The ‘gas test’ technique (Menke and Steingass, 1988) is becoming popular for predicting the feeding value of forages. However the relationships observed by Khazaal et al. (1995) between in vivo data and prediction measurements on grasses and legume hays were less good with the gas test technique than with the in situ technique. The present results are part of the work undertaken at the laboratory with the aim of improving the accuracy of the technique on various types of foods (Chenost et al., 1997).

Seventy-nine forages (24 green (G) grasses, 38 grass hays (H), five G lucernes and 12 lucerne H) of known in vivo organic matter apparent digestibility (OMD) out of which 45 (17 G grasses, 16 grass H, five G lucernes and seven lucerne H) of known voluntary dry-matter intake (DMI), both measured with sheep, were chosen based on the range of variation of their OMD (from 0-490 to 0-808) and of their crude protein content (CP from 48 to 253 g/kg DM).

Fermentable energy (FE) was defined in the latest United Kingdom metabolizable protein system as energy available for microbial protein growth and multiplication in the rumen (Agricultural and Food Research Council, 1992). In this system the FE value was calculated by subtracting the metabolizable energy (ME) of fat and ME of any fermentation products from the ME of the food. This estimation is indirect and largely based on whole tract digestion data, thus it can not take into account the influence of rumen outflow, fermentation and degradation rate and digesta retention time in the rumen. In addition, this approach does not take into account undegradable protein and /or starch. As a result, the precision of the FE values are questionable. Therefore a precise, accurate, rapid, cheap and direct technique to measure FE from food ingredients should be developed. Such a technique should consider the factors mentioned above. This work evaluated the ability of four techniques to estimate the FE value of concentrate foods for ruminants.

The in situ and in vitro techniques have been adopted to estimate the degradability of organic matter (OM) in the rumen on the basis that this provides an estimate of ATP for microbial protein synthesis. However this assumption may be incorrect since ATP production requires the fermentation of degraded carbohydrate and Beever (1993) has shown that some degraded hexose can be used synthetically without ATP production. In addition, degraded OM from protein is likely to produce less ATP than the same amount of degraded carbohydrate. The gas production (GP) technique measures end products of fermentation and may be a better guide to ATP production. On the assumption that the in situ and in vitro techniques provide satisfactory estimation of OM degradability, the work discussed here used the GP technique to estimate the effective unfermentable OM fraction of the degraded OM (EUFDOM) for a range of concentrate foods.

The in situ dry matter disappearance technique (Ørskov and McDonald, 1979) evaluates forages for their rate and extent of degradation in the rumen. However, this method does not allow the evaluation of a large number of samples at one and the same time and therefore which limits screening of treatments applied to forages. The in vitro gas production method is faster and allows handling of many samples per batch; therefore, gas production could be an alternative to the use of nylon bags if the response to treatments between the two methods is similar among treated forages. The objective of this experiment was to compare results obtained with both the gas production and the nylon bag techniques for forages treated with four levels of maceration and conserved as hay or silage.

Ruminants in many less developed countries may consume poor quality roughages such as straws, stovers and senescent native pasture as a major part of their diet, particularly during the dry season when high-quality forages are in short supply. The majority of these roughages are high in fibre, low in protein and the intake of digestible nutrients often is not enough to meet maintenance requirements. Intake and digestibility of poor-quality roughages may be increased by supplementation. The response to supplementation can be attributed to an increase in the supply of nitrogen and/or readily fermentable carbohydrate, resulting in an increase in rumen cellulolytic micro-organisms and therefore enhanced fibre degradation.

Gliricidia sepium (Jacq.) Steud. is widely used as a high-protein supplementary food for ruminants in many parts of the tropics. Native to Central America and Mexico, it has become widely naturalized elsewhere but the introduced populations are mostly of unknown origin and uncertain quality. Studies of the genetic resources from the native range, under a research programme at OFI, have shown great variation between populations in yield (Dunsdon and Simons, 1996). The objective of the research described here was to investigate whether there is also important genetic variation in fodder quality, to inform decisions on future distribution of ‘superior’ germplasm of G. sepium under the OFI programme.

The most extensively used methods to characterize and predict nutritive values of forages are fibre fractionation according to Van Soest (Van Soest et al., 1991), in situ degradability (Ørskov and McDonald, 1979) and gas production (Menke and Steingass, 1988; Pell and Schofield, 1993). The aim of the present experiment was to establish whether these methods could be used to characterize Mediterranean foods.

The growth of gut anaerobic fungi is usually determined in batch culture by gravimetric measurements of dry-matter (DM) loss, or by quantification of fermentation end-products (Lowe et al., 1987). These techniques involve destructive sampling of the cultures and require many replicate cultures in time-course experiments. Moreover, techniques using DM loss are only able to measure the end-point of degradation of substrate and cannot follow the growth over the entire fermentation period. Using a pressure transducer, Theodorou et al. (1994) developed a simple and sensitive gas production procedure to determine the fermentation kinetics of ruminant foods. This procedure can also be used to follow the growth of anaerobic fungi on soluble and particulate substrate in batch cultures Theodorou et al. (1995).

The gas production (GP) technique has been developed at a number of laboratories throughout the world as a means of providing information on the rate and extent of the rumen fermentation of foods. However, each laboratory has developed its own method for obtaining a GP profile. The incubation may occur in gas-tight syringes (e.g. Hidayat et al., 1993; Khazaal and Ørskov, 1994) or in culture bottles using pressure transducers that are either manually operated (Theodorou et al., 1994) or automated (e.g. Pell and Schofield, 1993; Cone et al., 1996). Clearly, the method adopted to measure gas production could have quite a significant effect on the profile obtained. In a previous study (ring test 1; J. A. Huntington, unpublished) identical samples of five different foods (barley, soya-bean meal, ‘poor’ hay, ‘good’ hay and cellulose) were supplied to eight different laboratories. Each laboratory obtained GP profiles of each food using their own particular technique. These were then fitted to the model proposed by France et al. (1993). As might be expected there was quite substantial variation between laboratories. It was therefore decided to repeat the study (ring test 2) but to adopt a more standard procedure to determine the extent of variability between laboratories that is inherent in this technique.

Protein degradation in the rumen determines the supply of dietary nitrogen (N) to microbes and undegraded amino acids for direct absorption. The current systems of feeding ruminants identify rumen degradable protein (RDP) from undegraded protein (UDP) which is digestible post ruminally (Agricultural and Food Research Council (AFRC), 1992; Huntington and Givens 1995). The in sacco method has been useful to estimate RDP, UDP and rate of degradation of proteins in ruminants. However, its use is constrained by the fact that it requires surgically modified animals, is laborious, inconsistent and does not differentiate between proteins that disappear from the bag or which are digested by microbes. It is therefore important to develop rapid, robust and reproducible alternatives (in vitro) to simulate ruminal digestion of proteins. Chaudhry (1998) has shown a potential in Streptomyces griseus protease (enzyme) to estimate ruminal digestion in vitro given its consistently high proteolytic activity with purified proteins. This study examined the validity of this enzyme to estimate degradability of food proteins.

Buffer, enzyme and foods A potassium phosphate buffer (buffer) with a pH 6-8 was used to prepare protein suspensions and a solution of the protease (P5147, SIGMA). Samples of sunflower meal (SF), maize-gluten meal (MG), distillers’ dark grains (DG) and field beans (FB) were dried and ground through 1 or 4 mm sieves for in vitro enzyme and in sacco studies respectively.

New feeding systems for ruminants are based on the estimation of the quantity of protein that can be absorbed by the small intestine. This protein has two origins: dietary or microbial. The measurement of food protein which escapes rumen degradation is probably the major element in the evaluation of food protein value. The in situ incubation in nylon bags (Ørskov and McDonald, 1979) is the technique widely utilized to determine rate and extent of protein degradation. However, the technique is difficult to manage as it requires cannulated animals and can furnish reproducible data only utilizing standardized procedures. Although it is possible to standardize the amount and particle size of food sample, bag characteristics, incubation times and calculation model, it is, on the other hand, very difficult to standardize the basal diet (in our opinion to evaluate the diet only in function of forage/concentrate ratio and crude protein content is inadvisable). Thus for many years various alternative methods which are easier to manage and to standardize (chemical, enzymatic and microbiological procedures) have been developed (Nocek, 1988), to replace the in situ method. The most commonly used enzyme is an endoprotease from Streptomyces griseus (Krishnamoorthy et al, 1983; Poos-Floyd et al., 1985; Aufrère et al, 1991, Infascelli et al, 1993). However, some of the authors (Infascelli et al., 1993 plus Poos-Floyd et al., 1985) agree that this bacterial protease is of little consequence when predicting protein food degradation due to its unspecificity. In addition Poos-Floyd et al., 1985) suggest discarding the endoprotease from Streptomyces griseus due to the variability of results. On the contrary, the latter authors, evaluating rumen protein escape and proportion of degraded nitrogen (N) with enzymatic methods contemporarily, found ficin, a tiolic enzyme, preferable. Recently, Tomànkovà and Kopêcný (1995) have developed a method using another tiolic enzyme, bromelain, to predict rumen protein degradability. Kosmala et al. (1996) in agreement with Poos-Floyd et al. (1985) indicate ficin as the protease to estimate rumen degradability. Because these enzymes allow measurement of crude protein solubility at fixed incubation times, we think that the latter should not be necessarily correlated with the effective degradability that originates from combining degradability kinetics with rumen outflow rate. Thus, the aim of the present work was to compare the bromelain and ficin methods for estimating N solubility, particularly with regard to the variability of results.

Cellulose and hemicellulose are the major structural polysaccharides of plant cell wall. The efficient utilization of these polysaccharides by ruminants is often restricted by the presence of lignin. Cellulose and hemicellulose are hydrolysed by a group of enzymes called cellulases and hemicellulases. The present paper describes the cellulase and hemicellulase assay methods and their potential applications.

Carboxymethyl (CM)-cellulose, Avicel, cellobiose, xylobiose, p-nitrophenyl-p β-D-glucoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), p-nitrophenyl-β-D-xyloside (pNPX) and p-nitrophenyl- α-L-arabinofuranoside (pNPAf) were from Sigma. Birchwood xylan and filter paper are from Carl Roth GmbH and Co., Germany and Whatman International Ltd, UK, respectively. H3P04-Swollen cellulose and 4-O-methyl-α-D-glucuronyl-xylotriose (mGpA-Xyl3) were prepared as described (Wood, 1988; Khandke et al., 1989a).

Rumen fermentability of raw materials and foods can be evaluated by different means. Recently a large interest has been generated by the use of in vitro gas production associated with the degradation and fermentation kinetics of forages and concentrates. Measuring and modelling gas production have been studied (Beuvink, 1993) and implemented by automation (Theodorou et al., 1994). This approach has been applied to a variety of foods with different purposes: estimating the effect of maturity on alfalfa and brome hay digestion (Stefanon et al., 1996), evaluating the role of genetic variation on perennial ryegrass rumen fermentation (Loo et al., 1994) and comparing rice-straw fermentability (Williams et al., 1996).

This research has been designed to rank maize silages according to their fermentability and to evaluate possible correlation to the near infrared reflectance spectroscopy (NIRS) predicted wet chemistry values and in vivo calculated organic matter digestibility (OMD).

Browse plants are generally regarded as important food resources for ruminants in the tropics due to their high levels of nitrogen. Particularly during the dry season livestock eat browse which may contain considerable amounts of phenolic compounds including tannins at levels of up to half of the dry matter (Reed, 1986). Tannins are known to react with proteins to form rumen undegradable or totally indigestible complexes.

A uniform food fraction is assumed to have the same true digestibility irrespective of source and level in the food. In the test of uniformity developed by Lucas (1964) and applied by Van Soest (1967) the digestible fraction per unit food is regressed upon its concentration in the food. The slope of the line gives its true digestibility, the intercept, the potential metabolic fraction and the variation around the regression line explains the extent of uniformity of the fraction.

In the Dutch protein evaluation system (Tamminga et al., 1994) microbial protein synthesized in the rumen is estimated from the fermentable organic-matter (FOM) content. The reference method to determine FOM is by in situ incubations of food in the rumen of fistulated animals. However, this is a cumbersome, time-consuming and expensive procedure. In practice, FOM is estimated from the digestible organic-matter content (DOMD) corrected for crude fat (Cfat), rumen escape protein (EP) and starch (ES) and the fermentation products (FP), using tabular values. Apart from being indirect, this approach takes little or no account of the variability within foods. Although an in vitro method based on rumen fluid may give a good prediction of in situ FOM for the majority of the foodstuffs (Cone et al., 1994), an enzymatic method is preferable for practical reasons. From the organic-matter (OM) degradation by a mixture of amylase and other carbohydrate degrading enzymes at 39°C during 0, 2, 6 and 24 h, Cone et al. (1996) obtained a moderate correlation with in situ FOM for 29 grass and grass silage samples (R2 = 0.88; residual s.d. = 37 g/kg dry matter (DM)) and 80 samples of concentrates, maize and clover (R2 = 0.77; residual s.d. = 42 g/kg DM). In this study a modified version of the enzymatic method of de Boever et al. (1994), normally used to predict the in vivo OM digestibility and energy value of concentrates, was tested for its reliability to predict in situ FOM of compound foods and forages.

In the last few years there has been an increasing concern regarding the use of antibiotics in ruminant feeding and the potential for selection of antibiotic-resistant pathogen micro-organisms. Some authors (Martin and Streeter, 1995; Callaway and Martin, 1996) suggested that organic acids (aspartate, fumarate, malate) potentially provide an alternative to currently used antimicrobial compounds. Several in vitro studies (Martin and Streeter, 1995; Callaway and Martin, 1996) showed that incorporation of DL-malate into fermentations with both Selenomonas ruminantium HD4 and with mixed ruminal micro-organisms resulted in changes in final pH, methane and volatile fatty acids (VFA) that are analogous to ionophore effects. Nisbet and Martin (1993) hypothesized that malate acted as an electron sink for hydrogen. However, the mechanism of action is not well known. Malate is a key intermediate in the succinate-propionate pathway and therefore could stimulate propionate production. The objective of this study was to study the effects of DL-malate and propionate on the in vitro rumen fermentation of a 50:50 foragexoncentrate diet.

The study was carried out using the rumen simulation technique (RUSITEC) following the general incubation procedure described by Czerkawski and Breckenridge (1977). The complete unit consisted of eight vessels with an effective volume of 700 ml each. The vessels inocula (solid and liquid) were obtained from three ruminally fistulated ewes given a diet consisting of 700 g alfalfa hay and 300 g concentrate per kg dry matter (DM) (Table 1) and transferred to the RUSITEC system within 30 min of the 1st day of the experiment. The flow through the vessels was maintained by continuous infusion of artificial saliva at a rate of 533 ml/day. Each vessel received daily a nylon bag containing 7 g alfalfa hay, 7 g concentrate and 0·10 g vitaminsminerals mix. From the 1st day of incubation three vessels received daily 5·62 mmol DL-malate (disodium salt; Sigma-Aldrich Quimica, S.A., Spain), three vessels received 5·62 mmol of propionate (monosodium salt; Sigma-Aldrich Quimica, S.A., Spain) and two vessels received no addition (control). DL-malate and propionate were weighed into the nylon bags and carefully mixed with the food.

The estimation of rumen microbial protein synthesis is one of the main points in the nitrogen (N)-rationing systems for ruminants, as microbial protein provides proportionately 0.4 to 0.9 of amino acids entering the small intestine in ruminants receiving conventional diets (Russell et al., 1992). Methods of estimating microbial protein synthesis rely on marker techniques in which a particular microbial constituent is related to the microbial N content. Marker : N values have generally been established in mixed bacteria isolated from the liquid fraction of rumen digesta and it has been assumed that the same relationship holds in the total population leaving the rumen (Merry and McAllan, 1983). However, several studies have demonstrated differences in composition between solid-associated (SAB) and fluid-associated bacteria in vivo (Legay-Carmier and Bauchart, 1989) and in vitro (Molina Alcaide et al, 1996), as well in marker : N values (Pérez et al., 1996). This problem could be more pronounced in the in vitro semi-continuous culture system RUSITEC, in which there are three well defined components (a free liquid phase, a liquid phase associated with the solid phase and a solid phase), each one having associated microbial populations.

The objective of this experiment was to investigate the effect of using different bacterial isolates (BI) on the estimation of microbial production of four different diets in RUSITEC (Czerkawski and Breckenridge, 1977), using (15NH4)2 SO4 as microbial marker, and to assess what effects any differences would have on the comparison of microbial protein synthesis between diets.

This study was conducted in conjunction with an in vitro experiment described by Carro and Miller (1997). Two 14-day incubation trials were carried out with the rumen simulation technique RUSITEC (Czerkawski and Breckenridge, 1977). The general incubation procedure was the one described by Czerkawski and Breckenridge (1977) and more details about the procedures of this experiment are given elsewhere (Carro and Miller, 1997).

Rumen microbes utilize mainly ammonia as a nitrogen (N) source for their growth but some species also use a variety of amino acids (AA) or peptides. Several studies have shown both an enhanced fibre digestion and efficiency of rumen micro-organisms when N sources such as AA, peptides and protein were provided in addition to ammonia (Griswold et al.,1996). However, no difference either in digestion or in growth of rumen microbes was found in other in vivo(Fujimaki et al.,1989) and in vitro (Kernick, 1991) studies. The objective of this experiment was to investigate the effects of protein, peptides, AA and ammonia on microbial growth and fermentation of an all fibre basal diet in a semicontinuous culture system (RUSITEC; Czerkawski and Breckenridge, 1977).

For the past years in Europe, the need for reliable alternative techniques to animal experimentation has been urged by ethical and legal prescriptions. In ruminant nutrition, dual outflow stirred-tank reactors (Hoover et al., 1976; Merry et al.,1987) have proved to be suitable for the accurate control of environmental conditions to study the metabolism of rumen microbes. Their ability to support a varied ciliate population, however, is uneven. As protozoa are major agents in the rumen microbial community, investigating the best set of general operating conditions to maintain their population at high levels is an important issue.

The study was conducted in six dual outflow continuous fermenters (working volume of 1.11) supplied with 30 g/day of a pelleted diet made of 750 g orchard-grass hay and 250 g ground barley per kg. The vessels were inoculated with the rumen contents from four wethers, each equipped with a rumen cannula and given a similar diet. They were continuously infused with an artificial saliva containing: Na2HPO4.12H2O 6.157 g/l, NaHCO3 5.268 g/l, KHCO3 0.597 g/l, NaCl 0.305 g/l, HC1-cysteine 0.4 g/l and alcalinized to pH 10.0 with 1.63 g/l NaOH. The fermentation broth was separately complemented with 31.7 mg CaCl2, 47.5 mg MgCl2 and 0.755 g (NH4)2SO4 per day.