Fiber in forage is a major source of dietary energy and affects intake and digestibility of forages, but less than 50% of the fiber is readily digested and utilized by ruminant animals [31]. Neutral detergent fiber (NDF) describes highly digestible cell fiber material. Forages with high NDF concentrations and lignified cell walls limit feed intake [32] because the animal feels “full”. The NDF concentration increases rapidly during a rapid decrease in dry matter digestibility after alfalfa matures beyond the vegetative stage. Acid detergent fiber (ADF), on the other hand, describes cell wall material that either is hard to digest or never digests. Forage legumes have more rapidly digestible cell wall than grasses of similar maturity, but the potential extent of cell wall degradation in legumes is generally low [33]. Legume cell walls contain more pectin but less cellulose and hemicellulose compared to grasses [34] which accounts for the initial rapid digestion in legumes. Grasses have vascular bundles distributed throughout the parenchyma of stem cross-sections, whereas the vascular tissues in legumes form a discrete and continuous ring around the stem which expands through cambial activity [35] that impedes ruminal microbe access for digestion. Histological staining for pectin and lignin indicated that tissues may differ more dramatically in their cell wall composition in legumes than grasses [22,36]. Within grass, leaf tissue makes up the greatest proportion of the plant. These leaves have thin-walled, non-lignified mesophyll tissue [37], and mesophyll tissue is found to be completely degraded by rumen microbes [38]. In Alfalfa, where the proportion of stem tissues is larger than grass, the fiber has higher lignin content and thus lower digestibility (40–50%) compared to high fiber digestibility (60–70%) in grasses [31]. Alfalfa has been reported to contain greater lignin and lesser cell wall concentration than grass when alfalfa and grass had the same digestibility [39].

A previous study [22] divided alfalfa stem components into four major categories in relation to cell wall development: chlorenchyma, cambium, secondary phloem, and primary xylem parenchyma consisting of thin, non-lignified primary walls. The pith parenchyma with thin-walled tissue undergoes little cell wall thickening and lignification after stoppage of stem elongation while the collenchyma, epidermis and primary phloem tissues form thick primary walls without lignin at the end of stem elongation. The primary phloem and secondary xylem undergo thickening of secondary cell wall and are highly lignified, after stem elongation is ceased. Alfalfa stems contain diverse tissue types that include thin, non-lignified walls; minimal wall thickening with lignification; thick walls that do not lignify; and thick cell wall that lignify [22]. Tissues which are non-lignified and pectin-rich, such as collenchyma, are considered as rapidly and completely degradable compared to tissues which are lignified and xylan-rich, such as secondary xylem fiber [40]. The non-lignified wall are completely degradable regardless of the thickness, but lignified tissues have variable degradable pattern based on the lignin distribution in the cell wall [19]. Less than 10% of the cell wall has been found to be degradable in alfalfa with thick primary and secondary wall of xylem fiber [19]. Non-lignified epidermis, collenchyma, chlorenchyma, cambium, and primary xylem parenchyma were found to rapidly and completely degrade within the first 8 h of fermentation [19]. Non-lignified secondary walls of the primary phloem fiber completely degrades in 24 h, while the lignified pith parenchyma and secondary xylem fiber were 9.1 to 65.5% degradable even after 96 h, and the primary and secondary xylem vessels were completely nondegradable [19]. However, in grasses, the thick and lignified sclerenchyma tissue were found to be extensively degradable when fermented for 48 h with rumen microbes [36].

Grass cell walls are more degradable than legume cell walls during 72 h to 96 h fermentation [33,41]. Cell wall degradability may be negatively affected by the cross-linkage of matrix components far greater than lignin concentration alone [42]. Although non-lignified alfalfa stems degrade two to five times faster than nonlignified mesophyll grass tissues, lignified alfalfa stem tissues degrade less when compared to reported lignified grass stem sclerenchyma [19]. The differences of cell wall degradation between alfalfa and grass tissues could be associated with the cell wall lignification and polysaccharide composition of individual tissues.

Dietary NDF, regardless of plant source, have been identified as predictors of enteric methane (CH₄) production. As digestibility of fiber increases, so does intake and ruminal fermentation, resulting in increased methane production [43]. However, the effect of carbohydrate type (structural or non-structural) on methane production is relatively less important at low intake levels [44]. Studies that have tried to explain how the NDF intake affects methane emissions have been inconclusive. Some independent studies have found that changes in NDF impact methane production without changes in intake [45,46], while others have found no difference in methane production for either changes in intake or NDF [47]. A meta-analysis of trials investigating NDF and methane in beef cattle found that NDF content alone does not explain enteric methane emissions. However, the quality and intake of the feed does impact methane emissions [48]. Therefore, by improving digestibility and nutritive value resulting in improved feed efficiencies will result in a reduction of methane emissions.

Cell walls make up 23 to 90% of the plant mass [49] and are composed mainly of cellulose, hemicellulose, lignin, and other components, such as pectin and protein. Cell wall digestibility is variable and is negatively related to lignin concentration which is the primary limiting component of cell wall digestion [42]. Cell wall fibers that have high lignin and are linked to other structural carbohydrates are negatively associated with dry matter intake, dry matter digestibility, and animal performance [50].

Cellulose constitutes the largest portion of cell wall accounting for 40 to 50% of plant dry matter [51]. The yield of the stem and concentration of cellulose in the cell wall component of forage (fiber fraction) increase as alfalfa matures [40]. Although increases in stem mass and cellulose concentration in the cell wall theoretically increase the potential yield of digestible energy [26], the increase in lignin deposition during stem maturation and tight linkages of lignin with cellulose microfibrils substantially reduce microbial degradation of lignocellulosic biomass [52,53]. Rumen microflora have less than 50% access to plant fiber fractions [21] because of the heteromatrix complex formation between low-digestible lignin and high-energy cellulose [54]. The digestibility of cellulose is reduced when cellulose and lignin form these complex structures [55]. However, cellulose and hemicellulose are completely degraded when they do not have any bound lignin [56].

Hemicellulose is the second largest component of the alfalfa cell wall, constituting 15 to 20% of the forage dry matter [57]. Hemicellulose digestibility is more affected by cellulose digestion than lignin, despite the fact that hemicellulose and lignin being covalently linked [58]. Grass hemicellulose is more digestible by ruminants compared to cellulose, whereas the reverse is true for legumes [59]. Hemicellulose is characterized by complex structures built using monosaccharides such as xylose, galactose, mannose, and arabinose. Xylans have the slowest rate and extent of digestion [19,60] of all the hemicellulose components. A very strong negative relationship between xylose concentration and alfalfa in vitro degradability was reported indicating the vital role of xylan in inhibiting alfalfa digestibility [61]. Although xylans in legume cell walls are slowly degradable, its concentration in legumes is less than that in grasses [40]. A study found that xylan from alfalfa cell walls was the least digestible cell wall carbohydrate by sheep [62] and the same can be assumed for cattle. Low digestibility in mature grasses could be due to the linkage of xylan and lignin [62] that occur during plant maturity. No information is available about the effect of binding between xylan and cellulose on the degradation of these carbohydrates in the rumen [63].

Lignin composes approximately 15% of the total dry matter of the alfalfa cell wall component, which limits cell wall degradation and forage digestibility by rumen microbes [64,65]. Lignin binds cellulose [66], xylose, arabinose, and mannose of heteroxylans of hemicellulose [67,68], which increase as alfalfa matures [69]. Cell wall digestibility is limited by lignin through the combination with cell wall components [70,71] and through the inhibition of phenolic acids such as p-coumaric acid, ferulic acid, and sinapic acid on rumen microbes [66,72,73]. These acids have inhibitory effects on rumen fungi, which limits the ability of fungi to degrade fibers in alfalfa and bermudagrass (Cynodon dactylon L. Pers.) [74]. Jung and Fahey [73] found that in vitro digestibility of cellulose and starch was depressed when supplied with p-coumaric, ferulic, salicylic, and vanillin acids. Some studies stated that lignin is indigestible [75,76,77,78] owing to the lack of known ruminal anaerobic fermentation [79], whereas others found that lignin is partially digestible in the abomasum with little change in the intestines [80]. During ruminal digestion of lignin, methane (22–29%), and carbon dioxide (65–69%) gases are produced, suggesting that ruminal digestion is relatively efficient compared to anerobic digesters [81]. Some studies have divided lignin into non-core lignin and core lignin monomers or polymers and number of covalent bonds [18,82]. Non-core lignins are monomers [83] and normally have one covalent linkage between a phenolic compound and either core lignin or hemicellulose [84], whereas core lignins are condensed and polymeric [85] and usually have two or more covalent bonds between monomers within its molecule [83]. Depending upon the lignin covalent links with carbohydrates, lignin can protect about 1.4 times its own mass of cell wall carbohydrates from microbial fermentation [86]. Another study found that lignin can protect two times its own mass of cell wall carbohydrates from microbial fermentation [87].

Alfalfa stems contain a variety of tissues with different patterns of cell wall development. The process of lignification varies between plant tissues. Some tissues, such as mesophyll in leaves, never lignify, whereas secondary xylem and other tissues accumulate high concentrations of lignin. About 6–9% of the dry weight of the whole alfalfa plant and 20% of the cell wall is lignin [88]. The lignin pathway was altered in alfalfa through decreasing the expression of two genes involved in the biosynthesis of coniferyl and sinapyl alcohol, which are considered as the main building blocks of lignin [28]. These changes in lignins were able to reduce 20% lignin within the plant which resulted in 2–5% increase in digestibility of the tissue. In contrast, conventional breeding takes more than 15 years of selection, which has resulted in a 2–3% increase in cell wall digestibility [89]. The relationship between in vivo dry matter digestibility and lignin was found to be significantly negative in alfalfa, when measured as acid detergent lignin [90].

Pectin is a non-fibrous carbohydrate that accounts for 10–12% of the cell wall matrix in alfalfa stems. Pectin is completely digestible and is the cell wall polysaccharide most rapidly degradable by rumen microbes [60,91,92,93]. Selectively increasing easily digestible carbohydrates that make up the alfalfa cell wall, such as pectin, is a method to increase carbohydrate availability and hence improve protein utilization and alfalfa digestibility by ruminants [94,95,96]. About 20–35% and 1.0–10% of extractable pectic polysaccharides on a cell wall basis are present in legumes and in grasses, respectively [57]. Alfalfa leaves contain higher pectin concentrations than stems [60,97], and pectin concentration declines as stems mature [32,97]. However, pectin does not lower ruminal pH via lactate production, an intermediate product derived from microbial starch catabolism in the rumen, despite being a readily degradable source of energy in the rumen [98,99].

Neutral detergent soluble fiber (NDSF), in which pectic polysaccharides are the predominant components, can be used to estimate pectin concentration in alfalfa [97]. Variation in NDSF has been found in alfalfa [97,100]. Similarly, significant genetic variability for NDSF was reported in two alfalfa populations [101,102]. In addition, NDSF concentration was found to be negatively correlated with NDF, ADF, and acid detergent lignin (ADL) concentrations, whereas it positively correlated with in vitro dry matter digestibility (IVDMD) in alfalfa [102]. Similar results were found related to genetic improvement for NDSF concentration in five alfalfa populations, in which NDSF was found to be negatively correlated with total cell wall concentration (CW), and proportions of neutral detergent fiber (NDF), cellulose, and lignin in the CW, and positively correlated with crude protein concentration and IVDMD [103].

As a perennial plant, alfalfa may proceed from the vegetative stage to seed production multiple times per growing season and over multiple years. The mass and height of alfalfa increases with plant maturity and the latter is associated with an increase in the number of stem internodes [40]. While the diameter of stem internodes continues to increase with plant maturity, the elongation of stem internodes ceases after approximately 21 days according to some reports and remains stable, or decreases in length over time [22,40]. The alfalfa leaf-to-stem ratio declines over time as stems proliferate with branching and defoliation due to leaf shading and foliar diseases. A defoliation event, such as grazing and mechanical harvest, can effectively reset alfalfa growth back to the vegetative stage.

In tandem with plant morphological development, the patterns of cell wall development of the different alfalfa stem tissues have been observed using microscopy. Alfalfa stem tissues follow different developmental pathways as they mature, which have implications for the degradability of stem cell walls. While some tissues, such as thin-walled chlorenchyma and thick-walled collenchyma, remain non-lignified and thus completely degradable in the presence of rumen microbes at all maturity stages; others like primary phloem fibers and pith parenchyma become lignified once stem elongation ceases and cambial activity begins [19,22,40]. Lignification occurs only in tissues with thickened secondary walls, including pith parenchyma, primary phloem fibers, and xylem tissues, with lignin deposition beginning in the primary cell wall and then proceeding into the secondary cell wall [22]. Xylem primary and secondary tissues immediately lignify making them impervious to degradation by rumen microbes [40]. Secondary xylem tissues arising from cambial activity comprise an increasing proportion of the stem as alfalfa matures, which helps to explain why stem lignin content increases with maturity [40]. At the same time, the concentration of pectin decreases while cellulose and hemicellulose concentrations increase in stem tissues contributing to the decline in alfalfa stem digestibility [40].

Two common ways to increase digestibility of fiber in alfalfa are conventional breeding [31] and genetic engineering as reduced-lignin types [29]. Conventional breeding that used selection traits targeting the stems rather than the total biomass was found to have a greater impact on alfalfa digestibility owing to the presence of highly lignified fiber in stems [104]. Several studies have successfully used traditional breeding methods and genetic techniques to improve the digestibility in alfalfa cultivars. For example, the Hi-Gest alfalfa trait was developed by conventional breeding to improve total forage digestibility through increased leaf to stem ratio [105,106]. Conventional breeding improved fiber digestibility in alfalfa stem cell walls without diminishing dry matter yield, where populations were developed by recurrent phenotypic selection and evaluated by enzyme-released glucose as a proxy trait for fiber digestibility, indicating that selection was effective [107]. Alfalfa plants differed in stem NDF and ADL as a proportion of NDF (ADL/NDF) in another breeding program, in which plants were identified with either low or high rapid and low or high potential IVNDFD [25,30]. In contrast, the HarvXtra alfalfa (reduced lignin) was developed by genetic modification to improve nutritive value mainly through altering ADL and in vitro neutral detergent fiber digestibility (IVNDFD) in the stems [108,109]. Similar leaf-to-stem ratio and biomass yield between HarvXtra and conventional alfalfa cultivars was found through the evaluation of morphological characteristics in a single-location study [109]. One study conducted in multiple locations found higher leaf-to-stem ratio and increased stem digestibility but lower biomass yield in HarvXtra compared to conventional alfalfa [110]. Alfalfa breeding programs should emphasize stems when comparing nutritive value among whole plants, leaves, and stems because of the presence of the larger value of narrow-sense heritability for feeding-value traits in the stems [111], and they are the rate-limiting step for overall forage digestibility [23].