Influence of tannic acid application on alfalfa hay: in vitro rumen fermentation, serum metabolites and nitrogen balance in sheep

Alfalfa protein is poorly utilised by ruminants due to its rapid degradation in rumen. The objective of the study was to assess the influence of spraying tannic acid (TA) on chopped alfalfa hay on in vitro rumen fermentation and nitrogen (N) retention by sheep. Alfalfa hay with and without TA was fed to sheep to determine nutrient digestibility and N balance. TA was sprayed on chopped alfalfa at three concentrations to determine its effect on in vitro fermentation of dry matter (DM) and N balance in sheep. Final TA concentrations were 0, 30, 60 and 90 g TA per kg DM. The control was sprayed with the same amount of water but without TA. In vitro DM degradation and the production of gas, ammonium-N (NH4-N) and short-chain fatty acid (SCFA) were measured. TA-sprayed alfalfa and the control were fed to sheep to determine nutrient digestibility and N retention. Addition of TA had no influence on the extent and rate of gas production but significantly decreased NH4-N concentration at 30 (P < 0.05), 60 and 90 (P < 0.0001) g/kg DM. Addition of polyethylene glycol (PEG) to TA-sprayed alfalfa increased NH4-N to a level comparable to non-TA-sprayed alfalfa. Spraying of alfalfa with TA significantly decreased (P < 0.05) isovalerate but did not affect the total and individual SCFA acid production. Tannic acid significantly (P < 0.05) reduced in vitro true degradability of DM (IVTD) after 24 h incubation at levels of 60 and 90 g TA per kg DM. Neutral-detergent fibre digestibility (dNDF) after 24 h (P < 0.01), 60 and 90 (P < 0.0001) g TA per kg DM. The effect of TA on either IVTD or dNDF was not significant (P > 0.05) after 48 h of incubation. There was a strong linear relationship between percentage increase in gas production due to PEG and protein precipitation capacity (R2 = 0.94). N digestibility was significantly reduced with all three levels of TA additions. However, the proportion of urine-N to total N output was reduced by adding 60 g (P < 0.05) and 90 g (P < 0.01) TA per kg DM. Serum metabolites and liver enzymes were not affected by TA (P > 0.05). Higher faecal N as the TA level increased indicates incomplete dissociation of tannin-protein complexes post ruminally. Factors affecting dissociation of tannin-protein complexes need further study.     

The bacterial and archaeal community structures and methanogenic potential of the cecal microbiota of goats fed with hay and high-grain diets

The cecum plays an important role in the feed fermentation of ruminants. However, information is very limited regarding the cecal microbiota and their methane production. In the present study, the cecal content from twelve local Chinese goats, fed with either a hay diet (0% grain) or a high-grain diet (71.5% grain), were used to investigate the bacterial and archaeal community and their methanogenic potential. Microbial community analysis was determined using high-throughput sequencing of 16S rRNA genes and real-time PCR, and the methanogenesis potential was assessed by in vitro fermentation with ground corn or hay as substrates. Compared with the hay group, the high-grain diet significantly increased the length and weight of the cecum, the proportions of starch and crude protein, the concentrations of volatile fatty acids and ammonia nitrogen, but decreased the pH values (P?<?0.05). The high-grain diet significantly increased the abundances of bacteria and archaea (P?<?0.05) and altered their community. For the bacterial community, the genera Bifidobacterium, Prevotella, and Treponema were significantly increased in the high-grain group (P?<?0.05), while Akkermansia, Oscillospira, and Coprococcus were significantly decreased (P?<?0.05). For the archaeal community, Methanosphaera stadtmanae was significantly increased in the high-grain group (P?<?0.05), while Methanosphaera sp. ISO3-F5 was significantly decreased (P?<?0.05). In the in vitro fermentation with grain as substrate, the cecal microorganisms from the high-grain group produced a significantly higher amount of methane and volatile fatty acids (P?<?0.05), and produced significantly lower amount of lactate (P?<?0.05). Conclusively, high-grain diet led to more fermentable substrates flowing into the hindgut of goats, resulting in an enhancement of microbial fermentation and methane production in the cecum.     

Continuous fermentation of soy sauce by immobilized cells of Zygosaccharomyces rouxii in an airlift reactor

Summary The optimum conditions for continuous alcohol fermentation of soy sauce with immobilized Zygosaccharomyces rouxii cells were investigated using an airlift reactor. The optimum pH and temperature of the fermentation were 4.5–5.5 and 25°–27.5° C, respectively. Ethanol content in the fermented liquid was increased with increasing height to diameter ratio of the reactor and the ratio of air to nitrogen in the supplied gas (total supplied gas: 0.08 vvm). A notable decrease in ethanol content was observed when only nitrogen gas was supplied. The products fermented by supplying air (0.02 vvm) had a higher conent of aroma components than that by supplying only nitrogen gas, and the aroma of the former products was similar to that of conventional soy sauce. This alcohol fermentation using an airlift reactor was continued for about 50 days without problems even if conditions such as residence time and aeration were altered.     

The effect of high levels of molasses in combinations with hay on digestibility of organic matter,microbial protein synthesis and volatile fatty acid production in vitro

Fermentation in vitro of $\text{0}\text{100}$, $\text{10}\text{90}$, $\text{40}\text{60}$ and $\text{70}\text{30}$ cane molasses/hay mixtures in media containing urea, trace minerals and vitamins showed a progressive improvement of digestible organic matter in vitro (INVDOM) of hay as the level of molasses increased. Replacement of urea by soya bean meal induced a lower INVDOM of hay in the mixture with 70% molasses.Structural carbohydrates (hay) induced higher microbial synthesis per unit digestible organic matter (DOM) than simple sugars (molasses).The results observed emphasized the importance of adding urea to molasses for improved digestion of hay and a higher percentagge of propionic acid in the rumen. The effect of formulation of molasses/hay rations with soya bean meal or urea on the pattern of fermentation is discussed.     

Scrotal,testicular and semen characteristics of young Boer bucks fed winter veld hay: The effect of nutritional supplementation

The aim of the work was to determine the effects of feeding winter veld hay plus a supplement on scrotal, testicular and semen characteristics in young Boer goat bucks. Fifteen Boer goat bucks (6–8 months of age), were allocated to two groups and fed ad libitum for a period of 29 days. The WH group (winter hay or control group; n = 8) received a chopped diet consisting of grass hay, predominantly Themeda triandra grass (cut during the winter) from natural pastures (veld). The WH + S group (winter hay plus supplement; n = 7) received the same diet consisting of Themeda triandra veld hay and supplemented with maize meal, molasses meal and urea. Body weights and feed intake were recorded, as well as scrotal, testicular and semen characteristics. Results indicate a detrimental effect of winter veld hay feeding on characteristics such as sperm cell abnormalities (43% in the WH group versus 24% in WH group), testicular volume (156 ml in the WH + S group versus 104 ml for WH animals) or scrotal circumference (20.7 cm in WH + S group versus 17.7 cm in WH group). It is essential to supplement the nutrition of small ruminants under winter environments, to maintain scrotal, testicular and semen characteristics, especially if the animals are to be used in the subsequent breeding season.     

Effects of groundnut stover or Bermudagrass hay supplementation to doe kids on winter hardwood range

Goats in north-central Texas raised on rangeland often face winter forage quantity and quality deficits that may be mitigated by feeding hay or stover. Groundnut (Arachis hypogea) stover (8% CP, 35% ADF, 43% NDF and 8% acid detergent lignin (ADL) DM basis) and bermudagrass (Cynodon dactylon) hay (12% CP, 33% ADF, 73% NDF, and 5% ADL DM basis) were tested at Stephenville, Texas as 0.0, 0.5, or 2.0% BW supplement/substitution diets on 18 kg Boer X Spanish doe kids browsing native hardwoods (8 ha⁻¹). Both hay and stover were fed ad libitum in a traditional feedlot, using a complete formulated feed ration as a control. Trials ran for 10 weeks from January to March in 2003 (134 mm rainfall) and in 2004 (182 mm rainfall). Goats receiving 0.5% and 2% BW bermudagrass or 2% BW groundnut stover had greater ADG than those in the control and 0.5% BW groundnut paddocks (P < 0.05). Goats fed complete ration in the drylot had greater (P < 0.05) ADG than those eating either hay or stover ad libitum. Bermudagrass hay rejected by goats in the hardwood trial was 20% lower in CP, 7% greater in NDF, 8% greater in ADF, and 9% greater in ADL than the original fed hay; groundnut stover refusals were 21% lower in CP, 12% greater in NDF, 19% greater in ADF, and 20% greater in ADL concentration than the stover when fed. Supplementing goats on hardwood range with bermudagrass hay or groundnut stover may improve ADG when browse is scarce or of poor quality.     

Enrichment, Isolation, and Cultural Characteristics of Marine Strains of Clostridium botulinum Type C

Terrestrial strains of Clostridium botulinum type C, designated 468 and 571, were used to screen various media for growth and sporulation at 30 C. Of the various formulations tested, only egg meat medium fortified with 1% additions of yeast extract, ammonium sulfate, and glucose (FEM medium) gave good growth and satisfactory sporulation. FEM medium was used to recover four marine type C isolates from inshore sediments collected along the Atlantic, the Gulf of Mexico, and the Pacific coasts of the United States. The isolation techniques involved repeated transfer of cultures showing type C toxin in FEM medium and purification by a deep tube method. The medium used for purification was beef infusion-agar supplemented with 0.14% sodium bicarbonate and 0.1% l-cysteine hydrochloride. l-Cysteine was adopted in preference to sodium thioglycolate, because some lots of the latter were definitely inhibitory for growth. The addition of bicarbonate markedly increased viable spore counts of both the marine and terrestrial strains. Various cultural and biochemical characteristics of the marine and the terrestrial strains were compared. With the exception of some variations in their fermentation patterns, both groups showed similar characteristics. Of 23 fermentable compounds tested, the terrestrial strains attacked only glucose and mannose. The marine strains fermented glucose, mannose, galactose, and ribose actively; dextrin, inositol, maltose, and melibiose were weakly fermented.     

Growth of Neocallimastix sp. Strain R1 on Italian Ryegrass Hay: Removal of Neutral Sugars from Plant Cell Walls

The anaerobic fungus Neocallimastix sp. strain R1 was grown for up to 5 days on a medium containing autoclaved Italian ryegrass hay as the carbon source. Culture supernatants and digested cell walls were harvested at 12-h intervals. Supernatants were analyzed for the fermentation products formate and acetate, and residual cell walls were analyzed for dry-matter and neutral-sugar losses. Fungal growth was accompanied by the digestion of plant cell walls and the accumulation of fermentation products in culture media. Dry-matter losses were accounted for by removal of four major neutral sugars (arabinose, galactose, glucose, and xylose) from the plant cell walls. First-order reaction kinetics could be used to describe the loss of each sugar. All cell wall sugars, including arabinose and galactose, which are not fermented by Neocallimastix sp. strain R1 were removed simultaneously. Although the rates of removal of individual sugars were similar, there were significant differences in their extents of removal: the extent of removal of arabinose exceeded that of the other three sugars, and xylose was the least digestible. This study provides the first account of simultaneous (nonpreferential) removal of neutral sugars from plant cell walls by an anaerobic fungus. Although in vitro techniques were used, these results indicate a potentially significant role for the anaerobic fungi as fiber digesters in the rumen.     

Comparative fermentation studies of industrial strains belonging to four species of solvent-producing clostridia

Industrial and culture collection strains of solvent-producing clostridia, classified as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum were utilised in a comparative study of fermentation performance in a laboratory fermentation medium, a molasses fermentation medium, and a maize fermentation medium under standardised culture conditions. At least one representative strain was selected from each of the sub-groups within the four species. Preliminary evaluations were first undertaken for the three different fermentation media to determine the most appropriate media formulations, carbohydrate concentrations, and culture conditions for comparison of the solvent-producing ability of these strains. Standardised fermentation media and culture conditions were then selected for each of the comparative fermentation studies. These included TYA medium containing 4% glucose, a supplemented molasses medium containing 6% fermentable sugars, and a supplemented maize mash medium containing 8% maize. Additional comparative fermentation studies on industrial strains belonging to two species of solvent-producing clostridia were carried out in molasses containing higher concentrations of fermentable sugars, and the sugar concentrations supporting maximum levels of solvent production were determined. Although all the strains tested grew in the maize fermentation medium and degraded starch, only a few strains produced consistently high solvent levels. Optimum starch utilisation and solvent production was obtained at a maize concentration of 80 g/l. Pretreatment of the maize by milling or saccharification decreased the buffering capacity of the medium and resulted in decreased solvent production. Decreasing the time used to gelatinise the starch had little effect. Solvent yields and concentrations obtained in this study were compared with various published data in the scientific and patent literature and appeared to closely simulate the results obtained in the industrial fermentation process. The fermentation performances of individual strains could provide useful comparative data for the selection and development of strains for use on various commercial fermentation substrates.     

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.     

Effect of pentoses and pentitols on fermentation of hay by mixed populations of ruminal microorganisms

Consecutive batch culture, a technique which involves sequential transfer of cultures to fresh medium at regular intervals, was used to establish mixed ruminal-microbial populations in an anaerobic medium containing highly digestible hay. Once volatile fatty acid production was stable, perturbations were imposed in consecutive cultures by the addition of one of each of the following pentoses or analogous pentitols: l-arabinose, d-lyxose, d-ribose, d-xylose, l-arabitol, d-arabitol (lyxitol), ribitol, and xylitol. With the exception of d-lyxose, the addition of pentoses caused marked increases in propionate and valerate production, and except for d-arabitol, pentitol addition caused increases in butyrate and valerate production. On transfer to and continued incubation in the control medium, volatile fatty acid production reverted to preperturbed levels. The presence of pentitols and pentoses significantly reduced the endpoint pH of cultures and the proportion of hay that was fermented. With all added substrates, the response to the perturbation was at its maximum within one incubation (i.e., within 48 h). Similarly, the variables being monitored all returned to control levels within one incubation. On the basis of these results, it is suggested that changes were related to the need to maintain a redox balance within anaerobic cultures rather than any significant changes in the microbial population that was present.     

Fate of inoculated Escherichia coli in hay

AIMS: To monitor the fate of inoculated Escherichia coli in dry and moist hay of various types, under laboratory conditions. METHODS AND RESULTS: Wheat, vetch and clover hay were used as received or wetted to 250-300 g kg(-1) moisture. The hay was inoculated at about 10(6) CFU g(-1) with a kanamycin-resistant E. coli strain that expresses the green fluorescence protein, and was stored in small open glass jars that were covered with aluminium foil. Three jars per treatment were sampled on days 1 and 3, or 4 and 7, or 8, 20 and 50, respectively, after the initiation of the experiments, and the numbers of E. coli in the hay were determined. The results indicated that E. coli disappeared from both dry and moist hay by 7-8 days after inoculation. However, in a few cases colonies that were presumed to be E. coli developed after incubation in Luria broth medium. CONCLUSIONS: The tagged E. coli strain usually disappeared rapidly from both the dry and the moist hay, in spite of the high level of inocula used. However, in some cases a few, possibly injured E. coli might have persisted, and could be detected after incubation in a rich growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is part of a risk assessment associated with sewage irrigation of forage crops in Israel. The results indicate that E. coli added to the hay is not likely to pose a health risk to cattle or to humans. Nevertheless, more research with natural strains of E. coli and other enteric pathogens that might be more adapted to forage conditions is warranted in order to ensure the safety of sewage-irrigated crops.     

粘质沙雷氏菌产灵菌红素培养基的筛选

目的：确定菌株S418产生灵菌红素的最优培养基配方及其的分类地位。方法：以花生粉为基础培养基,通过单因素试验和四因素三水平正交试验筛选出了菌株S418产灵菌红素的最佳培养基配方;根据该菌株的16S rRNA基因序列系统发育树分析初步确定了菌株S418的分类地位。结果：培养基最优配方为：花生粉2%,花生油0.5%,L-脯氨酸1%,硫酸镁0.025%。在28℃、pH7.5、250r/min振荡培养24h,灵菌红素产量达67.92mg/L。菌株S418初步鉴定为粘质沙雷氏菌（Serratia marcescensS418）。结论：花生粉培养基是一种适合粘质沙雷氏菌产灵菌红素的优良培养基。     

Comparative Study of Sugar Fermentation and Protein Expression Patterns of Two Lactobacillus plantarum Strains Grown in Three Different Media

A comparative study of two strains of Lactobacillus plantarum (REB1 and MLBPL1) grown in commercial medium (MRS broth), cucumber juice, and liquid pig feed was performed to explore changes to the metabolic pathways of these bacteria, using a proteomics approach (two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry) combined with analyses of fermentable sugars and fermentation end products. The protein expression showed that even with an excess of glucose in all media, both strains could metabolize different carbohydrates simultaneously and that hexoses could also be used via a phosphoketolase pathway with preferential expression in liquid feed. Sugar analyses showed that the fermentation of sugars was homolactic for all media, with some heterolactic activity in liquid feed, as shown by the production of acetate. Cucumber juice (the medium with the highest glucose content) showed the lowest hexose consumption (10%), followed by liquid feed (33%) and MRS broth (50%). However, bacterial growth was significantly higher in cucumber juice and liquid feed than in MRS broth. This discrepancy was due to the growth benefit obtained from the utilization of the malate present in cucumber juice and liquid feed. Despite different growth conditions, the synthesis of essential cellular components and the stress response of the bacteria were unaffected. This study has improved our understanding of the mechanisms involved in the growth performance of an appropriate lactic acid bacterium strain to be used for food and feed fermentation, information that is of crucial importance to obtain a high-quality fermented product.     

Effect of Azotobacter strains on sugar beet callus proliferation and nitrogen metabolism enzymes

The effect of five Azotobacter chroococcum strains and nitrogen content in nutrient media on callus growth of two Beta vulgaris L. cultivars were investigated, as well as the activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in inoculated callus tissue. On medium with full nitrogen content (1 N) the inoculation with A. chroococcum strain A2 resulted in the highest calli mass, while strains A8 and A14 maximally increased NR activity. On media with 1/8 N the highest effect on calli growth, GS and GDH activity had the strain A8. The strain A2/1 significantly increased callus proliferation on medium without N. Asymbiotic association between sugar beet calli and Azotobacter depended on genotype/strain interaction and was realised in presence of different nitrogen levels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of different carbohydrates on in vitro fermentation activity and bacterial numbers of porcine inocula under osmotic stress conditions

The bacteria of the intestine have to cope with varying osmotic conditions in their ecosystem. In this in vitro study, the modified Hohenheim gas test (HGT) was used to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum, when fermenting different carbohydrates (inulin, corn starch, potato starch, cellulose, pectin), under normal buffered and osmotic stress conditions (elevated medium salinity). After 24 h of fermentation, gas, ammonia and short-chain fatty acid (SCFA) production was measured, and the cell numbers of total eubacteria, Lactobacillus spp., Bifidobacterium spp. and enterobacteria were analysed, using real-time polymerase chain reaction. There was a significant reduction in gas production after 24 h when comparing osmotic stress conditions with normal buffered conditions, and there were also differences among carbohydrates under both conditions. The content of SCFA was significantly lower when comparing osmotic stress with normal buffered conditions. Under osmotic stress, inulin and corn starch increased (p < 0.05) cell numbers of total eubacteria, while Bifidobacterium spp. and enterobacteria were higher (p < 0.05) when corn starch and pectin were fermented, respectively, in comparison to the other carbohydrates. The in vitro system of the modified HGT appears to be suitable to scrutinise effects of carbohydrates on the metabolic activity and composition of the microbial community under osmotic stress conditions, as they might occur during situations of osmotic diarrhoea.     

Effect of eight growth media upon fermentation profiles of ten anaerobic bacteria

A study was made of the influence of eight growth media upon the fermentation end-product patterns obtained with ten species of anaerobes. Acidic and neutral end-products of fermentation were analysed by gas chromatography and the results were examined statistically by computer. Differences in end-product profile were at least as great between different media used to grow a single anaerobic species, as between different anaerobes grown in a single type of medium. When individual fermentation end-products produced by a single anaerobe species were examined, statistically significant differences were found between different media for individual end-products. It is concluded that standardisation of growth medium is essential in diagnostic laboratories concerned with identification of anaerobes with the aid of gas chromatography.     

Production of γ-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel

A newly isolated strain of Cunninghamella echinulata grown on glucose produced significant quantities of biomass and cellular lipids in media with high C/N ratio. The oil yield from glucose consumed increased after nitrogen exhaustion in the growth medium, but gamma-linolenic acid (GLA) content in cellular oil systematically decreased during the lipid accumulation process. When lipid accumulation was completed, GLA concentration in the cellular lipids progressively increased. The highest GLA production (720 mg/l) was achieved in medium with a C/N ratio equal to 163. C. echinulata was also able to grow on orange peel. The C/N ratio in the orange peel decreased from 50 to 26 during solid-state fermentation. Maximum oxygen uptake was observed during assimilation of reducing sugars, whereas a polygalacturonase activity was detected after reducing sugars had been exhausted. The maximum GLA production was 1.2-1.5 mg/g of fermented peel, calculated on a dry weight basis. After enrichment of the pulp with inorganic nitrogen and glucose, an increase in the production of oil and GLA was observed.     

Role of Megasphaera elsdenii in the Fermentation of dl-[2-C]lactate in the Rumen of Dairy Cattle

Since Megasphaera elsdenii ferments a variable part of dl-lactate to butyrate, measurement of the percentage of dl-lactate fermented to propionate via the acrylate pathway in rumen contents will underestimate the participation of M. elsdenii in the dl-lactate fermentation. The percentage of dl-[2-C]lactate fermented via the acrylate pathway and the percentage of dl-lactate fermented to butyrate can be measured with C-FT (Fourier transform)-nuclear magnetic resonance. On the average, the contribution of M. elsdenii to dl-lactate fermentation in the rumen of dairy cattle was found to be 74% (standard deviation, 13%), but differed with animal or diet. After feeding a cow readily fermentable carbohydrates, the contribution of M. elsdenii to the fermentation of dl-lactate increased as a consequence of catabolite repression in other dl-lactate-fermenting bacteria.     

Variation of proteolytic activity in ewe during in vivo digestion of two pea-based diets differing by their fermentability characteristics.

In order to study the effects of a small difference in starch and nitrogen availability on proteolysis, two different diets were supplied to four ewes fitted with rumen fistulae. They differed in the ratio of fermentable nitrogen over fermentable energy. with 144 g of fermentable nitrogen (FN) per kg of fermentable energy (FE) for diet I and 126 g FN x kg(-1) FE for diet II. The diets were constituted of 700 g hay grass, 200 g ground pea and either 100 g ground wheat (diet I) or 100 g corn starch (diet II). After two weeks of an adapting period to the diets, rumen content was sampled after feeding over time. The rate of disappearance of soluble proteins was 2.5 times higher with diet II and ammonia concentrations were significantly lower (from -28 to -43%) with diet II. Total proteolytic activity, by considering all the bacterial compartments, was significantly higher with diet II (+40 EU/mL x h(-1)): changes in the total proteolytic activity in the particulate and the liquid phases of the rumen could explain the difference observed between the two diets. Moreover, with diet II, exopeptidase activities increased more in the liquid phase, especially leucine aminopeptidase and Dipeptidyl peptidase I (DPP-I), and the diversity of endopeptidase activities increased in the particulate phase. These two facts could account for the higher total proteolytic activity in the rumen content with diet II.