Ruminal degradability and intestinal digestibility of individual amino acids in mixed diets with different crude protein levels measured by the modified in vitro three‐step and mobile nylon bag technique

The ruminal degradability and intestinal digestibility of dry matter (DM), crude protein (CP) and amino acids (AA) in three total mixed rations with different CP levels were estimated using the modified in vitro three‐step procedure (TSP) and mobile nylon bag (MNB) technique on growing lambs. The ruminal effective degradability of DM and CP did not respond with increasing dietary CP level. However, the intestinal digestibility of DM was significantly increased with increasing dietary CP level estimated by TSP (P < 0.05) or MNB method (P < 0.01). Intestinal digestibility coefficients of CP determined by TSP were lower than those of the MNB method. Histidine was extensively degraded by rumen micro‐organisms, while tyrosine was the most anti‐degradable AA among the samples. The ruminal AA degradability exhibited no significant differences except for threonine, tryptophan, alanine, aspartic acid and proline for the three diets. Similarly, only a few AAs (i.e. histidine, methionine, tryptophan, aspartic acid and cysteine in TSP; histidine, tryptophan, aspartic acid and serine in MNB) had significant differences in their intestinal digestibility; in addition, values of MNB were lower than that of the TSP method, indicating that intestinal digestibility of DM seems to be overestimated in TSP, while that of CP might be overestimated in the MNB method.     

The evaluation of the mobile nylon bag technique for determining the apparent protein digestibility in a wide variety of feedstuffs for pigs

Studies were conducted with four (50 to 70 kg) barrows to evaluate the mobile nylon bag technique (MNBT) for determining the apparent protein digestibility (AD) in a variety of feedstuffs. One-gram samples of each feedstuff, ground through a 1.0-mm mesh screen, were enclosed in nylon bags (25 X 40 mm; 48 microns mesh). Following predigestion (.01 N HCl; 4,000 Folin Intestinal Pepsin Units/liter), four bags of each sample were inserted into each pig via a duodenal T-cannula. The indigested contents were pooled within pig and feedstuff prior to crude protein analysis. The AD of each feedstuff was determined previously with four barrows according to the conventional method (CM), based on a 5-d total collection of feces following a 14-d adaptation period. The following results, AD (%), were obtained with the MNBT (n = 4) and CM (n = 4), respectively. Barley, 70.1 vs 81.2 (P less than .05); corn, 72.3 vs 82.1 (P less than .05); wheat, 79.6 vs 86.4 (P less than .05); corn gluten feed, 73.9 vs 70.9; rice bran, 57.3 vs 57.4; wheat middlings, 70.8 vs 79.4 (P less than .05); soybean meal, 89.9 vs 93.0; a mixture of soybean meal and hulls, 86.9 vs 88.8; coconut expeller, 65.0 vs 69.8; fababeans, 79.5 vs 78.5; peas, 87.6 vs 88.1; meat meal, 83.2 vs 84.0; complete diet (4.3% crude fiber), 76.3 vs 83.6 (P less than .05); and complete diet (13.6% crude fiber), 56.5 vs 55.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

利用尼龙袋法比较3个全株玉米品种青贮前后肉牛瘤胃降解特性

本试验旨在利用尼龙袋法比较不同品种全株玉米青贮前后瘤胃降解特性的差异.试验采用2×3双因素试验设计,选择玉米7367号、玉米7377号、玉米7387号3个全株玉米品种为研究对象,采用尼龙袋法比较3种全株玉米青贮前和青贮后的营养成分和瘤胃降解特性,旨在探讨适合河北省种植的全株玉米青贮品种.结果表明:1)青贮前玉米7377...     

Effect of diet composition and feeding pattern on the prececal digestibility of starches from diverse botanical origins measured with the mobile nylon bag technique in horses

This trial was conducted to determine the extent of prececal starch digestibility depending on the botanical origin of starch and on diet characteristics (i.e., composition and feeding pattern). The prececal disappearance of six substrates (oats, barley, corn, horse bean, potato, and wheat) was measured in four cannulated horses fed (as-fed basis) 11.8 g/kg BW of a high-fiber (HF) or high-starch (HS) pelleted feed and 10.0 g/kg BW of meadow hay using the mobile bag technique (MBT). The daily feeding pattern was either three meals (two meals of pellets and one meal of hay) or five meals (three meals of pellets and two meals of hay). The experimental procedure was a 2 x 2 factorial arrangement tested in a Latin square design. After 2 wk of adaptation to the diet, collections were made on 5 d. Thirty nylon bags, composed of five bags of each substrate, were intubated to each horse during the ingestion of the morning meal. Bags were collected in the cecum, using a magnet, at 9 h postintubation. In spite of strong interindividual differences, approximately 80% of the intubated bags were collected. On average, the mean retention time of the bags was 6.2 h (+/-0.17). Regardless of the feeding pattern, the transit of the bags was faster when the fiber content of the diet was higher (P = 0.003). Likewise, regardless of the meal composition, transit was also faster when the ration was split into five daily meals (P = 0.001). The DM disappearance, corrected with particulate losses (DMD(c)), differed depending on the substrate tested (33.5, 57.1, 63.8, 67.7, 78.6, and 86.2% for potato, horse bean, oats, barley, corn, and wheat, respectively; P = 0.001). The DMD(c) of corn, barley, and potato was higher when HS was fed (P = 0.020); regardless of the substrate, DMD(c) was higher with five daily meals (P = 0.001). The starch disappearance (StarchD(c)) was different depending on the substrate (P = 0.001; 36.1, 71.2, 86.6, 89.2, 99.0, and 99.7% for potato, horse bean, barley, corn, wheat, and oats, respectively). Whatever the substrate, StarchD(c) was higher when HS was fed (P = 0.007), but it was not affected by the feeding pattern of the diet. Although passage rate was modified and feed intake was different, the botanical origin of starch was the main factor that affected prececal starch disappearance in horses.     

瘤胃尼龙袋法测定10种饲料过瘤胃淀粉量和淀粉瘤胃降解率

以3头安装有永久性瘤胃瘘管的利木赞×鲁西黄牛杂交阉牛为试验动物,采用尼龙袋法对大麦、麸皮、玉米、玉米DDGS、苹果粕、大豆皮、豆粕、苜蓿干草、青贮饲料、羊草等10种饲料原料中淀粉的瘤胃降解率和过瘸胃淀粉含量进行测定.结果表明,10种饲料的过瘤胃淀粉量依次为60.37、17.98、216.23、9.23、14.18、6.13、18.22、8.11、3.78 g/kg和2.77 g/kg;其淀粉瘤胃降解率依次为90.57%、92.32%、64.37%、78.95%、84.18%、86.30%、70.93%、66.43%、72.19%和65.08%.总之,大麦等能量饲料的过瘤胃淀粉含量较高,苜蓿等粗饲料的过瘤胃淀粉含量较低,副产品饲料和蛋白质饲料过瘤胃淀粉含量居中.     

ORIGINAL ARTICLE: Effects of variety,sowing season and maturity stage on ruminal phosphorus release from whole maize stover and its morphological fractions

An in situ degradation technique was used to investigate the effects of variety, season and maturity stage on ruminal Phosphorus (P) release from whole maize stover and morphological fractions from five varieties of maize (conventional maize, CM; sweet maize, SM; waxy maize, WM; high oil maize, HOM; and fodder maize, FM). Maize plants were harvested in 2005, 17 and 31 days after tasselling and manually separated into leaf blade, leaf sheath, stem and husk. Results showed that the values of rapidly released P fractions (a), slowly released P fractions (b), rate constant of P release for fraction b (c) and effective disappearance (ED) of P were significantly influenced by variety, sowing season and maturity stage (p < 0.05). The ED of P in whole maize stover among the varieties decreased in the following order: FM (89.8%) > HOM (87.9%) > CM (87.0%) > SM (86.9%) > WM (83.9%). Advanced maturity stage increased (p < 0.001) the a and ED values of P in the leaf blade and leaf sheath, but decreased (p < 0.001) these parameters in the husk. The a fraction and ED of P were higher (p < 0.001) for maize sown in spring than those sown in summer. Significant (p < 0.05) interactions among variety, sowing season and maturity stage were noted in the a and ED values of P. Effective disappearance of morphological fraction varied significantly (p < 0.001) and followed the order: stem (90.3%) > leaf sheath (88.2%) > leaf blade (85.2%) > husk (84.9%). In conclusion, most of P in maize stover could be released in the rumen. The potential pollution resulting from P excretion in faeces may be alleviated, when maize stover is largely used as the main forage source of ruminants.     

Cell wall degradation of tropical and temperate forage grasses measured by nylon bag and in vitro digestion techniques

Four tropical and four temperate grasses were subjected to nylon bag fiber degradation study with fistulated Holstein cattle as well as in vitro digestion with broad spectrum cellulase, and the degradability of cell wall carbohydrate and lignin for the respective forage grasses were determined. The degradability of neutral detergent fiber and acid detergent fiber for the same plant materials appeared to be similar, but that of acid detergent lignin was constantly lower. As for the plant materials used in the present study, Italian ryegrass, orchardgrass and guineagrass showed high degradability of fibers in nylon bag incubation, while setaria, cogongrass and wheat showed smaller degradability. In both nylon bag incubation and in vitro digestion experiments, the forage samples showing relatively lower degradability exhibited notably lower disappearance of xylose than that of glucose or arabinose. It was also observed that dry matter degradability correlated negatively with the initial content of esterified and/or etherified p‐coumaric acid. These results were assumed to be caused by the difference in resistance towards rumen microbial degradation due to the concentration of lignin‐carbohydrate complex.     

Maturity of coastal bermudagrass and alfalfa affects ruminal in situ and total tract dry matter and phosphorus disappearance in cannulated steers

Variability of phosphorus (P) availability among forage species and plant maturity is largely ignored when formulating ruminant diets. To determine if variability in P availability changes with forage species and/or maturity, ruminal in situ and total (ruminal+post-ruminal) dry matter (DM) and phosphorus disappearance (PD) from alfalfa (ALF; Medicago sativa) and coastal bermudagrass (CB; Cynodon dactylon) harvested at four stages of maturity was measured in cattle. Forages were hand clipped at 14, 21, 28 and 35 days after first cutting. Ruminal in situ DM disappearance (DMD) and PD were measured after 24 h ruminal incubation in Dacron bags. Total tract DMD and PD were measured using the mobile nylon bag technique. Disappearance of DM and P were greater (p≤0.05) in the rumen than post-rumen for both species regardless of maturity; however, 80 g PD/kg DM in 35-day ALF (9% of total PD) and 224 g PD/kg of 35-day CB (38% of total PD) occurred post ruminally. Alfalfa DM disappeared to a greater (p ≤ 0.05) extent than CB and showed 5% greater total tract PD at 14 days and 13% more at 35 days compared to the grass. Alfalfa total tract PD decreased (p ≤ 0.05) 5.4% from 14- to 35-day maturity while the decrease was far greater for CB, 12.4%. Results from this study indicate that ruminant nutritionists should take into account forage species and maturity when calculating PD in diets; these details can be used to aid in formulating more precise rations that reduce fecal-phosphorus excretion into the environment.     

瘤胃尼龙袋法和体外产气法评定反刍动物饲料的营养价值的比较

试验通过评定菜籽粕、玉米、米糠、苜蓿、玉米青贮和玉米秸秆的营养价值来比较瘤胃尼龙袋法和体外产气实验。结果表明:各种饲料的干物质有效降解率分别为52.90%、54.27%、65.70%、40.76%、37.24%和38.17%,精饲料干物质有效降解率(EDMD)中,a部分占比例较高(其中米糠达到64.51%);每克饲料DM的渐进产气量(A)分别为105.00、180.51、109.30、172.71、147.76和148.66 mL,粗饲料的产气量普遍高于精饲料;两种方法中,产气参数A、B与EDMD均呈负相关(r=-0.55和r=-0.61)。从速率曲线来看,降解速率曲线主要反映了b部分的降解情况,而饲料产气的速率曲线却能明显反映出两相的发酵情况。     

ORIGINAL ARTICLE: Physiological values and factors affecting the metacarpal bone density of healthy feedlot beef cattle as measured by dual‐energy X‐ray absorptiometry

The metacarpal bone mineral density of 136 healthy feedlot beef cattle of four different breeds (Charolaise, Limousine, Irish Crossbreed and Slovakian Crossbreed) raised and fed on standard conditions was measured by means of a dual‐energy X‐ray absorptiometry technique in an ex vivo study design. The average reference values (mean ± SD) of bone mineral density (BMD) for animals aged between 12 and 22 months and weighing between 236 and 546 kg have been reported and the effects of (i) breed, (ii) gender, (iii) age and (iv) body weight on bone mineral density have been considered. A significant difference (i) among different breeds and (ii) between genders resulted, whereas a high correlation between bone density and (iii) age and (iv) body weight was detected within the same breed and gender, with body weight being the most important factor affecting BMD. A modern new technological insight into the study of bovine bone physio‐pathology is proposed.     

ORIGINAL ARTICLE: Seasonal quality profile and production of foliage from trees grown on degraded cropland in arid Uzbekistan,Central Asia

Feed shortages hamper livestock rearing and thus impede the development of rural livelihoods in Central Asia. The production and in vitro quality of foliage from Ulmus pumila, Elaeagnus angustifolia and Populus euphratica on degraded cropland were examined to determine the potential of these species to supplement diary cattle diets. Leaf dry matter (DM) production of the species, respectively, averaged 6, 8 and 17 t DM/ha, 4 years after planting. Over seasons and years, crude protein concentrations (g/kg DM) ranged within 151–257 for E. angustifolia, 70–241 for U. pumila and 92–187 for P. euphratica. The metabolizable energy concentrations (MJ/kg DM) were the highest in U. pumila and ranged within 9–10, followed by 7–10 of E. angustifolia and 7–9 of P. euphratica. The organic matter digestibility (%) ranged within 58–70, 54–66, and 51–66, respectively, for these species. These indicators combined denoted a medium‐to‐good feed quality of E. angustifolia and U. pumila leaves as a cheap protein supplement to roughages. The foliage of P. euphratica was the least suitable. The seasonal profile of in vitro indicators revealed the highest feed quality in spring but early fall seems most appropriate for forage collection given the peak leaf production and an adequate quality.     

Rumen fermentation and degradability in buffalo and cattle using the in vitro gas production technique

An in vitro trial was conducted to investigate the effect of different inoculum sources (buffalo vs. cattle) on rumen fermentation and degradability. Incubations were carried out using rumen fluid obtained from buffalo or cattle fed the same diet [60% grass hay and 40% concentrate; 18 kg dry matter (DM)/day]. The fermentation kinetics of eight feeds commonly used in ruminant nutrition (alfalfa hay, barley meal, beet pulp, corn meal and silage, ryegrass hay and silage and soya bean meal s.e.) were studied with the in vitro gas production technique and rumen fermentation parameters (substrate disappearance, pH and volatile fatty acids production) were determined after 120 h of incubation. The linear relationship indicates that the microbial metabolic pathways of the two inocula for all the substrates were qualitatively similar, albeit often quantitatively different. In this in vitro study, a significant influence of rumen inoculum (buffalo vs. cow) on fermentation and degradability of the examined substrates was found. The differences in buffalo and cattle rumen fermentation can be explained with a different microbial activity of the two ruminant species, because of different amount of microbial population or microbial population constituted by different species of bacteria and protozoa.     

ORIGINAL ARTICLE: Echinacea purpurea and Allium sativum as immunostimulants in fish culture using Nile tilapia (Oreochromis niloticus)

The study was conducted to evaluate the efficiency of echnicacea (E) and garlic (G) supplemented diets as immunostimulant for tilapia (Oreochromis niloticus). Seven treatments were designed including a control (C). Fish were fed on 35% protein diet at a rate of 3% body weight per day. Echinacea (1.0 ppt) and garlic (3%) were incorporated in the feed, which was administered for periods of 1, 2 and 3 months (summer season), followed by basal diet for 4 more months (winter season). Neutrophil adherence and haematocrit values increased in both supplemented groups with prolonging period of application. The neutrophils adherence was significantly increased in all treatments except group administered echinacea for 1 month. The lymphocytic counts were significantly (p < 0.004) elevated that resulted in a significant increase in the total leucocytic count in groups administered echinacea for 1 and 2 months when compared with the control and/or other treatments. The gain in the body weight and specific growth rate was significantly increased in all supplemented groups (p < 0.004) during summer, but remained without any significant increase after winter. The survival rate was significantly high (>85%) in all the supplemented groups. The percentage of protection, after challenge infection using pathogenic Aeromonas hydrophila was the highest in groups supplemented with echinacea and garlic for 3 months after summer and winter seasons. It could be concluded that echinacea and garlic improve the gain in body weight, survival rate and resistance against challenge infection. Both compounds showed extended effects after withdrawal and improved resistance to cold stress during the winter season. However, a full commercial cost benefit analysis is necessary before recommending their application in aquaculture.     

Comparison of the dietary fiber digestibility and fermentability of feedstuffs determined by conventional methods and in vitro gas production technique in pigs

Abstract

This study evaluated the accuracy of an in vitro 2-step method combined with an in vitro gas production technique (2+IVGPT) for the estimation of dietary fiber (DF) digestibility coefficient and fermentability of feedstuffs in the large intestine of growing pigs. Digestibility coefficients of corn, wheat, soybean meal, and wheat bran, estimated by the in vitro methods, including the 2-step, 3-step, 2+IVGPT, and in vivo were compared. The fermentation characteristics of feedstuffs were also determined through the 2+IVGPT. The results showed that the in vivo DF digestibility coefficient of wheat bran was 0.39, which was similar to the result estimated by the 2+IVGPT (0.40). In regard to the fermentation characteristics, the lowest maximum rate of gas production (R_max) was obtained for wheat bran (11.0 ml h^?1). To conclude, in vivo total tract digestibility of DF in the fibrous feedstuff (e.g. wheat bran) could be accurately estimated by the 2+IVGPT.     

贮存时间和方法对乳猪袋装流质饲料中维生素和微生物含量影响研究初报

为开发袋装流质饲料,将自来水和哺乳乳猪全价配合粉料按4:1比例装入蒸煮袋内后用封口机封口,浸泡40min后立即将蒸煮袋放入温度为90℃的蒸饭箱内在常压下用蒸汽蒸煮40min,并分别测定第1、15和30d哺乳乳猪袋装流质饲料中的维生素和微生物含量;另外,将哺乳乳猪袋装流质饲料开袋后分别放在猪场内和冰箱内(6℃),测定第24h后袋内哺乳乳猪流质饲料微生物含量。结果表明:蒸煮和延长贮存时间会降低袋装流质饲料中维生素的含量,同时蒸煮可有效杀灭饲料中的微生物,但随着贮存时间的延长,有些微生物的活性增强。     

ORIGINAL ARTICLE: Inulin‐coated butyrate increases ileal MCT1 expression and affects mucosal morphology in the porcine ileum by reduced apoptosis

Carbohydrates, which were not digested in the jejunum, will be fermented by micro‐organisms to short chain fatty acids. These are transported by the monocarboxylate transporter 1 (MCT1) through the gut wall and serve as fuels for colonic cells. To deliver butyrate to the distal part of the intestine, inulin with a low precaecal digestibility was chosen as a coating material. Approximately 150 g of inulin‐coated butyrate (containing 81 g butyrate) per day was fed to pigs (mean weight: 97 kg) over a period of 6 days after an adaptation period of 6 days with linear increasing amounts of butyrate. The following observations compared to controls were observed: (1) coating was digested microbially in the ileum; (2) MCT1‐mRNA showed a higher expression in the ileum; (3) apoptosis was reduced in the ileum but mitosis was not changed; and (4) length of villi increased by approximately 25% in the ileum. Feeding inulin‐coated butyrate resulted in an increased ileal surface. Delivery of butyrate to the colon requires a more resistant inulin‐coating.     

Optimum grape pomace proportion in feedlot cattle diets: ruminal fermentation,total tract nutrient digestibility,nitrogen utilization,and blood metabolites

Because of its high content of polyphenolic compounds, the dietary inclusion of grape pomace (GP) in ruminant diets can reduce reactive nitrogen (N) and methane emissions and enhance the shelf life and beneficial fatty acids (FAs) content of meat. However, the dietary inclusion of GP beyond a threshold that is still to be determined for feedlot cattle can also compromise nutrient supply and, thus, growth performance. This study investigated the optimum proportion of GP in finishing cattle diets. Nutrient intake and apparent total tract digestion, ruminal pH and fermentation, estimated microbial protein synthesis, route of N excretion, and blood metabolites were measured. Six ruminally fistulated crossbred beef heifers (mean initial body weight ± SD: 714 ± 50.7 kg) were used in a replicated 3 × 3 Latin square with 21-d periods. Dietary treatments were 0%, 15%, and 30% of dietary dry matter (DM) as GP, with diets containing 84%, 69%, and 54% dry-rolled barley grain, respectively. There was a linear increase (P = 0.07) in DM intake and quadratic change (P ≤ 0.01) in neutral detergent fiber (NDF) intake. There was a quadratic change (P ≤ 0.04) in apparent total tract DM, NDF, and crude protein digestibility as dietary GP content increased. However, there were no treatment effects (P ≥ 0.18) on total ruminal short-chain FA concentration and duration and area pH < 6.2, 5.8, and 5.5. Although N intake did not differ (269, 262, 253 g/d; P = 0.33) across dietary treatments, feeding GP led to a tendency for a quadratic change (P ≤ 0.07) in ruminal ammonia-N and plasma urea-N concentrations. Total N excretion also changed (quadratic, P = 0.03) because of changes (quadratic, P = 0.02) in fecal N excretion as urinary excretion of N and urea-N did not differ (P ≥ 0.15) across treatments. Feeding GP led to quadratic changes (P ≤ 0.01) in fecal excretion of fiber-bound N. Microbial N flow and apparent N retention also changed (quadratic, P ≤ 0.04) as dietary GP proportion increased. In conclusion, responses to dietary GP proportion were mostly quadratic with indications that nutrient supply as reflected by changes in apparent total tract nutrient digestibility, microbial N supply, and apparent N retention could be compromised beyond a 15% dietary inclusion level.     

ORIGINAL ARTICLE: mRNA abundance of adiponectin and its receptors,leptin and visfatin and of G‐protein coupled receptor 41 in five different fat depots from sheep

Adipose tissue (AT) expresses adipokines, which are involved in the regulation of energy expenditure, lipid metabolism and insulin sensitivity. Visceral (v.c.) and subcutaneous (s.c.) depots largely differ concerning their metabolic characteristics as to the control of lipolysis and the sensitivity to insulin. The adipokines adiponectin, leptin and visfatin influence lipolysis and insulin sensitivity. Signalling by G‐protein coupled receptor 41 (GPR 41) stimulates leptin release via activation by short‐chain fatty acids. We hypothesized that the metabolic differences between v.c. and s.c. fat depots may also apply to the expression of adiponectin, its receptors, leptin, visfatin, insulin receptor (IR) and GPR 41. Therefore, we aimed to compare the mRNA expression of adiponectin, leptin and visfatin, of the adiponectin receptors 1 and 2 (AdipoR1/2) and IR as well of GPR 41 between several s.c. and v.c. fat depots in sheep. Samples from 10 rams were collected at slaughter (40 kg BW) from three s.c. depots, i.e. close to sternum (s.c.S), close to withers (s.c.W), and at the base of tail (s.c.T), and from two v.c. depots, i.e. from perirenal (v.c.P) and omental (v.c.O) fat. The mRNAs of both adiponectin receptors, as well as IR and putative GPR 41, were higher expressed in v.c. fat than in s.c. fat (p ≤ 0.05). Leptin mRNA abundance was greater in s.c. than in v.c. fat (mean ± SEM: s.c.: 2.55 ± 0.81; v.c.: 0.66 ± 0.21) and also differed among the five separately measured fat depots. Our results show differences in mRNA abundance for leptin, AdipoR1 and R2, as well as for IR and GPR 41 in s.c. compared with v.c. fat, thus confirming the need for individual consideration of distinct fat depots, when aiming to characterize adipose functions in ruminants.     

Effects of three methane mitigation agents on parameters of kinetics of total and hydrogen gas production,ruminal fermentation and hydrogen balance using in vitro technique

Methane (CH₄) can be mitigated through directly inhibiting methanogen activity and starving methanogens by hydrogen (H₂) sink. Three types of mechanism (i.e. bromoethanesulphonate (BES), nitrate and emodin) and doses of CH₄ mitigation agents were employed to investigate their pathways of CH₄ inhibition. Results indicated that both BES and emodin inhibited CH₄ production and altered H₂ balance, which could be accompanied by decreased dry matter disappearance (DMD), fractional rate of gH₂ formation, volatile fatty acid (VFA) production, ability to produce and use reducing equivalences and molecular H₂, and increased final asymptotic gH₂ production, time to the peak of gH₂, discrete lag time of gH₂ production and fermentation efficiency. However, emodin decreased gas volume produced by rapidly fermentable components of substrate and the rate of fermentation at early stage of incubation, while BES supplementation inhibited gas volume produced by both rapidly and slowly fermentable components of substrate and the rate of fermentation at middle or late stage of incubation. The nitrate supplementation inhibited CH₄ production without affecting VFA profile, because of its dual role as H₂ sink and being toxic to methanogens. Nitrate supplementation had more complicated pattern of fermentation, VFA production and profile and H₂ balance in comparison to BES and emodin supplementation.