Glucose and fructose are present as free monosaccharides in plants (57, 58) at concentrations generally below 10 g/kg DM, although they can be up to 61 g/kg DM in spring grass (57). Glucose can account for 20–90% of total non-structural carbohydrates in the leaves of tropical grasses (59) and 11–36 g/kg DM (60) and 10–20 g/kg DM (58) in some cool-season grasses. Fructose is not detectable or is only present in very small amounts in the leaves of tropical grasses (59) and between 8 and 22 g/kg DM (60) and 5–12 g/kg DM (58) in the leaves of cool-season grasses. Free sugars are estimated to degrade in the rumen at a rate of 40–60%/h (61).

Monosaccharides are the basic units of disaccharides, oligosaccharides and polysaccharides, and in plants include pentoses (5-carbon sugars) such as xylose and arabinose, and hexoses such as glucose, galactose, rhamnose, mannose, fructose, glucuronic acid, and galacturonic acid. Many of these can occur in a variety of different polysaccharides. For example, in lignified secondary cell walls of dicotyledons, xylose occurs mostly in xylans, but in primary walls of dicotyledons, it occurs mostly in xyloglucans, with small amounts in pectic polysaccharides. Similarly, in primary cell walls of dicotyledons arabinose, galactose, rhamnose, and galacturonic acid occur mostly in pectic polysaccharides. These associations result in different rates and extent of digestion of monosaccharides from the polysaccharides. The ratio of xylose to arabinose has been suggested as an indicator of the potential digestion of forage cell walls (62). This would apply particularly to dicotyledonous forages, but even in grasses where heteroxylans containing arabinose occur in both primary and lignified secondary cell walls, the proportion of arabinose in these polysaccharides is greater in primary cell walls, which also contain pectic polysaccharides containing arabinose (44). Therefore, it is important to know the monosaccharide composition of carbohydrates in forages.

Di- and- oligo-saccharides, non-fibre polysaccharides and soluble fibre are part of NFC, which are rapidly degraded in the rumen (20–60%/h) and to a large extent (61) but vary in concentrations, monosaccharide composition and structure (see next section), which might affect CH₄ emissions differently.

The main disaccharide present in forages and feeds is sucrose, and the main oligosaccharides are raffinose, stachyose and verbascose, but the contents of sucrose (<40 g/kg DM) and oligosaccharides (<40 g/kg DM) are generally low across a range of feeds including whole plant cereals (57). However, the concentration of sucrose in grasses reached up to 62 g/kg DM in the leaves of Festuca arundinacea, between 24 and 42 g/kg DM in Agropyron (red), L. perenne, Dactvlis glomerata, Phleum pratense, and Bromus inermis, but was <9 g/kg DM in Bromus tectorum (60), 8–18 g/kg DM in F. arundinacea, Lolium multiflorum × F. arundinaceae, Thinopyrum ponticum, Thinopyrum intermedium, and B. inermis (58) and 24–42 g/kg DM in P. pratense (63). The simple sugars in fresh fodder beet underground storage organs and leaves consist of 15–20% sucrose (64).

Fructans are present in the vegetative parts of temperate grasses ranging from 19 to 383 g/kg DM, typically 25–50 g/kg DM (58). Fructans have a linear chain of β-2,6-fructosyl-fructose residues and can have a branched structure by both β2-1 and β2-6 linkages in some plant species. The side and branch chains of fructans have a α-1,2-linked residue as a terminal unit in most cases, which is a non-reducing end (65, 66). Fructans with a low degree of polymerisation can be extracted with boiling 80% ethanol, and fructans with a high degree of polymerisation can be extracted with boiling water (67). Fructans are rapidly and completely fermented in the rumen (68). In the 1970s, a high fructan content was found in perennial ryegrass plants of specific populations, leading to the breeding of cultivars called generically “high sugar grass” (69), which have received considerable attention for their putative benefits to animal production (29, 69, 70). Inulin is also a fructan, sometimes called inulin-type fructan, but it is a linear β 2-1 fructan with a basic unit of α-D-glucose linked to 1-2 β-D-fructose β (1, 5) bond (66). Inulin is widely present in other forage crops like the tubers of Jerusalem artichoke (Helianthus tuberosus) (71) and the roots of chicory (66).

Starch is a carbohydrate composed of glycosidically linked glucose units and composed of two types of polysaccharides: linear amylose and branched amylopectin. Amylose has a linear chain only, and the length of the chain is several hundred glucose residues (72). Amylose occasionally contains α-1,6 linked glucose. Amylopectin has a chain of α1,4-linked glucose residues with side chains linked by α1,4,6-linked glucose residues (72). The side chain sometimes has terminal glucose which is a reducing sugar (72, 73). A large number of the macromolecular chains in starch granules are organised in crystalline structures, with three patterns of crystalline structures occurring: A-type pattern common in cereal starches, B-type pattern present in some tuber starches and cereal starches, and C-type pattern is common in legume starches. B-type crystalline starches with larger granules are more resistant to enzymatic breakdown than A-type crystalline starch with small granules (74). Starch is rapidly degraded in the rumen (20–40%/h; 51), with the physical structure surrounding starch granules being the main barrier to degradation (73). Starch concentration in vegetative parts of forages is very low, typically ranging from 10 to 33 g/kg DM (58), with 40 g/kg DM in the leaves of Lolium temulentum (75), 34 g/kg DM in lucerne leaves, 3 g/kg DM in lucerne stems, 4 g/kg DM in timothy hay (76), 2–3 g/kg DM in timothy leaves (63) and 5–17 g/kg DM in forage brassicas (Sun et al., unpublished data). Starch is the main storage polysaccharide in grains and some tubers such as potatoes and sweet potatoes, but not in the underground storage organs of swedes, turnips, and fodder beet.

Pectic polysaccharides are present in the primary cell walls of all forage plants and are located particularly in the middle lamella (77). The pectic polysaccharides in the primary walls of grasses (family Poaceae) have a similar structure to those in dicotyledonous primary walls (78), but they occur in much lower proportions in Poaceae (Table 1). The principal monosaccharide components of pectins are galacturonic acid, rhamnose, arabinose and galactose (79). There are three main types of pectic polysaccharide domains: homogalacturonan (HG), rhamnogalacturonan I (RG I), and rhamnogalacturonan II (RG II) (80) and a small amount of a fourth domain, xylogalacturonan (XGA), occurs in some walls (44, 78). The XGA is composed of a backbone of 4-linked α-D-galactosyluronic acid residues to which xylose residues are attached at the C(O)3 position of about half of the galacturonic acid residues (78).

The HG is a linear homopolymer of 4-linked α-D-galactosyluronic acid residues. Methyl-esterification of galactosyluronic acid residues often occurs at the C-6 carboxyl group; acetyl groups are also present at C-2 and/or C-3 in some plants (79). Two HG chains with blocks of more than 10 unesterified galacturonic acid (GalA) residues can form a dimer via Ca²⁺-cross-linking (80). RG I consists of a backbone of alternating (1, 7)-linked α-D-galacturonic acid and (1, 5)-linked α-L-rhamnose residues and has several different side-chains attached to the C4 of rhamnose. These side-chains are mainly composed of arabinose and galactose chains (arabinans and galactans, respectively) linked by (1, 8)-linkages and by (1, 7)-linkages, respectively (81). Type I arabinogalactan side-chains have (1, 7)-β-galactan chains with limited substitution by short arabinose-containing oligomers (82). RG II is quantitatively a minor constituent of primary cell walls in dicotyledons (83), and it has been isolated from the primary cell walls of the Poaceae. It is released as low-molecular-weight polysaccharides, containing about 30 different glycosidic linkages with at least 12 different glycosyl residues, after treating with endo-polygalacturonase (78).

Cellulose, a simple linear polymer of (1, 7)-linked β-D-glucosyl residues, is the most abundant plant polysaccharide and forms the basic structure of all plant cell walls (84). Cellulose occurs as crystalline microfibrils about 3 nm in diameter containing 18 cellulose molecules aligned side to side (85, 86). In secondary lignified cell walls, some of the cellulose microfibrils may aggregate into structures known as macrofibrils (87). Primary cell walls contain ~20–30% cellulose, and lignified secondary walls contain ~40–60% cellulose (88). Cellulose typically accounts for about 35~50% of plant dry matter (89).

Other non-cellulosic polysaccharides have traditionally been called hemicelluloses and comprise a group of polysaccharides that are extracted from cell walls with alkaline solution after initial treatments with water and chelating reagents (90). They include xyloglucans, heteroxylans, heteromannans, and β-glucans and are bound to cellulose by hydrogen bonds. Their structures vary greatly in the walls of different cell types and different plant species. Xyloglucans (XGs) are the principal other non-cellulosic polysaccharides of dicotyledonous primary cell walls (91), accounting for up to 20–25% of the primary cell walls (92). The main backbone is composed of 4-linked β-D-glucosyl residues, most of which are linked to α-D-xylosyl residues of side chains with α-(1, 9)-glycosidic linkage and these xylosyl residues can be linked to fucose, galactose, and less commonly arabinose (92).

Fucogalactoxyloglucans have been found in the primary walls of most dicotyledonous plants except for those of solanaceous plants (e.g., potato), which contain arabinoxyloglucans (93). In the walls of the monocotyledon family Poaceae (grasses), the xyloglucans do not contain arabinose and fucose (94). The content of xyloglucans in members of the Poaceae family is small, accounting for only 2–5% of the walls (95).

Heteroxylans include arabinoxylans, glucuronoxylans, and glucurono-arabinoxylans. They have a backbone of 4-linked xylose residues with short side chains of arabinose, glucuronic acid and 4-O-methyl-glucuronic acid residues (96). The xylose residues in the backbone may be O-acetylated, and in the Poaceae the arabinose residues may be esterified with ferulic or p-coumaric acid (96, 97). Heteroxylans are present in small proportions in the primary walls of dicotyledons, but they are abundant in the Poaceae. Cellulose microfibrils in the walls of Poaceae may be interlocked mainly by glucuronoarabinoxylans (GAXs). Unbranched GAXs are cross-linked with each other or to cellulose via hydrogen bonds. Branched GAXs are unable to form crosslinking between two GAXs or GAX to cellulose since arabinose and glucuronic acid side groups prevent the formation of hydrogen bonds (95). Galactoarabinoxylans have 4-linked xylan backbone with α-L-arabinofuranose residues and/or short chains containing arabinose and galactose attached at C3- or C2- positions. They are found in perennial ryegrass (98, 99) and cocksfoot grass (Dactylis glomerata) leaves (98).

Heteromannans galactoglucomannans (GGM) are found widely in a small amount, and they have a backbone of (1, 7)-linked β-D-mannose and β-D-glucose residues.

(1,3:1,4)-β-Glucans or written as(1 → 3), (1 → 4)-β-glucans are also known as mixed-linked glucans or β-glucans and consist of unbranched and unsubstituted chains of (1,3)- and (1,4)-β-glucosyl residues with varying ratio of (1,4)-β-D-glucosyl residues to 1,3)-β-D-glucosyl residues (100). They have been found in the primary cell walls of Poaceae, and contain about 30% 3-linked residues and 70% 4-linked residues (101). In cereal grains, they occur particularly in the walls of the aleurone, and starchy endosperm and their contents vary markedly, largely depending on species (102), and these polysaccharides are particularly abundant in barley (103). (1,3:1,4)-β-Glucans are thought to form a gel-like matrix in cell walls between the reinforcing cellulose microfibrils (102).

Czerkawski and Breckenridge (52) conducted in vitro rumen simulation technique (RUSITEC) incubations with 26 carbohydrates. These carbohydrates resulted in similar CH₄ production per unit of carbohydrate fermented, except for rhamnose. Rhamnose resulted in distinguishable CH₄ production, but this monosaccharide is only a minor component in plants (44). However, carbohydrates differed in their extent and rate of fermentation: glucose, fructose and sucrose were fermented rapidly; L-arabinose, xylose galactose, and mannose were fermented at an intermediate rate; and glucuronic acid, galacturonic acid, and fucose fermented at a slow rate (52). Carbohydrates with a slow fermentation rate have a higher probability of escaping the rumen without being fermented (104), which could result in lower CH₄ emissions. Pacheco et al. (105) speculated that the escape of soluble cell contents could explain why CH₄ produced per unit of digestible organic matter was less in forages with a greater content of water.

Sucrose concentration is generally low in forages, but it can be high in some feeds, for example, as the main storage carbohydrate in some roots like those of fodder beet. Its fractional fermentation rate in the rumen was about 1,200–1,404%/h (106), which is much higher than for starch [e.g., 30%/h for barley starch (107)]. In a sheep trial conducted by Huhtanen and Robertson (108), with 400 g/kg DM of sugar-beet pulp replaced by sucrose, maize starch or xylose, CH₄ emissions were similar for sheep supplemented with sucrose, maize starch or xylose [6.6% of gross energy (GE)], but lower than those fed sugar-beet pulp (7.1% of GE intake). Sucrose was more rapidly fermented than steam-flaked maize starch in vitro at two pre-determined pH levels (pH 6 and 7) (109). Methane emissions per unit of degraded organic matter (OM) were similar for sucrose and starch at pH 6, but more CH₄ was produced from sucrose than from starch at a pH of 7. At pH 6, sucrose led to a higher molar proportion of propionate and less butyrate than starch, while at pH 7, the fermentation profile was similar for both sucrose and starch. At both levels of pH, sucrose produced more CH₄ and more SCFA (109). These findings suggest that pH in the rumen will interact with carbohydrate type leading to varying levels of CH₄ production in response to carbohydrate supplementation.

Golder et al. (110) found that supplementation of the diet with crushed triticale grain decreased rumen pH and increased total SCFA, acetate, butyrate and propionate concentrations compared with the control and these changes were even more extreme when triticale grain plus fructose were supplemented. Although CH₄ emissions were not measured, the large drop in rumen pH suggests a reduction in CH₄ emissions could be expected.

Pectic polysaccharides present in forage are methyl esterified to varying degrees. The hydrolysis of methyl esters from pectic polysaccharides produces methanol (CH₃OH), which is stoichiometrically converted to CH₄ as 4CH₃OH → 3CH₄ + CO₂ + 2H₂O. The theoretical molar ratio of CH₄ produced from methanol (CH₄/CH₃OH) is 0.75. When pectin or methanol was infused into the rumen of sheep, the measured conversion ratio was 0.77, suggesting all methanol infused was converted to CH₄ (111). Thus, forages containing large amounts of highly methyl-esterified pectic polysaccharides might result in higher CH₄ emissions.

Poulsen et al. (54) reported no differences in CH₄ production among sugar beet pectin, maize starch, wheat starch and chicory inulin incubated in vitro (batch culture), but the extent of fermentation may change the final CH₄ production. Nevertheless, pectin resulted in a higher molar proportion of acetate, and a lower proportion of butyrate and inulin lowered acetate and increased butyrate, compared with starch from either maize or wheat. These differences in SCFA suggested that different amounts of hydrogen were produced and consequently would have led to different amounts of CH₄ released per unit of fermented substrates. In the RUSITEC-based study by Zhao et al. (112), CH₄ emissions were lower with inulin than with starch, and inulin resulted in a reduced molar proportion of acetate and acetate: propionate ratio and increased butyrate proportion, which suggests a potential for reduced CH₄ emissions by feeding inulin. Consisted with this result, rumen fermentation of fructose, which is a product of inulin degradation in the rumen, resulted in less acetate and more butyrate compared with starch in steers (113).

In pectic polysaccharides, HG is a major component and has a backbone of galacturonic acid residues esterified with methyl groups to different degrees. The cleavage of methyl groups from HG may release methanol which is used for CH₄ formation by some microbes (111). Geerkens et al. (114) compared mango peels rich in pectin, de-pectinised mango peels, apple pectin and citrus pectin in vitro (batch culture) mixed with hay (Table 2). Mango peels produced more CH₄ than hay when expressed as either CH₄/gas production (GP) or CH₄/SCFA, while de-pectinisation of mango peels resulted in reduced CH₄, suggesting that pectin may cause an increase in CH₄ production. However, CH₄/GP dropped, and CH₄/SCFA was similar or dropped when citrus or apple pectin was incubated compared with hay. This suggests that the structure of pectic polysaccharides affects CH₄ formation. This result is different from the finding of Czerkawski and Breckenridge (52) in which the degree of methyl esterification was not considered. When high and low degrees of methyl esterification were compared, low esterified pectin produced less CH₄ than high esterified pectin for both apple and citrus pectins (114). This supports the hypothesis that methyl group cleavage from pectin leads to increased CH₄ formation, with methanol as an intermediary.

Between 22 and 94% of dietary starch eaten is digested in the rumen depending on total starch intake and starch source (115). The size and structure of starch granules and the pattern of enzyme-resistant crystalline starches may affect their degradation rate in the rumen (73). For example, starch from potatoes is degraded faster than that from barley and oats (115).

Dairy cows fed diets containing lucerne hay and concentrate (45:55 w/w) based on wheat grain had lower CH₄ yield (11.1 g CH₄/kg DMI) compared with cows fed concentrates based on maize grain (19.5 g/kg DMI) (116). Average daily ruminal pH was similar between cows fed either type of grain, but the duration when ruminal pH was below 6 was twice as long for wheat grain supplemented cows compared with maize grain supplemented cows. Wheat and maize have different starch structures, leading to faster degradation for wheat starch than for maize starch with values of 103.8%/h for wheat grain and 8.1%/h for maize grain reported by Moharrery et al. (115). The faster degradation rate of wheat starch might result in a longer duration of pH below 6, which in itself may inhibit methanogen activity and therefore contribute to lower CH₄ emissions in cows fed wheat grain as found by Moate et al. (116). Lactating dairy cows fed diets based on grass-clover/maize silage (60% diet DM) and either maize cob silage, highly rumen digestible rolled barley grain or sodium hydroxide wheat grain, with a high rumen escape starch (at 25% of diet DM), had similar CH₄ yield and total tract starch digestibility (117).

Besides starch type, grain processing also affects the degradation rate of starch in the rumen (73), which might alter the rumen fermentation profile and rumen pH and could therefore change CH₄ emissions. Lactating dairy cows fed diets with 60% grass silage and 40% concentrate containing either slowly degradable native maize grain (5%/h) or rapidly degradable gelatinised maize grain (16%/h) at either 270 or 530 g/kg concentrate had similar CH₄ yield and rumen pH but an increased rate of starch fermentation and increased level of starch reduced CH₄ produced per unit of estimated rumen-fermentable organic matter (118).

Thus, the amount of starch digested in the rumen and the rate of digestion will affect the rumen fermentation pattern and rumen pH, whilst starch escaped from the rumen has little chance to be converted to CH₄ (Figure 3). The proportion of starch degraded in the rumen, the rate of degradation in the rumen, and rumen escaped starch need to be considered when determining the CH₄ mitigation potential. The complex interactions between the rate of rumen fermentation, pH and the passage rate of starch from the rumen could have resulted in inconsistent results reported in the literature attempting to link the supplementation level of concentrates with the amount of CH₄ emissions. Rumen passage rate is affected by many factors including supplement concentrates in the diet, whereas it could result in a shift of dissolved hydrogen and consequently rumen fermentation pattern and CH₄ emissions (20). Another aspect to consider is the effect of feeding concentrates on fibre digestion. Supplementation of large amounts of grain suppresses the degradation of fibre, which will result in less hydrogen produced and, therefore, less CH₄. For instance, a recent study conducted by Bougouin et al. (119) showed that cows fed with starch-rich diets based on grass silage has comparatively lower enteric CH₄ emission than fibre-rich diets based on grass silage. The main reasons for reduced methanogenesis may be a reduction of the rumen protozoa population (by 36%) and a shift in rumen fermentation toward propionate at the expense of butyrate. However, increasing the level of starch should be carefully applied as a high amount of starch feeding can lead to ruminal acidosis. According to Nozière et al. (120), the chemical treatment of grains can be used to prevent rumen acidosis by reducing the digestibility of the starch inside the rumen. Furthermore, introducing buffers (e.g., sodium bicarbonate or plant buffering capacity) to starch-based diets reduces the prevalence of rumen acidosis by stabilising rumen pH and improving feed digestion (121, 122).

Hindrichsen et al. (53) compared feeds with different major carbohydrates represented by different substrates including oat hulls (lignified fibre), soybean hulls (non-lignified fibre), apple pulp (pectin), sugar beet pulp (hemicelluloses and pectin), guar gum (galactomannan), Jerusalem artichoke tubers (fructan), molasses (sucrose) and wheat (starch). They were incubated in RUSITEC fermenters mixed with forage (a mixture of maize silage, grass silage and hay) at a ratio of 1:1, on a DM basis, and CH₄ emissions (mmol CH₄/g degraded OM) were lower for oat hulls, guar gum and wheat than for molasses, with other supplements intermediate (Table 3). Multiple-regression analysis of CH₄ emission data with chemical composition revealed a negative correlation with lignin and a positive correlation with total sugars (53). The same diets, except sugar beet pulp and guar gum, were fed to 12 Brown Swiss dairy cows with forage: concentrate ratios of 1: 1 (123). Methane emissions per unit of DMI were only lower for cows fed oat hulls (Table 3), which might have resulted from a lower digestibility due to the high degree of lignification of fibre in oaten hulls. Additionally, CH₄ emissions per unit of DM were numerically 10% lower for cows fed the Jerusalem artichoke tubers than cows fed wheat. When emissions were expressed as per unit of digested OM, the difference among diets disappeared (123).

Methane emissions were 16% higher for sheep fed 1.0 kg molassed sugar-beet pulp (containing 166 g sugars and 166 g cellulose) than for sheep fed 1.0 kg chopped hay (125 g sugars and 302 g cellulose) (124). Methane emissions were 8% higher for lactating dairy cows fed grass-clover silage based diet supplemented with molasses (g/kg diet DM, 250 g starch, 32 g sugar, 300 g NDF) vs. wheat grain (g/kg diet DM, 5 g starch, 240 g sugar, 278 g NDF) (125). Giger-Reverdin and Sauvant (126) analysed a CH₄ emission dataset from male sheep fed 22 different feed classes, which resulted in the separation of feeds into four CH₄ yield groups being: high—peas and faba beans; medium—grains, sugar beet pulp and soybean meal; low—silages, hays and straw; very low—distillers grains. However, there was substantial variation in CH₄ yield within feed class, and the proportion of the test feed in the diet and type of basal forage diet were not mentioned.

A recent study conducted by Jonker et al. (127) found that dry cows grazing fodder beet (Beta vulgaris) supplemented with perennial ryegrass based pasture silage (6 kg DM/cow/day) produced 28% lower CH₄ yield (g/kg DM intake) than cows that grazed forage kale (Brassica oleracea) supplemented with barley straw (Hordeum vulgare) (3 kg DM/cow/day). Fodder beet has a very high concentration of readily fermentable carbohydrates, which may affect rumen fermentation and thereby reduce the CH₄ emission. Børsting et al. (122) found that feeding sugar beet molasses instead of wheat increased enteric CH₄ production and yield. This might have been because a high sugar concentration in sugar beet molasses increased the proportion of butyrate in rumen liquid leading to greater H₂ formation, which will increase the enteric CH₄ production (20) when the rumen pH is maintained at the normal level (109).