be complementary to other tannin assays (Makkar et al., 1995c; Makkar, 1989; Makkar, 2000) in evaluating the nutritional quality of tanniniferous feeds.
Significance of bound tannins and the efficiency of microbial protein synthesis. The above approach of incubating tannin-containing feeds in the presence and absence of PEG also enables studies to be made on the nutritional significance of both extractable and unextractable (bound) tannins. Addition of PEG during the incubation of tannin-rich NDF led to an increase in gas production, suggesting that tannins released as a result of NDF degradation by rumen microbes are biologically active and have the potential to influence rumen fermentation (Makkar et al., 1997b). Similar results were obtained on incubation of tannin-rich browses made free of extractable tannins by repeated use of 70 % aqueous acetone.
Another application of this method is to study the effect of tannins on the partitioning of nutrients between microbial protein and SCFAs or gases or to study the efficiency of rumen microbial protein synthesis. Using DAPA, purines and 15N approaches for measuring microbial mass it has been shown that in the presence of PEG, the degradabilities of substrate and microbial mass production were higher, the efficiency of microbial protein synthesis was lower (Makkar et al., 1995a, 1998a; Getachew et al., 2000b). Similar results have also been obtained by other approaches based on the gas method in which the rate of ammonia uptake is taken as the efficiency of microbial protein synthesis (Makkar et al., 1998a), and using nitrogen balance approach (Getachew et al., 2000b). Conversely, efficiency of microbial protein synthesis is expected to be higher in the presence of tannins. The net microbial mass production would depend on the balance between decreased degradable dry matter and higher microbial mass production per unit of dry matter digested in presence of tannins.
This method has also been used to develop strategies to enhance the microbial mass production through enhancing efficiency of microbial protein synthesis for tannin rich feeds. Incorporation of PEG in small amounts and scattered over the incubation period increased the efficiency of microbial mass production (Getachew et al., 2000d) which translated into the inclusion of PEG in fed blocks. The licking of these blocks by animals resulted in slow release of PEG in the rumen which produced higher animal productivity compared to when the same amount of PEG was given to animals in one portion (Ben Salem et al., 1999).
Protein degradability. Although the 24-h in vitro degradable nitrogen values obtained from tannin-rich browse and herbaceous legumes were lower than those reported for low quality roughages (Getachew et al., 1998b; 2000b), the relatively high crude protein content of these browses and herbaceous legumes could play a significant role in supplying rumen degradable nitrogen. In the presence of a tannin-inactivating agent, PEG, their in vitro degradable nitrogen values were raised. The difference between these values observed in the presence and absence of PEG indicates the amount of protein protected by tannins from degradation in the rumen (Getachew et al., 2000b). Whether the protein protected by tannins from microbial degradation is fully available to animals post-ruminally requires further research. Raab et al. (1983) reported a close relationship between in vivo and in vitro values when incubation was terminated after 17 h. When normal proteins feed was tested about 80 % of the 24 h value was degraded in the first 8 h incubation whereas in protected protein feed only 60 % of the 24 h value was degraded in this time (Raab et al., 1983). The appropriate incubation time for in vitro degradability studies in the presence and absence of PEG for tannin-rich feeds may depend on the nature of protein and tannins, which should be identified. Also, this method for quantifying feed proteins that have been protected by tannins from degradation in the rumen needs to be validated in vivo.
Measurement of feed proteins during fermentation using polyacrylaminde gel electrophoresis coupled with the use of an image analyser could be another attractive approach for measuring the rumen degradability of nitrogen, and for studying the influence of various natural plant products (e.g., tannins, saponins, alkaloids; (Makkar et al., 1997a, 1998a,b).
