Maximizing the Feed Value of High Fibre Forages to Ruminants: The Impact of Ammonia Fibre Expansion Technology (AFEX) and Novel Recombinant Fibrolytic Enzymes on Digestion and the Rumen Microbial Population

Introduction

The world population is expected to increase by ~2 billion, reaching 9.7 billion in 2050 (UN, 2019). Compared to 2012, this growth of the global population and affluence of emerging economies is predicted to promote a 40 to 55% increase in global demand for meat and dairy products by 2050 (FAO, 2018). Land resources available to expand production is limited, and there is increasing societal pressure to reduce the degradation of natural resources. In addition, animal production, in particular beef production, has been criticized based on the use of grain that could be used as food for humans, the use of arable land to produce animal feed (instead of producing food for humans directly), and its contribution to greenhouse gas (GHG) emissions (Mottet et al., 2017). Hence, meeting this increase in the demand for livestock products will require more sustainable production practices and efficient utilization of feeds.

Lignocellulosic cereal crop residues, such as straw and corn stover, are low-cost and widely available forage sources; however, their utilization in ruminant diets is limited due to their low digestibility (< 500 g/kg; Beauchemin et al. 2019). Cereal crop residues have intrinsically low feed value because cereal crops are grown mainly focusing on maximizing grain yield, with little consideration for the residual of forage portion of the crop. Cereal crop residues are characterized by having high fibre (> 70% NDF) and lignin (> 7.0% ADL), and low protein (< 6.5% CP) content (Beauchemin et al. 2019), traits associated with its low digestibility, and consequent low feed intake (Wilson and Mertens, 1995). The cell wall carbohydrates (fibre) are the main component of these cereal crop residues accounting for up to 80% of the organic matter, and the extent of degradation by rumen microorganisms of this fraction is directly related to the amount of energy available for animal production (Jung and Allen, 1995). These carbohydrates, particularly hemicellulose, are associated with a variety of side groups including acetyl, ferulic/coumaric acid and glucuranoic acids, with covalent linkages to lignin. The lignin that links to these carbohydrates is not degraded in the rumen as this process requires oxygen, which is lacking in the rumen. Hence, the lignin/phenolics complexes reduce digestibility by restricting the access of rumen microbes and enzymes to the carbohydrates within plant cell walls.

Development of technologies to maximize the conversion of low quality forages, like cereal crop residues, into energy within the rumen has the potential to improve the overall efficiency of ruminant production systems from both economic and environmental standpoints. This review will focus on two technologies, ammonia fibre expansion (AFEX) and recombinant fibrolytic enzymes, to improve the ruminal fibre digestion of low quality forages. Many studies in the last 60 years have evaluated the ability of alkali treatments (NaOH, NH₃, Ca(OH)₂, KOH, and CaO) to improve ruminal digestion of corn cobs and stalks, and wheat, barley, rice and oat straws (Klopfenstein and Owen, 1981). However, variability in the results, and safety, environmental, technical, and economical concerns have limited the widespread adoption of alkali treatment of forages for ruminants. Similarly, much work has been done evaluating the effect of adding fibrolytic enzymes to ruminants diets with responses found to be inconsistent, an outcome that has also limited the adoption of this technology (McAllister et al., 2001; Beauchemin et al., 2003; Adesogan et al., 2014; Meale et al., 2014; Arriola et al., 2017). Recent investments in these technologies for the production of biofuels from lignocellulose, and advancements in our ability to study the rumen microbiome and express novel recombinant enzymes have created new opportunities for improving the utilization of low quality forages by ruminants.

Ammonia Fibre Expansion Technology (AFEX)

Process

Originally developed for the pre-treatment of biomass for cellulosic ethanol production, AFEX is an ammonia thermochemical pre-treatment of recalcitrant lignocellulosic biomass. The AFEX treatment is a dry-to-dry process where steam and alkaline anhydrous ammonia are applied to the fibrous substrate in a reaction chamber at high pressure (200-400 psi) and temperature (80-150°C) for < 1 h, followed by a rapid pressure release and ammonia recovery by steam stripping (Mor et al., 2018). The potential health and safety hazards associated with ammonia volatilization, that have limited the use of traditional ammoniation on farm, have been greatly improved by the development of AFEX technology as most (∼98%) of the ammonia is recovered for re-use (Teymouri et al., 2005; Mor et al., 2018). Similar to other alkali treatments, AFEX treatment of cereal crop residues can promote cleavage of the hydrogen, α-ether and ester bonds between lignin and hemicellulose, and the linkages between hydroxycinnamic acids (i.e,. p-coumaric and ferulic acids) and hemicellulose, consequently disrupting the plant cell wall structure (Xiao et al., 2001). By decreasing the crystallinity and promoting the swelling of cellulose, alkali treatments promote the attachment of rumen microbes to plant cell walls, a process which is essential for ruminal fibre degradation. Compared to other alkali treatments, AFEX is a more intensive process (Bals et al., 2010), that partially solubilizes hemicellulose, enhances the fragility of the fibre, and increases the surface area available for microbial attachment. This has been hypothesized to improve the accessibility of carbohydrate-active enzymes (CAZymes) to the biomass, increasing the rate and extent of ruminal fibre degradation (Beauchemin et al., 2019).

As a result of ammoniation, crude protein concentration of AFEX-treated cereal residues has been shown to increase anywhere from 81 to 312% compared to the untreated forages (Bals et al., 2010; Blümmel et al., 2018, Beauchemin et al., 2019; Saleem et al., 2019; Passetti et al., 2020). The greater CP content with AFEX treated forages can reduce the need for protein supplementation in the diet of ruminants, as this non-protein N can be used to meet the soluble N requirement of the rumen microbes and contribute to microbial protein synthesis (Scott et al., 2011). The solubilisation of hemicellulose by AFEX is evident by the reduction in NDF without any changes in ADF content. Our previous studies have shown that NDF content of AFEX treated crop residues was reduced by 15 to 24% (Beauchemin et al., 2019; Saleem et al., 2019; Passetti et al., 2020).

Rumen Microbial Population

Mammalian herbivores, including ruminants, do not express the cellulolytic or hemi-cellulolytic enzymes that are involved in degrading ingested plant material. Herbivores rely on a complex gut microbiome composed of a diverse range of bacteria, fungi, and protozoa to perform this function. The rumen microbiome is comprised of bacteria (up to 10¹¹ cells/mL), protozoa (10⁴ -10⁶ cells/mL), fungi (10³ -10⁶ zoospore/mL), methanogens (10⁶ cells/mL) and bacteriophages (10⁷ -10¹⁰ particles/mL) that have evolved to specialize in degrading lignocellulosic biomass (Morgavi et al., 2013). The ruminant host is dependent on the enzymes produced by this microbial community to breakdown complex fibrous substrates and convert them into fermentable sugars.

When feed enters the rumen it is rapidly colonized by bacteria and fungi (Edwards et al., 2008; Piao et al., 2014; Huws et al., 2016). There have been extensive studies on the process of feed colonization (McAllister et al., 1994; Edwards et al., 2008; Huws et al., 2013; Huws et al., 2016). The currently accepted model of feed colonization was proposed by Huws et al. (2016) and involves a multi-step process. The epiphytic microbes attached to the surface of feed are rapidly displaced by rumen bacteria within the first hour that feed enters the rumen. This leads to the formation of a stable, “primary colonizing” community within the first 4 hours feed is in the rumen. This diverse primary colonizing community metabolizes readily accessible carbohydrates and nutrients in feed (Huws et al., 2016; Terry et al., 2020). As these resources are depleted, some members of the primary community are displaced and a less diverse “secondary colonizing” community develops. The secondary colonizing community is specialized in the degradation of recalcitrant carbohydrates locked within a cellulose and hemicellulose matrix (Piao et al., 2014; Huws et al., 2016; Terry et al., 2020). Most studies examining feed colonization in the rumen have employed metagenomics or metataxonomic approaches focusing on changes in the makeup of the microbial community. Most recently, a study used metatranscriptomics to examine the biological function of the colonizing communities and found that microbial sub-populations co-operate, compete and rely on each other during the colonization of feed in the rumen (Huws et al., 2021). The secondary colonizing community expresses high levels of CAZymes involved in the degradation of structural carbohydrates whereas the primary colonizers display a diverse range of metabolic processes. Interestingly, the dominant microbes that are found in both stages of digestion were observed to alter biological activity over time and displayed metabolic plasticity in response to the changes in nutrient availability that result as feed is degraded in the rumen (Huws et al., 2020). This work has shed light on the complexity of niche specialization displayed amongst rumen microbes.

Our team has examined how AFEX treatment of a variety of crop residues impacts the rumen microbial community and feed colonization in the rumen (Passetti et al., 2020; Ribeiro et al., 2020; Terry et al., 2020). Terry et al. (2020) found that colonization of crop residues was biphasic regardless of feed pretreatment with AFEX technology or fibrolytic enzymes. AFEX pretreatment was found to alter the function and composition of the primary colonizers, but not the secondary colonizers. The AFEX treated forages had an increased abundance of bacteria and pathways associated with simple carbohydrate degradation, reiterating that AFEX treatment increases the availability of a diverse range of polymers found in hemicellulose, pectin, and potentially lignin that can be rapidly metabolized by bacteria in the primary colonizing community. A related study examining AFEX treated rice straw also observed alterations in early colonization (Passetti et al., 2020). The bacterial community colonizing AFEX-treated rice straw was more similar to that colonizing high quality alfalfa hay as compared to untreated rice straw. The similarity between the microbial community colonizing alfalfa and AFEX-rice straw is reflective of the fact that AFEX increased the rapidly digestible fraction of rice straw (Passetti et al., 2020). As the duration of incubation increased the composition of phyla colonizing all forages became more similar, again illustrating that AFEX appears to alter the early stages of fibre digestion in the rumen. Ribeiro et al. (2020) examined the effect of replacing alfalfa hay with AFEX treated wheat straw on the metabolism and performance of sheep. Unlike the studies focusing on the colonizing communities, the impact of AFEX on the rumen microbiome were not as obvious. Intriguingly, the similarity between the bacteria associated with these feeds indicate that AFEX enhanced the digestibility of wheat straw to a level comparable to high quality alfalfa hay. AFEX also impacted the protozoal population in the rumen as it increased the total rumen protozoa counts in lambs as compared to an alfalfa hay diet. This observation was suggested to be due to the sensitivity of rumen protozoa to decreases in rumen pH below 5.6 (Dehority, 2003). The higher level of protozoa associated with the diet containing AFEX treated straw was attributed to a higher minimum rumen pH (5.44 vs. 5.38) and a reduction in the amount of time ruminal pH was below 5.6 (0.59 vs. 1.03 h/d) compared to the alfalfa diet (Ribeiro et al., 2020).

Digestion and Performance

Bals et al. (2010) observed that AFEX was much more effective at increasing digestion of late harvest switchgrass as compared to traditional ammoniation methods (206% vs. 56% increase, respectively). Blümmel et al. (2018) investigating the impact of AFEX on 10 different crop residues, using a ruminal batch culture in vitro method, observed that the mean apparent DM digestibility increased by 28% (493 vs 630 g/kg) after 24 h of incubation. Using the rumen simulation technique (RUSITEC) we observed that DM disappearance of barley straw improved 35% (62.4% vs. 46.2%) and total daily VFA production improved by 17.5% (58.5 vs. 49.8 mmol/d) with AFEX (Griffith et al., 2016). Similarly, AFEX treatment of wheat straw increased DM (69.6 vs. 38.3%), NDF (65.6 vs. 36.8%), and ADF (61.4 vs. 36.0%) disappearance and total daily VFA production (53.7 vs. 38.2 mmol/d) and the molar proportion of propionate, while it decreased the proportion of acetate and the acetate-to-propionate ratio in the RUSITEC (Saleem et al. 2019). These results show that the impact of AFEX treatment of cereal residues on DM and NDF digestibility is approximately 2 times greater than that reported for traditional ammoniation of cereal straws (~15%; Fahey et al., 1993).

Beauchemin et al. (2019) evaluated the impact of AFEX on the in situ ruminal degradability of four crop residues (barley, wheat and rice straw, and corn stover). Effective ruminal NDF degradability (+28 to +126% depending upon the crop residue) was greatly increased by AFEX, mainly by increasing the potentially degradable fraction and decreasing the undigested fibre fraction. The AFEX treatment of the crop residues also increased the NDF fractional rate of degradation with the exception of barley straw, and decreased the undigested NDF (uNDF) fraction by 48 to 78% (Beauchemin et al., 2019). Passetti et al. (2020) also observed similar effects, with AFEX doubling the in situ ruminal DM and NDF degradability of rice straw after 48 h of incubation. The substantial increase in the NDF slowly degradable fraction (B) and potentially degradable fraction (A+B), and the decrease in uNDF shows the remarkable ability of AFEX to increase the proportion of NDF available for digestion in the rumen. The uNDF values observed by Beauchemin et al. (2019) for AFEX treated crop residues (i.e.,< 200 g/kg) were similar to values reported for high quality roughage such as alfalfa hay, corn silage and beet pulp (Soufizadeh et al., 2018). The uNDF is the NDF fraction of the diet that is not digestible by rumen microbes, even if its residency within the rumen is infinite (Huhtanen et al., 2007). As intake by ruminants fed high forage diets is often controlled by rumen fill and the rate of disappearance, a higher uNDF intake limits feed intake. The use of uNDF content of forages and diets as an indicator of physically effective NDF, gut fill, digestion, passage dynamics and intake has received increasing interest, and nutritional models have included it as an important factor in defining the intake and digestibility of forages (Ellis et al., 1999; Van Amburgh et al., 2015). Lower uNDF content of diets can decrease gut fill, increase feed passage rate through the rumen and increase intake. However, it can also decrease the physically effective fibre content and rumination time (Cotanch et al., 2014). Thus, diets containing AFEX crop residues may also need to incorporate other sources of roughages as physically effective fibre to ensure optimum rumen function and prevent ruminal acidosis.

Animal feeding trials with AFEX-treated forages are scarce. Initial studies conducted by Weimer et al. (2003) replaced 7% of the alfalfa hay in the diet of lactating dairy cows with AFEX treated rice straw and observed a 1.3 kg increase in milk production (38.3 vs. 39.6 kg/d). More recently, Mor et al. (2018) replaced wheat straw with increasing quantities of AFEX treated wheat straw pellets in a high forage diet (70% wheat straw DM basis) for lactating Murrah buffalo and KaranFries cattle. The AFEX diets increased cows milk production, with no changes in body weight. Buffalo fed a wheat straw diet lost body condition, while buffalo fed AFEX treated wheat straw did not. Including 50% of the diet DM as AFEX treated wheat straw pellets in the diet as substitute for untreated wheat straw increased total tract DM digestibility from 52.4 to 61.4% (17.2% increase) and milk production from 7.5 to 9.4 kg/d (25.3% increase). Mor et al. (2018) concluded that AFEX treatment of wheat straw increased the energy available for lactating dairy cattle without effecting palatability.

Passetti et al. (2020) evaluated the growth performance and digestibility of ewe lambs fed pelleted diets with 25% forage inclusion in the diet DM as alfalfa hay, rice straw, or AFEX treated rice straw. Lambs fed rice straw showed similar growth to those fed alfalfa based diets. However, lambs fed AFEX rice straw diets had lower DMI (-9%), ADG (-20%), and feed efficiency (-12%) than lambs fed untreated rice straw diets. AFEX rice straw diets promoted similar DM but higher NDF and ADF total tract digestibility than rice straw diets. This reduction in performance with AFEX treated rice straw were not expected based on the improvements in the ruminal in situ DM and NDF degradability as a result of AFEX treatment of rice straw (Passetti et al., 2020). Untreated rice straw was expected to limit intake as a result of gut fill compared to AFEX rice straw diets. However, this was not observed likely due to the processing of the untreated rice straw, which was ground to pass through a 4 mm screen and pelleted, reducing the particle size and its residence time in the rumen. This most likely reduced the negative feedback loop on intake as a result of rumen fill, increasing the DMI and ADG of lambs fed rice straw diets.

In a second study, we looked at the effect of replacing alfalfa hay pellets with AFEX wheat straw pellets (30% of the diet DM) on the digestibility and performance of lambs (Ribeiro et al., 2020). An increase in DMI of 7.4%, and improved ADG in the first 42 d (327 vs. 304 g/d), but not over the entire feeding period (~94-d) was observed by feeding AFEX wheat straw diet compared to an alfalfa based diet (298 and 305 g/d, respectively). As a result, feed efficiency for the full feeding period was reduced (5.7%) for the AFEX straw diet compared to the alfalfa diet. Digestibility of DM was similar between diets, but digestibility of CP was reduced, and despite the higher fibre content of the AFEX diet, the digestibility of NDF and ADF increased as AFEX replaced alfalfa. There was no difference between diets in final BW as lambs were fed to a constant weight of 50 kg. However, days on feed were longer for the alfalfa-based diet than the AFEX diet (97 vs. 91 d). The fact that lambs fed AFEX wheat straw exhibited similar performance to the alfalfa diet (high quality forage), attests to its ability to improve the nutritional value of low quality forages such as straw for ruminants.

Acetamide

During the AFEX treatment of crop residues some acetamide (CH₃CONH₂) is formed by the reaction of acetyl linkages within the plant cell wall with ammonia (Chundawat et al., 2010). Acetamide is a concern as it was shown to cause liver cancer in rats when consumed at high levels for prolonged periods (WHO, 1999). Currently, acetamide can be naturally found in products such as milk, beef and thermally processed foods such as roasted coffee beans at concentrations of up to 0.4 mg/kg (Vismeh et al., 2018). Acetamide has only recently been considered a food contaminant but the maximum levels of contamination in food that are safe for human consumption have not been defined be regulatory agencies (Bals et al., 2019). Concentration of acetamide in AFEX treated cereal crop residues was shown to range from 4 to 7 mg/g of DM (Bals et al., 2019). However, the amount of acetamide in AFEX treated cereal residues are much lower than those shown to increase the incidence of liver carcinoma (23.6 mg/g of DM) in rats (Jackson and Dessau, 1961; Fleischman et al., 1980).

Acetamide is slowly metabolized by microorganisms (Arner, 1964). Mor et al. (2019) raising the possibility that ruminal bacteria could metabolize acetamide to acetate and ammonia, reducing the risk of it accumulating in meat or milk. The results from our previous study agree with this concept, as blood plasma acetamide concentrations were reduced after 2 weeks of feeding AFEX diets to lambs (Ribeiro et al., 2020). However, in our study the concentrations of acetamide in the diaphragm of lambs after slaughter were still greater for AFEX (10.1 mg/kg) compared with the control treatment (1.7 mg/kg). In a second study, we observed that acetamide in blood plasma greatly declined by removing AFEX rice straw from the diet of lambs 7 days before slaughter, but the concentration in the diaphragm only declined slightly (2.7 vs 2.1 mg/kg; Passetti et al., 2020). These studies show that the concentration of acetamide in lamb is increased by feeding AFEX diets, although the levels are far below those that were found to cause cancer in rats (Fleischman et al., 1980).

Recombinant Fibrolytic Enzymes

Selection Process

Taking advantage of the advancements in our ability to study the rumen microbiome (e.g. nonculture techniques) and express novel recombinant enzymes, we have recently conducted a large project aiming to address some of the issues that limit the effectiveness of fibrolytic enzymes in ruminant diets. In this project we used the current knowledge of the various carbohydrate degrading enzyme families [glycoside hydrolase families (GH); i.e. families of enzymes involved in the hydrolysis and/or rearrangement of glycosidic bonds] in the rumen and our previous experience with enzyme activities associated with increased ruminal fibre degradation, to screen and select effective pure recombinant fibrolytic enzymes that would work under ruminal conditions (i.e. pH, temperature, and synergism with a mixture of ruminal enzymes). The recombinant enzymes were first selected on the basis of synergy with rumen enzymes using a high throughput in vitro microassay (Badhan et al., 2014) that measured sugar release (glucose + xylose) when enzymes were incubated with barley straw. The best candidates were then screened using a standard ruminal batch culture technique and a semi-continuous culture system, Rumen Simulation Technique (RUSITEC), for their ability to increase the degradation of barley straw (Ribeiro et al. 2018). Based on this work we selected a recombinant enzyme that was then expressed and purified in large quantities to conduct in vivo studies with cattle (Ran et al. 2019) and sheep (Ribeiro et al. 2020).

Rumen Microbial Population

Few studies have been conducted to examine the impacts that the inclusion of enzymes in ruminant diets has on the rumen microbiome. It is possible that inclusion of enzymes may alter the availability of substrates for the rumen microbes and/or expose areas on the feed surface for colonization by rumen microbes. In our work to develop a recombinant feed enzyme adapted for the rumen environment we examined the impact of feed enzymes on feed colonization, rumen metabolism and animal performance (Ribeiro et al., 2020, Terry et al., 2020). Pretreatment of crop residues for 24 h prior to feeding did not alter the colonization process of either untreated or AFEX treated corn stover or barley straw (Terry et al., 2020). The addition of an enzyme mixture to an artificial rumen system also did not result in large shifts in the rumen microbiome; however, there was a significant change in the abundance of the bacteria Ruminococcus flavefaciens, and F. succinogenes, two important rumen fibre degrading bacteria (Saleem et al., 2019). In a study with growing lambs, the addition of a recombinant xylanase was found to alter the abundance of rumen bacteria and rumen protozoa (Ribeiro et al., 2020). It was observed that Christensenellaceae, a bacterial family with an important role in biofilm (i.e. syntrophic consortium of microorganisms that associates to each other and attaches and colonizes the surface of the substrates to promote digestion) formation and rumen degradation of starch and fibre (Mao et al., 2015; De Mulder et al., 2017), increased with enzyme treatment (Ribeiro et al., 2020). There have also been studies that have observed changes in the composition of the rumen archaeal community when enzymes are included in the diet (Zhou et al., 2011). Fermentation of rice straw with a mixture of Bacillus subtilis, Enterococcus faecalis, cellulase, and xylanase for 14 d was observed to alter the composition of the rumen microbiome and increased acetate and propionate levels in the rumen (Hu et al 2020). The pretreatment of feeds with enzymes has been long suggested to promote the release of reducing sugars and other hydrolysis products, promoting chemotactic response that stimulate microbial attachment to feed particles (Cheng and McAllister, 1997; Beauchemin et al., 2003; Giraldo et al., 2007). We have previously observed increased bacterial colonization of feed in an in vitro rumen simulation system as a result of pretreatment of diets with enzymes (Wang et al., 2001; Ribeiro et al., 2015, 2018). Additionally, our recent work shows that the profile of bacteria attached to feed could be modified as a result of enzyme treatment (Ribeiro et al., 2020). This enzyme effect on the rumen microbial community and on the specific microbes involved in biofilm formation isstill not well understood. It is likely that the enzyme and the method of enzyme application has an influence on the efficacy of treatment. The variability in the numbers and composition of the bacterial community attached to feedstuffs during ruminal digestion may help explain the variation in animal responses observed when feedstuffs are pretreated with enzymes. Future studies aimed at examining the impact of feed enzymes on ruminants should continue to evaluate their impact on biofilm formation and the rumen microbiome. A better understanding of this process may help develop enzyme treatments that can promote the attachment of ruminal microbes and enhance feed degradation in the rumen.

Digestion and Performance

In our recent work using a microassay, 1 recombinant endoglucanase (EGL7A, from GH7) and, 2 recombinant xylanases (XYL10A and XYL10C, from GH10) were selected for further evaluation in ruminal batch cultures and RUSITEC systems (Ribeiro et al., 2018). The selected enzymes consistently increased barley straw degradation in ruminal batch culture, but not in the semicontinuous culture RUSITEC system. Despite the lack of GH7 glycosyl hydrolases in the rumen, supplementation with an endoglucanase from this family did not improve the ruminal degradation of barley straw and suggested that this activity maybe redundant with other enzyme families that are present in the rumen. Only the recombinant enzyme, XYL10A, consistently improved substrate degradation in both batch culture and semi-continuous rumen fermentation systems.

Issues with scaling-up production of XYL10A made us select XYL10C the second best enzyme (best enzyme under the batch culture system) for the vivo studies using sheep and beef cattle. Supplementing the diet of beef cattle with this recombinant xylanase (XYL10C) increased in situ effective rumen degradability of AFEX treated crop residues by ~3%, but not with untreated crop residues (Beauchemin et al., 2019). The enzyme XYL10C did not improve total diet or fibre digestibility in wethers or beef heifers (Ran et al., 2019; Ribeiro et al., 2020), despite the fact that it was specifically selected for this trait in laboratory assays. XYL10C improved the ADG and G:F of lambs for the first 28 d of the performance study, but these improvements were not maintained throughout the feeding period (~94 d; Ribeiro et al., 2020). The rumen is often considered the most efficient biological system for degrading fibre (Flint et al., 2008), and making further improvements to this system presents a major challenge. The results from this lamb performance study suggest that this enzyme may promote growth in lambs early in the feeding period, but not after they were fully adapted to the diets. More studies looking at fibrolyitic enzyme application into feedlot beef cattle receiving and transition diets, and in high producing lactating dairy cow diets would be interesting to test this hypothesis.

Additive Effect of AFEX and Enzymes on Feed Digestion

The combination of physical/thermochemical pretreatment and enzyme application to fibrous substrates has been used to maximize sugar release before conversion to bioethanol and also to enhance fibre digestion in ruminants (Eun et al., 2006; Wang et al., 2004; Alvira et al., 2010; Sarks et al., 2016). Using different crop residues (barley straw, corn stover, rice straw, and wheat straw), we recently evaluated the potential of AFEX treatment of these residues and the application of a recombinant xylanase (XYL10C) to improve in situ ruminal degradability (Beauchemin et al., 2019). This study demonstrated the tremendous potential of AFEX treatment to improve the ruminal degradation and the feeding value of crop residues, and that the xylanase increased the effective rumen degradability of AFEX treated crop residues by an additional 3%. The increase in the effective rumen degradability AFEX crop residues by enzyme application was due to an increase in the rate of fibre degradation, but it did not increase the total potentially degradable fibre proportion. Enzyme application did not improve the rumen degradability of the untreated crop residues. The recalcitrance of the fibre in untreated crop residues likely hindered microbial and enzyme access to substrate, thereby limiting fibre degradation. This study showed that AFEX promoted greater accessibility of the exogenous enzyme to its substrate, consequently improving fibre digestion (Beauchemin et al., 2019). This work also shows the potential of combining these technologies to maximize the ruminal degradation of these feed residues.

Future Challenges and Conclusions

Although we have shown that AFEX technology can greatly improve the digestion and the feed value of low quality crop residues there is still much work to be done to scale up this technology to a commercial level. Developing central industrial facilities that can receive and process crop residues and produce AFEX treated feedstuffs in a cost effective manner presents a challenge, but the increased use of forages could increase the sustainability of the ruminant livestock industry.

Development of recombinant fibrolytic enzymes specific for ruminant diets has proven to be a challenging task. Our work suggests that the use of fibrolytic enzymes may have greater success in the earlier stages of the feeding period (< 30 d) when the ruminal environment is not fully adapted to the diets. The ruminal microbial community has evolved over millions of years and due to its redundancy, resilience, and ability to adapt to different feedstuffs, the development of new enzymes that can further improve ruminal fibre digestion is not a simple task. New recombinant rate limiting fibrolytic enzymes may only be effective until the next rate limiting enzyme starts to impede fibre digestion. A greater understanding of the ruminal microbial biofilm and the attachment process, and how enzymes could be used to manipulate this process may be a way to increase the efficiency of enzyme-based technologies in the future.

Combining different technologies to improve fibre digestion, like AFEX and recombinant enzymes, can maximize the energy extraction from recalcitrant fibre sources and likely improve the productivity, profitability, and sustainability of ruminant livestock systems.

Presented at the 2021 Animal Nutrition Conference of Canada. For information on the next edition, click here.