Using Conventional Ruminant Techniques and Molecular Spectroscopy to Study the Impact of Additive Fibrolytic Enzymes and Maturity Stage on Nutritional and Molecular Structural Changes of Legume and Legume-Cereal Intercropped Silage

Abstract

This chapter aims to I) provide research background and motivation on the impact of additive fibrolytic enzyme and maturity stage at harvesting on molecular structural changes and nutritional value of the cool-season legume silage and legume-cereal intercropped silage; II) provide recent research progress and development in whole plant faba bean (legume) silage and faba-oat (legume-cereal) intercropped silage. The reviewed projects include: I) effect of adding different levels of additive fibrolytic enzymes on utilisation of cool-season whole plant faba bean silage in ruminants to find an optimal dose level for this faba silage; II) effect of adding different levels of fibrolytic enzymes on utilisation of cool-season intercropped whole plant faba-oat (legume-cereal) silage in ruminants; III) effect of maturity stage at harvesting on nutritive quality of whole plant faba silage; IV) effect of frost damage on nutritive quality of whole plant faba forage in ruminant; V) feeding trial and dairy production performance, milk yield (ECM, FCM, fat yield etc.) with whole plant faba legume silage in early lactating cows to replace traditional barley and corn silages; VI) availability and utilisation of whole plant faba silage and intercropped whole plant faba-oat intercropped silage in ruminants; VII) using molecular spectroscopy to study nutrition and structure interaction of faba silage at cellular and molecular levels. Based on the scientific findings presented in this chapter, the following most important conclusions can be drawn: cool-season faba (legume) variety with different tannin levels impact not only nutrient profiles but also protein and carbohydrate-related molecular structure makeup. Additionally, the nutrient supply, bioenergy, degradation, digestion, and metabolic characteristics of cool-season faba silage and intercropped faba-oat silage were highly related independently and synergistically to molecular structure conformation. Furthermore, the nutrient utilisation and availability of cool-season faba silage and intercropped silage in ruminant livestock systems could be accurately predicted by the protein and carbohydrate molecular structures revealed with cutting stage vibrational molecular spectroscopy when they work together. Additive fibrolytic enzyme and maturity stage at harvesting significantly impacted both nutritional and molecular structural changes of legume and legume-cereal intercropped silage. Dairy production performance and milk yield (ECM, FCM, fat yield, etc.) studies showed that whole plant faba legume silage in early lactating cows could be used as an alternative silage to traditional barley and corn silages. The information described in this chapter gives better insight into cool-season legume silage and legume-cereal intercropping silage research progress in terms of inherent molecular structures, nutritive quality, animal production response, and molecular structure and nutrition delivery interactive relationship as well as impact by maturity stage and dosage levels of additive fibrolytic enzymes in the cool-season legume silage and intercropped legume-cereal silages.

1. Introduction

As new cool-season faba bean varieties (high-tannin, low-tannin, and zero-tannin) are developed and available in western Canada and production has been increasing in recent years [1, 2, 3, 4], utilisation of this faba legume as forage hay or silage is possible. To our knowledge, no systematic study on the nutrition quality of these cool-season whole crop faba beans as hay and silage has been found, and there is no study on whole crop faba and whole crop faba-oat intercropping silages from other research teams. Also, there is no research on the impact of maturity cutting stages: flower stage, mid-pod stage, and late-pod stage, on the feed nutritive quality of whole crop faba (legume) as hay and silage, as well as whole crop faba bean-oat intercropping forage (as silage or as hay) in dairy, sheep, goat, and beef cattle (all type of ruminants).

Recently, innovative mixed fibrolytic enzymes (FE) have been used to improve feed nutritive value and utilisation in dairy and beef cattle by increasing polysaccharide or feed fibre degradability in the rumen and digestibility in the whole gastrointestinal tract [5, 6]. These innovative fibrolytic enzymes are able to release trapped polysaccharides after breaking down chemical functional group bonds (e.g. ester bonds and ether bonds) between lignin and polysaccharides through hydroxycinnamic acid and ferulic acid bridges in the complex plant cell wall. However, there is no study on the optimal dosage of these innovative enzymes on whole crop faba (legume) silage and whole crop faba-oat (legume-cereal) intercropping silage in the literature [7].

Advanced synchrotron-radiation and Globar-sourced vibrational (micro)spectroscopy is capable of revealing internal structure features at cellular and molecular levels and simultaneously provides four kinds of important information: chemical composition, molecular structure, environment, and chemistry, within intact tissue with a highly spatial resolution [8, 9, 10, 11, 12, 13, 14, 15]. However, to date, these advanced vibration molecular techniques are still seldomly known to animal and feed scientists, particularly synchrotron technology. There is no study on using these advanced molecular spectroscopic techniques in legume silage (e.g. whole crop faba bean silage) and legume-cereal intercropped silages (e.g. whole crop faba bean-oat silage) in literature from other research groups.

The objective of this chapter is to provide research background and motivation on impact of additive fibrolytic enzymes and maturity stage at harvesting on molecular structural changes and nutritional value of the cool-season legume silage and legume-cereal intercropped silage; provide recent research progress and development in whole plant faba bean (legume) silage and faba-oat (legume-cereal) intercropped silage in ruminant system [4, 7, 16]. The information described in this chapter gives better insight into legume silage and legume-cereal intercropping silage research progress in molecular structure, nutrition and molecular structure interactive relationship, and animal production response to these cool-season legume silage or cool-season legume-cereal silage.

2. Recent research and progress in cool-season whole crop faba legume silage

2.1 Recent study on the effect of cutting stage and tannin content on nutritive quality of cool-season whole crop faba legume silage

Information regarding the utilisation of the cool-season whole crop faba legume silage in beef and dairy cattle is extremely limited [4]. Our team member, Guevara [4] carried out systematic studies to reveal the impact of faba legume tannin contents and newly developed cool-season varieties from Crop Development Centre (CDC, University of Saskatchewan) (Snowdrop variety with low-tannin content; SSNS-1 genotype with high-tannin content) and the impact of maturity cutting stages (at 88-days faba legume mid-pod stage; at 97-days faba legume late pod stage) on faba legume forage yield, nutrient profiles, bio-energy content (TDN, ME, and NE value) [17, 18], protein and carbohydrate subfractions and nutrient supply evaluated with the Cornell Net Carbohydrate and Protein System (CNCPS) [19, 20], rumen fermentation kinetics [21, 22, 23], potential rumen available nitrogen (ED_N) to rumen available energy (ED_E) synchronisation and degradation ratios [24, 25], intestinal digestibility of primary nutrients [26], metabolic characteristics [17, 22], and predict production performance in term of feed milk value (FMV) when utilisation of cool-season whole crop faba legume silage in lactation dairy cattle.

Our team member, Guevara [4] found that the yield on a dry matter (DM) basis of cool-season whole crop faba legume silage cut at the faba flower stage was lower than that at the late-pod stage (7.34 vs. 12.20 tons per ha). Guevara also found that there was much higher in the rumen pH, ammonia (NH₃) production, and volatile fatty acid (VFA) in terms of rumen acetic acid and propionic acid at the faba flower stage in the cool-season whole crop faba legume silage than that at the faba legume mid-pod stage and late-pod stage (5.39 vs. 4.35 and 4.51; 16.32 vs. 6.62 and 5.66% of total N; 6.33 vs. 2.35 and 1.70% DM; 1.44 vs. 0.04 and 0.06% DM, respectively). There was no significant difference in crude protein (CP) content among different cool-season silage varieties and among all three different maturity stages (CP: 22% of DM). However, there was no difference in net energy of lactation (NE_L3x) for dairy cows in cool-season silage when cut faba legume was at mid-pod and late-pod stages, but they were higher than cool-season faba legume silage when cut at flower stage (1.45 and 1.46 vs. 1.13 Mcal/kg DM). Starch content in cool-season faba legume silage was higher when cut at faba legume late-pod stage than that cut at faba legume mid-pod and flower stages (17.2 vs. 9.4 and 1.3% of DM). For fibre content in the cool-season faba legume silages, there was no difference between mid-pod and late-pod stages, but much lower when cut at faba flower stage (36.0 and 34.4 vs. 45.3% DM). The rumen degradation study by Guevara [4] showed that the rumen undegraded protein (RUP^NRC) was higher when cut at faba late-pod stage and rumen undegraded/bypass starch (BSt) was higher when cut at faba mid-pod stage (33 vs. 25 and 32 vs. 18 g/kg DM, respectively).

The results from Guevara’s [4] study in N to energy synchronisation showed a lower rumen available N to rumen available carbohydrates (ED_N/ED_CHO) overall ratio when cutting faba legume at the late-pod stage in comparison with faba silage cut at the mid-pod stage (−35 g/kg). The intestinal absorbable faba protein (IADP) and the total tract digested faba starch (TDST) were higher (84 vs. 61 g/kg CP and 175 vs. 95 g/kg DM, respectively) when cut at late-pod stage. Both the DEV/OEB and NRC protein systems showed that there was a lower in total truly digestible protein supply (DVE value: 59 vs. 68 g/kg DM), total metabolizable protein (MP: 67 vs. 73 g/kg DM) and feed milk value based on the DVE (1.20 vs. 1.37 kg milk per kg DM faba silage) or MP value (1.36 vs. 1.48 kg milk per kg DM faba silage) when faba legume silage was cut at mid-pod stage than that when cut at late-pod stage.

Then Guevara [4] concluded that in order to obtain high yield and high feed nutritional values, the cool-season faba legume forage should be harvested at the late-pod stage. In this late-pod stage, cool-season faba legume silage showed high predicted production performance. Therefore, the cool-season whole crop faba legume silage when cut at the late pod stage has the highest nutritional value and greatest potential to be used as an alternative ingredient in dairy and beef rations.

2.2 Recent study on the effect of frost damage on feed quality of cool-season whole crop faba legume forage for ruminants

Frost-damaged faba bean plants often happen due to the cold weather in western Canada. What nutritional value and how to utilise this frost-damaged faba bean is a question. Therefore, recently, our team, Guevara [4] systematically evaluated the impact of faba silage tannin contents (high level vs. low level vs. zero) and cool-season faba cultivars (CDC developed Snowdrop variety with lower level tannin content; CDC SSNS-1 variety with a high-tannin content) on physiochemical nutrient profiles, bioenergy value (TDN, ME, NE) for dairy and beef cattle [17, 18], faba protein and carbohydrate CNCPS subfractions and CNCPS nutrient supply [19, 20], rumen fermentation and degradation kinetics of rate and extent [21, 22, 23], potential rumen available N to rumen available energy synchronisation and hourly effective degradation ratios [24, 25], intestinal digestion of nutrients [26], metabolic characteristics (e.g. DVE, OEB, and MP values) [17, 22], and predicted animal production performance in terms of feed milk value of the cool-season frost-damaged whole crop faba legume hay harvested at 114 days of maturity stage [4].

In these studies, Guevara [4] reported that compared with cool-season low-tannin frost-damaged whole crop faba legume hay, the high-tannin frost-damaged faba legume hay was higher in organic matter and lower in acid detergent insoluble crude protein (ADICP: +2.5%DM and − 0.4%DM). However, there was no difference in starch and crude protein (CP) content at this maturity stage between the high- and low-tannin varieties with an average of 11.9% DM and 16.8% DM, respectively. The bio-energy values in terms of TDN, ME, and NE for dairy and beef cattle were also not different. However, there was a higher in fibre-bound protein (PB2) and lower indigestible protein (PC, +2.3 and − 3.1% CP) in the high-tannin frost-damaged faba hay than that in the low-tannin frost-damaged faba legume hay.

Rumen kinetic study [4] showed that the cool-season frost-damaged low lignin faba hay was higher in rumen bypass or undegraded protein with a RUP of +2.8% and lower in rumen undegradable neutral detergent fibre (NDF) fraction (U, −5.7%) compared to the frost-damaged high lignin faba hay. The intestinal phase study [4] showed that there was higher in various nutrient supplies and predicted production performance from the cool-season frost-damaged high lignin faba hay than that in the cool-season low lignin frost-damaged faba hay in terms of intestinal digested rumen undegraded DM (IDBDM, +15 g/kg DM), total metabolizable protein (MP, +4 g/kg DM), intestinal digestibility of rumen bypass or undegraded feed protein (dIDP, +7%), and feed milk value (FMV^NRC, +0.09 kg milk per kg DM faba hay).

Then Guevara [4] concluded that compared with the non-frost damage cool-season faba hay [4], both frost-damaged cool-season high- and low-tannin faba hay were lower in feed quality and nutritional supply at 114 days than the non-frost damage cool-season faba hay when cut at the faba flower stage (77 days), faba mid-pod stage (88 days), and faba late-pod stage (97 days). However, within the frost-damaged cool-season faba forage, the cool-season frost-damaged high-tannin faba hay which was harvested at a growth stage of 114 days had superior feed quality and nutritional value than that in the cool-season frost-damaged low-tannin faba hay which was harvested at the growth stage of 114 days in western Canada.

2.3 Recent studies in feeding trial and dairy production performance and metabolic characteristics with cool-season whole crop faba legume silage in high producing cows to replace conventional barley and corn silage

How to feed the cool-season whole crop faba legume silage and what animal production performance in comparison with conventional barley and corn silage in high lactating dairy cows are still not known from the literature. Therefore, our team [4, 27] conducted a dairy trial experiment to determine the impact of 50% and 75% partial silage replacements (T50, T75) in dairy cows’ rations and 100% complete silage replacement (T100) in dairy cows’ rations containing conventional corn and barley silages with the cool-season low-tannin Snowdrop variety of whole crop faba legume silage cut at faba late-pod stage (97 days old) on early lactating dairy cows (high production) in terms of dairy production performance of milk yield and components, DM feed intake and feed-milk efficiency, N balance, intestinal digestibility, rumen degradation and fermentation features, and metabolic characteristics as well as dairy cow feeding behaviour. This experiment that we used was a double 4 × 4 Latin square design (LSD) with four non-cannulated and four cannulated lactating cows). Each period of LSD lasted 25 days, including adaption and sampling collection.

Guevara et al. [27] and Guevara [4] reported that our results showed that the dairy cows fed T100 with 30.60% cool-season whole crop faba legume silage produced higher fat corrected milk yield (3.5% FCM) and higher energy corrected milk yield (ECM) than the cows fed a control diet of T0 with 18.37% corn silage +12.23% barley silage (+4.35 and + 3.48 kg/cow/d, respectively), but there was no significant difference in FCM and ECM when lactating dairy cows were fed T50 with 9.18% corn silage +6.12% barley silage +15.30% cool-season whole crop faba legume silage and fed T75 with 4.59% corn silage +3.06% barley silage +22.95% cool-season whole crop faba legume silage.

Our results also showed that when lactating dairy cows fed diets containing cool-season whole crop faba legume silage (T50, T75, T100) in comparison with control T0 produced higher milk fat yield (2.11 vs. 1.89 kg per cow per day). A feed efficiency study [4, 27] showed when lactating dairy cows consumed T75, FCM/DMI was higher than the lactating cows when consumed control T0 diet (2.21 vs. 1.91). There was no difference in starch digestibility of lactating dairy cows among the three cool-season faba silage-containing diets: T50, T75, and T100, but they were all lower than control T0 diet without any faba legume silage (92.65% vs. 96.13%).

Our dietary energy study results showed that the cow diets contained or included the cool-season whole crop faba legume silage (T50, T75, T100 diets vs. control T0 diet) significantly increased the total diet energy (1.91 vs. 1.65 Mcal per kg DMI), percentage of energy for cow body weight gain and total milk production (78.3 vs. 75.5% of total energy). The study showed similar rumen fermentation features in ammonia, VFA, and pH) among all the treatment diets (T0, T50, T75, and T100).

Then our team, Guevara et al. [27] and Guevara [4], concluded that the dietary inclusion of cool-season whole crop faba legume silage, which was cut at faba late-pod stage improves both fat and energy corrected milk yield, and also increases milk fat yield, and improves efficiency (FCM/DMI) without negatively affecting the DM intake. Consequently, our study [4, 27] showed that the cool-season whole crop faba legume silage cut at faba late-pod stage is a highly nutritive alternative feed which can improve dairy cow production performance in western Canada. In this feeding trail, we, Guevara et al. [27] and Guevara [4], also studied cost–benefit in terms of the income over feed cost (IOFC). The results showed a superior benefit to dairy farmers when using cool-season whole crop faba legume silage cut at late-pod stage to replace conventional barley and corn silages in high producing dairy cows [4].

3. Recent research and progress in cool-season whole crop faba and whole crop oat intercropping hay and silage

3.1 Recent study on the effect of maturity stage/cutting time on yield, chemical, and nutrient profiles, predicted production performance of cool-season whole crop faba and whole crop oat intercropping legume-cereal hay

The high protein and high starch content in whole crop faba legume forage make them suitable for ruminant diets. There is very limited information regarding the utilisation of cool-season whole crop faba legume hay for ruminants [16]. Therefore, our team member, Nagy [16], conducted experiments to study the impact of intercropping cool-season whole crop oat-faba for hay and the impact of the cutting stage on yield, chemical composition, and bio-energy profile [17, 18], protein and carbohydrate CNCPS fractions and CNCPS nutrient supply [19, 20], rumen degradation kinetics [21, 22, 23], N to energy synchronisation and degradation ratios [24, 25], intestinal digestibility [26], metabolic characteristics (e.g. MP, DVE, and OEB) [17, 22], and predicted dairy production performance of cool-season whole crop oat-faba (legume-cereal) hay. In our study [16], the oat and faba plant were intercropped and grown in three fields in Saskatchewan, Canada, and were cut at three growth stages for hay and silages: Cutting stage 1 with the oat plants at the inflorescence stage and the faba bean plants at the flat pod stage; Cutting stage 2 with the oat plants at the milk development stage and the faba bean plants at the milk pod stage; Cutting stage 3 with oat plants at the soft dough stage and the faba bean plants at the late pod stage.

The chemical compositions of cool-season whole crop faba-oat hay were determined using standard chemical analysis methods [28]. Bio-energy values and total digestible nutrients (TDN) were evaluated using the NRC chemical approach [17, 18], protein and carbohydrate subfractions and CNCPS nutrient supply [19, 20] were determined using the updated CNCPS 6.5 system. The rumen degradation was carried out using a standard in situ technique [21] with rumen cannulated lactating cows at our dairy research facility (RDTRF, Saskatoon, University of Saskatchewan, Canada). The rumen available N to rumen available energy potential synchronisation and hourly effective ED_N to ED_OM ratios were evaluated using Tas et al.’s method [24] developed by Wageningen University and Research, The Netherlands. The intestinal digestion was evaluated using the modified three-step in situ and in vitro method with pre-in situ 12 h incubation [26]. The truly digestible protein supply (DVE), protein degraded balance (OEB), net energy-based FMV, and metabolizable protein-based FMV [22, 23] were evaluated using both updated DVE/OEB and NRC nutritional systems.

Nagy [16] showed that cutting stages 2 and 3 had a higher DM hay yield than cutting stage 1. With increasing cutting stage, ash and soluble protein (SCP) were decreased from 14.1 to 9.6% DM and 13.3 to 9.8% DM, respectively. With increasing cutting stage, the starch, sugar, and non-fibre carbohydrate (NFC) contents in cool-season intercropped faba-oat hay were dramatically increased from 0.31 to 7.1% DM (starch), 6.6 to 13.0% DM (sugar), and 15.3 to 24.7% DM (NFC). The stage of cutting did not significantly impact NDF, acid detergent fibre (ADF), and acid detergent lignin (ADL). There was no difference among the three cutting stages in the cool-season whole crop faba-oat hay. In the TDN and bioenergy studies, the results showed that with extending cutting stage, the tdNFC, TDN value, NE for lactation, NE for growth, and NE for maintenance in both beef and dairy cattle increased from 12.7 to 22.1% DM, 49.1 to 56.0%DM (TDN), 1.01 to 1.18 Mcal/kg (NE_L3x), 0.51 to 0.69 Mcal/kg (NE_g), and 1.07 to 1.26 Mcal/kg (NE_m), respectively. These results suggest that the cool-season whole crop faba-oat (legume-cereal) hay can be used as a high-quality forage for both beef and dairy cattle in western Canada.

3.2 Recent study on the effect of maturity stage/cutting time on silage yield, chemical profile, energy and protein-based feed milk value and metabolic characteristics of cool-season whole crop faba-oat intercropping legume-cereal silage

Recently, Nagy [16] conducted a systematic study on the impact of maturity cutting stage and the intercropping of cool-season whole crop faba with whole crop oat legume-cereal silage on intercropped silage yield, nutritive value profiles, protein and carbohydrate CNCPS subfractions and CNCPS nutrient supply, bio-energy content, ruminal fermentation kinetics features, rumen available N to rumen available energy synchronisation and hourly effective degradation ratio, intestinal digestion, and truly absorbable protein supply in term of DVE and MP values to dairy cows. The cool-season CDC oat and faba bean were intercropped, grown in three fields, and were cut at three growth stages: Cutting stage 1 with the oat plants at the inflorescence stage and the faba plants at the flat-pod stage; Cutting stage 2 with the oat plants at the milk development stage and the faba plants at the mid-pod stage; Cutting stage 3 with oat plants at the soft dough stage and the faba plants at the late-pod stage. The chemical composition was determined using standard chemical analysis methods (e.g. AOAC), bioenergy values and TDN and its components (e.g. tdNDF, tdCP, tdFA, and tdNFC) were estimated using the NRC summary method, and protein and carbohydrate subfractions were determined using the updated CNCPS 6.5 system. The in situ techniques were used to determine rumen fermentation/degradation kinetic profiles with rumen cannulated lactating cows at our Rayner dairy station (RDTRF) at the University of Saskatchewan, Saskatoon, Canada. The rumen available N to rumen available energy synchronisation in terms of hourly effective degradation ratio - ED_N to ED_OM was determined using a method reported by Tas et al. [24] from Wageningen University and Research. The three-step in situ and in vitro method with pre-in situ incubation 12 h was applied to determine intestinal digestion of primary nutrients. The DVE/OEB system [22, 23] and NRC nutritional model [17] were used to determine the total truly digestible protein supply in the intestine and metabolizable protein (DVE, OEB, MP, etc.) and net energy-based FMV as well as metabolizable protein or DVE based FMV.

Nagy [16] reported that the cool-season whole crop faba-oat silage is higher in protein content in the 2nd and 3rd cutting growth stages than in the 1st cutting growth stage (20, 18 vs. 16% DM). The 3rd cutting stage had the highest TDN value (58 vs. 55, 48% DM). Additionally, the total MP and FMV^NRC were higher in the 2nd and 3rd cutting growth stages compared with the 1st cutting growth stage (MP: 65, 68 vs. 61 g/kg DM; FMV^NRC: 1.31, 1.38 vs. 1.23 kg milk per kg of intercropped silage, respectively). These studies suggest that cutting stages 2 and 3 of cool-season intercropped faba-oat silage resulted in higher nutritive values (TDN, MP) and better predicted production performance.

4. Recent research and progress in feed additive impact on cool-season whole crop faba legume silage and cool-season whole faba-oat (legume-cereal) intercropping silage

4.1 Recent study on the impact of adding innovative fibrolytic enzyme (FE) at different dose levels on short-term and long-term degradability of cool-season whole crop faba legume silage in ruminant systems

Fibrolytic enzymes (FE) can be used to improve nutrient availability in ruminants by releasing cell-wall trapped nutrients in the complex plant cell wall and increasing fibre degradability and digestibility in animals [5, 6]. However, our literature research shows positive and no-effective impacts on dairy cows [29, 30, 31, 32, 33, 34]. These results are due to several impacts such as dosage level, types of enzymes, conditions, diets, etc.

Recently, an innovative mixture of fibrolytic enzymes has been developed and it is able to release polysaccharides from complex cell walls after breaking down chemical bonds between lignin and polysaccharides [35]. Our team members, recently Yang et al. [36] and Yang [7] conducted several experiments to study the impact of adding the innovative fibrolytic enzyme (FE) at different dose levels on DM and NDF fibre degradability of the cool-season whole crop faba legume silage (cv. CDC Snowbird). We used both the Daisy^IIin vitro incubation method and in situ nylon bag method to evaluate the degradability of DM (DMD) and neutral detergent fibre (NDFD), and we also compared these two different methods in evaluating the in vitro degradability.

In our experiments [7, 36], seven doses of innovative fibrolytic enzymes (IFE) were applied to the cool-season whole crop faba silage samples, including 0 (as control), 0.25, 0.5, 0.75, 1, 1.25, and 1.5 mL of FETR per kg DM of cool-season faba silage. Yang et al. [36] and Yang [7] reported that with increasing enzyme dosage levels, DMD was cubically impacted and NDFD was quadratically affected by the innovative fibrolytic enzymes in our in situ animal experiment. In the in vitro study, the dosage level quadratically affected DM degradability and cubically affected NDFD. When comparing the two different methods (in vitro vs. in situ), it was found that there existed a strongly or satisfactory correlationship between in situ and in vitro methods with r = 0.98 for overall DMD and r = 0.84 for overall NDFD.

Both our in vitro and in situ results showed that the DMD and NDFD were greatly impacted by this innovative fibrolytic enzyme in the cool-season whole crop faba legume silage. Although the Daisy^IIin vitro technique showed some inconsistent and had a relatively larger variation when compared with the in situ nylon bag technique, it remains a rapid and useful tool to evaluate a large amount of samples or treatments in DM and neutral detergent fibre degradability with less cost, time, and labour.

4.2 Recent study on the effect of adding innovative fibrolytic enzyme (FE) at different dose levels on rumen fermentation characteristics and degradation kinetics of cool-season whole crop faba legume silage in ruminants

In this experiment, our team member, Yang [7] carried out an in situ animal trial to determine the impact of innovative fibrolytic enzymes (FETR) on DM and NDF fibre rumen fermentation and degradation kinetic characteristics of cool-season whole crop faba legume silage that we developed recently. In our study [7], the in situ animal trial was performed using two rumen cannulated Holstein cows in our dairy research station (RDTRF, the University of Saskatchewan, Canada) and the cool-season whole crop faba legume silage samples were treated with seven dosages of the innovative enzyme, including 0 (as control), 0.25, 0.5, 0.75, 1, 1.25, and 1.5 mL of FETR per kg DM of faba legume silage. In situ, rumen degradation residues and fermentation and degradation kinetics were determined.

Yang [7] reported that the innovative fibrolytic enzyme application linearly decreased DM degradation residue at 0 hour. Significant quadratic effects were observed at 3 hours (short-term) and 24 hours (long-term) of incubation. However, no significant differences in rumen degradation residues were found at other incubation time points. The rumen NDF degradation residue at 0, 6, and 24 hours was quadratically affected with the innovative fybrolytic enzyme addition, and a cubic impact was observed at 48-hour rumen incubation.

With increasing dosage levels of innovative fibrolytic enzyme, the rumen soluble fraction of DM (S_DM) was increased linearly in dairy cows from cool-season whole crop faba legume silage. The NDF rumen degradation kinetics were greatly affected by the innovative enzyme application by increasing the potentially degradable fraction (D_NDF) and effective degradable fibre content (ED_NDF) and reducing the undegradable fraction (U_NDF). Increasing dosage levels linearly increased the sum of washable and degradable (W + D) fractions and, therefore, linearly decreased the undegradable fraction. The dosage level of innovative enzyme also cubically impacted both rumen bypass NDF (BNDF) and effective degradable NDF (EDNDF).

Our results [7] indicated that the innovative fibrolytic enzyme significantly improved fibre fermentation and degradation for the cool-season whole crop faba legume silage. Yang [7] also suggested a further study in the near future to evaluate the impact of pre-treatment of the innovative fibrolytic enzyme derived from Trichoderma reesei on animal production performance (lactation), feeding behaviour, rumen function and metabolic parameters, intestinal and total tract digestibility in highly lactating dairy cows fed the cool-season whole crop faba legume silage as a main source of forage in comparison with conventional barley and corn silage.

4.3 Recent study on the effect of innovative fibrolytic enzyme (FE) at different dose levels on nutrient utilisation of cool-season whole crop faba bean-oat intercropping (legume-cereal) silage in ruminants

Recently, Nagy [16] conducted a study to analyse the impact of dosage level of innovative fibrolytic enzyme derived from Trichoderma Reesei (FETR) on in vitro fermentation and degradation kinetic features of intercropped cool-season whole crop oat-faba silage using rumen cannulated dairy cows. The cool-season CDC oat and CDC faba were intercropped and grown in three fields in Saskatchewan, Canada and were cut at the maturity stage with the whole crop oat at the soft dough stage and the whole crop faba at the late-pod stage of maturity. The degradation kinetics of primary nutrients were estimated using an in vitro technique with rumen liquid from rumen-fistulated lactating dairy cows. The in vitro rumen fermentation features and degradation kinetic characteristics of DM and fibres (both NDF and ADF), including rumen degradation rate (Kd), lag time (T0), potentially degradable fraction (D), rumen undegradable fraction (U), and rumen effective degradable fractions and content (ED) were evaluated. The treatment design was a one-way structure with 5 dosage levels of innovated fibrolytic enzyme (FETR: 0, 0.075, 0.15, 0.225, and 0.3 ml per litre). The experimental design was a RCBD with the dosage level as a fixed effect and animals and in vitro run as random block effects. The in vitro data were analysed using the mixed model procedure in SAS 9.4 with the analysis RCBD model. The orthogonal polynomial contrast (OPC) of SAS was used to study the relationship between dosage levels and in vitro degradation kinetics.

Nagy [16] reported that there were strongly significant interaction effects between enzyme dosage levels and incubation time for the degradability of DM (DDM), degradability of neutral detergent fibre (DNDF), and degradability of ADF (DADF). There was a cubic relationship between enzyme dosage levels and DDM (P = 0.02), a tended linear relationship with DNDF (P = 0.06), and a quadratic relationship with DADF (P = 0.04). The results [16] indicated that the dosage level of innovative fibrolytic enzyme and incubation time had a significantly synergistic effect on in vitro degradability of DM, NDF, and ADF in this intercropped cool-season whole crop oat-faba (legume-cereal) silage.

4.4 Recent study on the effect of fibrolytic enzyme (FE, exogenous) on lactational performance, milk yield (ECM, FCM, fat yield, etc), feeding behaviour, rumen fermentation and digestibility in lactating cows fed cool-season whole crop faba legume silage-based diet

In this study, our team members, Yang et al. [37] and Yang [7] also carried out studies to determine the impact of pre-treating cool-season whole crop faba legume silage based-diet with exogenous innovative fibrolytic enzyme derived from Trichoderma reesei (FETR) on animal production performance (lactation), intestinal and total tract digestibility, rumen degradation and fermentation features, energy partitioning, N balance, as well as eating behaviour in lactational dairy cows. This experiment was conducted with eight lactating Holstein dairy cows (body weight: 710 ± 44 kg; days in milk: 121 ± 17 days) with four different innovative enzyme dosage treatments, including 0 (as a control), 0.5, 0.75, and 1.0 ml of FETR per kg DM of cool-season faba legume silage diet) in a double 4 × 4 Latin square design (2-LSD). The innovative enzyme dosage applied to cool-season faba silage diet in this experiment was selected based on our previous studies. They showed a positive impact on the cool-season whole crop faba legume silage.

Yang et al. [37] and Yang [7] reported that with increasing dosage levels, the NDF digestibility was linearly responsive. The innovative enzyme dosage treatment with 0.5 mL of innovative enzyme per kg of silage DM had the highest NDF digestibility (48.5%) compared with other innovative enzyme treatments. The % milk fat and fat yield were significantly affected by innovative fibrolytic enzyme application. They were linearly differed among the innovative enzyme treatments, being the highest (4.35%, 1.82 kg/d) for low enzyme dosage groups. Compared with the control group (milk yield 41.5 kg/d with %4.35 milk fat), the innovative enzyme treatments linearly affected and tended to affected milk yields in terms of ECM, FCM. The innovative enzyme treatments also linearly impacted the RCM production efficiency (FCM kg per kg of DM intake) and cubically impacted the ECM production efficiency (ECM kg per kg DM intake).

In our studies [7, 37], we demonstrated the positive and beneficial effects of pre-adding and pre-treating the cool-season whole crop faba legume silage with a lower dose level (0.5, 1 mL of FETR per kg DM of cool-season faba silage based TMR) of innovative fibrolytic enzyme to lactation dairy cows which could benefit the development of a new and alternative feeding strategy in western Canada.

5. Recent research in using advanced vibrational (micro)spectroscopy for cool-season legume and legume-cereal silage research at cellular and molecular levels

The silage’s nutritional value and digestive behaviour are affected by not only the chemical composition profile but also molecular structure conformation, and biological component matrix. However, the wet-chemical analysis method fails to reveal internal molecular structure and component matrix due to processing and digestion in wet chemical analysis. Advanced synchrotron-radiation and Globar-sourced vibrational (micro)spectroscopy is capable of revealing internal structure features at cellular and molecular levels and simultaneously provides four kinds of important information: not only chemical composition, but also molecular structure, environment, and chemistry, within intact tissue with a highly spatial resolution [8, 9, 10, 11, 12, 13, 14, 15]. The detailed principle and methodology of using synchrotron-radiation and Globar-sourced vibrational (micro)spectroscopy have been reported before [8, 9, 10, 11, 12, 13, 14, 15].

Recently, our team [2, 3, 4] carried out various studies using advanced molecular spectroscopic techniques, either synchrotron-based [38] or global based molecular spectroscopy to reveal (1) the impact of cool season low-tannin (cv. CDC Snowdrop) and high-tannin faba varieties (cv. CDC SSNS-1); (2) the impact of maturity cutting stage at 88-d mid-pod cutting stage and 97-d late-pod cutting stage on inherent structure spectral profile of cool-season whole crop faba legume silage at a molecular level; (3) investigate the interactive association and relationship between molecular structural profiles and nutrient utilisation and availability in ruminant livestock systems [39].

Guevara [4] applied molecular spectroscopic technique by using ATR-FTIR vibrational spectroscopy to study protein and carbohydrate structure make-up for the cool-season faba legume silages and compare different cool-season faba varieties with different tannin levels. It was found that the cool-season low-tannin faba silage had a higher total carbohydrates (TC) spectral peak area at the late-pod cutting stage than at the mid-pod cutting stage with a difference of +3.45 AU. For the structural carbohydrates spectral area intensity (STC), the cool-season low-tannin faba silage was higher when cut at mid-pod cutting stage (difference: +4.11 AU) than the cool-season high-tannin faba legume silage at late-pod cutting stage.

Amides functional group study [4] showed that the low-tannin silage had decreased the amide I area (−1.40 AU) when cut at mid-pod cutting stage than that at the late pod stage. As to amide II structure profile, the cool-season high-tannin faba legume silage had higher amide II at the late pod cutting stage than the high-tannin silage cut at the mid-pod stage with different +2.50 AU.

We also carried out detailed PCA spectral analyses of all the carbohydrate-related spectral region (ca. 879–1485 cm⁻¹). The 83% of the total variation was explained by PC1. In this region, it includes NSTC, TC, and STC regions. The results showed dramatical difference in the cool-season whole crop faba legume silage when cut at mid-pod stage or cut at late-pod stage. It is interesting to find that starch level in cool-season faba legume silage has a strongly positive correlation with structural carbohydrate peak number four (STC4) spectral height intensity with r = 0.94.

Protein 2nd structure spectral profile study showed that total digestible nutrients TDN, bio-energy value (r = 0.76), and crude protein level (r = 0.62, 0.65) in the cool-season faba legume silage positively correlated to α-helix and β-sheet. The TDN and bio-energy values also strongly positively correlated (r = 0.85) with the amide I spectral area.

The rumen undegradable protein and rumen bypass starch were strongly correlated to the structural carbohydrate spectral peak number one (STC1) height in the cool-season faba legume silage (RUP; r = − 0.82; BSt; r = − 0.84), while, rumen undegradable protein (RUP; r = − 0.83, − 0.90) was strongly negatively correlated with amide I peak height (RUP; r = − 0.83) and STC area (RUP; r = − 0.90), as well as α-helix to β-sheet spectral peak height ratio (RUP; r = − 0.73).

The relationship between molecular structure and intestinal digestions and nutrient supply study showed that intestinal digested crude protein (IADP) and metabolizable protein (MP) in cool-season faba silage were strongly correlated with structural carbohydrates peak # one (STC1) spectral height (IADP; r = −0.90; MP; r = −0.92). For MP value, it also strongly positively correlated to the protein 2nd structure profile in terms of α-helix peak height, β-sheet peak height, and amide I area (r = 0.86, 0.86, 0.71, respectively). The cool-season silage feed milk value based on the DVE value (FMV^DVE) and silage feed milk value based on MP (FMV^NRC) were strongly correlated to cellulosic compound (CEC) spectral area (r = −0.95, −0.82, respectively).

Our results showed that cool-season faba silage starch content could be predicted using α-helix peak heights, amide I, and STC4 with good estimation power (R² > 0.96), but total digestible nutrients, net energy of lactation, and crude protein were predicted by above molecular structure profiles with no good estimation power (R² < 0.67). On the other hand, important rumen kinetics, intestinal and total tract digestibility, and metabolic features were highly related to spectral areas of STC, CEC, and amide which can be used to predict with good estimation power (R² > 0.74).

6. Summary and conclusion

Based on the scientific findings presented in this chapter, the following most important conclusions can be drawn:

1. Cool-season faba (legume) variety with different tannin levels impacts not only the nutrient profile but also protein and carbohydrate-related molecular structure makeup.

2. Additionally, the nutrient supply, bioenergy, degradation, digestion, and metabolic characteristics of cool-season faba silage and intercropped faba-oat silage were highly related independently and synergistically to molecular structure conformation.

3. Furthermore, the nutrient utilisation and availability of cool-season faba silage and intercropped silage in ruminant livestock systems could be accurately predicted by the protein and carbohydrate molecular structures revealed with cutting stage vibrational molecular spectroscopy when they work together.

4. Additive fibrolytic enzyme and maturity stage at harvesting significantly impacted both nutritional and molecular structural changes of legume and legume-cereal intercropped silage.

5. Dairy production performance and milk yield (ECM, FCM, fat yield) studies showed whole plant faba legume silage in early lactating cows could be used as alternative silage to replace traditional barley and corn silages.

Acknowledgments

We want to thank David A. Christensen, Bunyamin Tar’an (CDC, Plant Science), John McKinnon, Bart Lardner, Zhiyuan Niu, and Brent Barlow (CDC) for their technical assistance. This chapter is part of three graduate theses (project abstracts from VG, CN, and JY) and made revisions for book chapter.

Funding

The Chair (PY) feed research programs have been supported by the Ministry of Agriculture Strategic Feed Research Chair Programs, the Natural Sciences and Engineering Research Council of Canada (NSERC, Canadian federal government), the Saskatchewan Pulse Growers (SPG), the Prairie Oat Grower Association, SaskCanola, the Saskatchewan Agricultural Development Fund (ADF), AB Vista (UK), and Various Feed and Animal Industries etc.

Authors’ contributions

VG, CN, JY, JH, ME, WZ, TR, and PY wrote, reviewed, edited, and approved the book chapter.

Competing interests

The authors declare that we have no competing interests.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Abbreviations

BST	Rumen bypass starch
CDC	Crop Development Centre
CEC	Cellulosic compound
CNCPS	Cornell Net Carbohydrate and Protein System
DVE	total intestinal digestible protein supply with DVE/OEB system
ECM	energy corrected milk
ED_N/ED_CHO	rumen available N and rumen available carbohydrates (ED_N/ED_CHO) hourly effective degradation ratios
FCM	fat corrected milk
FE	fibrolytic enzyme
FMV	feed milk value
IADP	intestinal absorbable protein
ME	metabolizable energy
MP	total metabolizable protein
NEL	net energy of lactation
OEB	degraded protein balance
PCA	principal component analysis
RUP	rumen undegraded protein
SR-IMS or SR-FTIRM	synchrotron-based infrared microspectroscopy
STC	structural carbohydrate
TDN	total digestible nutrients
TDST	total tract digested starch
VFA	volatile fatty acid