3.1. Forage Yield and Lodging Resistance Score
Forage yield and lodging resistance score of Saskatoon and Lanigan sites are presented in
Table 3. No significant interactions (
p > 0.05) between cutting treatments, cultivars, and mixtures were found for forage yield, lodging resistance score, and nutritive value. Therefore, the main effects of cutting treatment and cultivar × mixtures were reported.
At the Saskatoon site, the 3-year average of the first-cut DMY was 1.70 ± 0.24 and 2.15 ± 0.28 Mg ha
−1 for HiGest and Grazeland, respectively. However, the differences between first cut DMY among monocultures have not reached significant level (
p = 0.231). Under second cut, Hi-Gest was numerically lower in TDMY (20% lower; 2.99 vs. 3.71 Mg ha
−1;
p = 0.114) relative to AC Grazeland. In the monoculture plots, second cut DMY tended to be lower (1.32 vs. 1.55 Mg ha
−1; 15% lower;
p = 0.07) in previously stage 3-cut than in previously stage 1-cut plots. There was no significant difference (
p > 0.05) between HiGest-HBG and Grazeland-HBG (
p > 0.05) in DMY, averaged at 2.13 ± 1.36 Mg ha
−1, 1.20 ± 0.493 Mg ha
−1, and 4.25 ± 1.88 Mg ha
−1 for first cut, second cut, and TDMY, respectively. In the binary mixtures, second cut DMY did not differ due to maturity stage of the previous cutting and averaged 0.98 ± 0.120 Mg ha
−1. In both monoculture and binary mixtures, there was no difference (
p > 0.05) due to alfalfa maturity. The Alfalfa-HBG binary mixture had greater (
p < 0.05) DMY and TDMY relative to the alfalfa monoculture for first cut (4.16 vs. 1.92 Mg ha
−1 and 5.15 vs. 3.35 Mg ha
−1, respectively). When analyzing the second cut, the binary mixture had lower (
p < 0.05) DMY relative to alfalfa monoculture (0.98 vs. 1.42 Mg ha
−1) (
Table 3). At Saskatoon, no differences in lodging were observed (data not shown) for the alfalfa.
At the Lanigan site, Hi-Gest exhibited similar (
p = 0.797; 2.98 ± 0.405 Mg ha
−1) DMY and lodging scores (LS) (
p = 0.723; 2.1 ± 0.23) to Grazeland (
Table 3) in 2019. The lack of differences detected among alfalfa cultivars in forage accumulation at Lanigan might have been influenced in part by several factors, including but not limited to the re-seeding that took place in the year following establishment, and large variation among cultivars, indicated by greater standard errors. Likewise, Grazeland-HBG and HiGest-HBG mixtures did not vary (
p = 0.622; 9.12 ± 0.647 kg ha
−1) in DMY and lodging resistance score (
p = 0.164; 2.1 ± 0.14). As alfalfa matured (stage 1 to 3), DMY and LS increased (
p < 0.05) (DMY: 1.94, 2.97, and 4.02 Mg ha
−1; LS: 1.0, 2.3, and 3.1; for stages 1, 2, and 3, respectively). The DMY tended to be greater (
p = 0.09; 35.4% more) in HiGest at stage 3, than in Grazeland at stage 2 (4.24 Mg ha
−1 vs. 3.13 Mg ha
−1). In binary mixtures, forages at maturity stage 1 produced lower DMY (6.58 Mg ha
−1) than those at maturity stage 2 (9.86 Mg ha
−1) and stage 3 (10.65 Mg ha
−1), whereas forages in stage 2 and stage 3 did not differ (
p = 0.119) in DMY. In binary mixtures, with advancing morphological development (stage 1 to 3), LS increased (
p < 0.05) and averaged 1.0, 2.1, and 3.9 for stage 1, stage 2, and stage 3, respectively. Monoculture alfalfa were also lower (
p < 0.05) in DMY, but were similar in LS (
p = 0.531) compared to their binary mixtures. Although not significant (
p = 0.217), HiGest-HBG at stage 3 yielded numerically greater (11% more) than Grazeland-HBG at stage 2 (10.65 Mg ha
−1 vs. 9.58 Mg ha
−1 DMY). Alfalfa cultivars in monocultures and in binary mixtures were not different (
p = 0.531) in LS (2.2 ± 0.23). Likewise, no differences (
p = 0.182) were observed between HiGest at stage 3 and Grazeland at stage 2 in LS, however, HiGest-HBG at stage 3 had greater LS (
p = 0.014) than Grazeland-HBG at stage 2. Overall, the results of the current study indicate that Hi-Gest differed very little from Grazeland in DMY in monocultures and binary mixtures at Lanigan (Black soil zone), although only 1 year of production was studied there; hence further research evaluating several years of data is needed for definite conclusions to be drawn. Nevertheless, this study demonstrated that HiGest alfalfa can be persisted well both in a monoculture and in binary mixtures in Saskatoon (Dark Brown soil zones) and Lanigan (Black soil zones) of Saskatchewan.
3.2. Nutrient Profile
Forage nutrient profiles at Saskatoon are presented in
Table 4. HiGest had greater (
p < 0.05) TDN (2.6% more, 68.4 vs. 66.6%, DM basis), relative forage quality (RFQ) index (14.9% greater; 170 vs. 148), as well as in vitro neutral detergent fiber digestibility after a 48-h incubation (NDFD
48h: 13.5% greater; 42.9 vs. 37.8%), but had lower ADF (9.1% lower; 26.3 vs. 28.7%) and NDF (5.4% more; 34.7 vs. 36.6%) compared to AC Grazeland.
In the first cut forage, HiGest also was numerically lower in ADL (10.2% lower; 5.9 vs. 6.5%;
p = 0.57) relative to Grazeland (
Table 3). HiGest did not differ (
p > 0.05) from Grazeland for CP (avg. 19.9 ± 0.33% DM), Ca (2.6 ± 0.04% DM), and P (0.17 ± 0.01%, DM).
However, at maturity stage 3, alfalfa samples had lower NDFD48h (11.8% lower; 38.2 vs. 42.7%; SEM = 1.80; p = 0.021), RFQ (14.8% lower; 149 vs. 171; SEM = 6.9; p = 0.014) than those at maturity stage 1. Otherwise, stages 1, 2, or 3 did not differ (p > 0.005) between each other. Differences in nutrient parameters among binary mixtures were minimal (p > 0.05) and inconsistent. As alfalfa growth stage advanced (stages 1 to 3), CP, as well as NDFD48h decreased (p < 0.05) (CP: 13.7, 12.3, 10.4; NDFD48h: 53.4, 49.9, 46.4% NDF; for stages 1, 2, and 3, respectively).
In binary mixtures, forages harvested in stage 1 were greater (p < 0.05) in P relative to those harvested in stage 3. Expectedly, a moderate correlation was detected between ADL concentration and forage yield both in monoculture (r2 = 0.37; p < 0.01) and in binary mixtures (r2 = 0.15; p < 0.01).
Ten percent bloom is an important time to cut alfalfa to maintain quality in conventional crop production. As evident from
Table 4, HiGest the stage 3 forage was similar with the Grazeland stage 1 in CP (i.e., HiGest maintains quality for longer time). Moreover, HiGest harvested in all three stages (10, 40, and 100% bloom) had greater RFQ (from 2.3 to 16.7% greater) relative to Grazeland harvested at 10% bloom. Later findings with similar patterns were also observed in TDN and NDFD
48h. Therefore, this increased forage quality of Hi-Gest is widening the harvest window and lengthening the time period when alfalfa can be harvested by livestock producers. Thus, increased harvest flexibility is one of the important advantages to using low-lignin HiGest alfalfa.
At the Lanigan site, HiGest was similar (
p > 0.05) in all measured nutrient profile parameters with Grazeland (
Table 5). For monocultures, the maturity stage at harvest influenced (
p < 0.01) nutrient parameters; forages harvested at maturity stage 3 had lower NDFD
48h (15.0% less; 38.1 vs. 43.8% DM), RFQ (21.7% less; 115 vs. 147), and P (14.3% less; 0.28 vs. 0.32% DM;), but had greater NDF (13.4% more; 45.7 vs. 40.3;) and ADL (36.4% more; 7.5 vs. 5.5% DM;) than those at maturity stage 1. As alfalfa maturity advanced (stages 1 to 3), CP, NDFD
48h, as well as TDN concentration decreased (
p ≤ 0.05) (CP: 23.1, 21.5, 20.0; NDFD
48h: 43.8, 43.2, 38.15; TDN: 65.5, 62.9, 60.8% DM for stages 1, 2, and 3, respectively;
Table 5), which was in agreement with the findings of Llamas-Lamas and Combs [
21] and Balde et al. [
22]. On the contrary, ADF had increased (
p < 0.05) (30.0, 33.4, 36.1% of DM for stages 1, 2, and 3, respectively) with the maturity of alfalfa. However, the Ca concentration was not affected (
p > 0.05) by the stage of maturity of alfalfa.
As shown in
Table 5, the lack of significant difference (
p = 0.831) in NDFD
48h among the alfalfa cultivars in the current study could potentially be the reason for the absence of any cultivar × maturity interaction. HiGest at stage 3 was similar (
p > 0.05) to Grazeland at stage 2 in eight out of nine nutrient profile parameters. Nevertheless, HiGest at stage 3 was greater in ADL (16.7% greater;
p = 0.02; 7.5 vs. 6.4% DM) and lower in P, as well as in NDFD
48h (12.0% less;
p = 0.008; 38.9 vs. 44.2% NDF) than Grazeland at stage 2 (
Table 5). Thus, delaying HiGest alfalfa harvest has increased forage mass, although forage quality was maintained. Differences (
p > 0.05) were not observed in nutrient profiles among binary mixtures (
Table 5). In the binary mixtures, as plant maturity advanced (stage 1 to 3), NDFD
48h decreased (
p < 0.05), however, ADL (
Table 5) concentrations were increased (
p < 0.05). The results of the current study showed that binary mixtures with AC Success HBG had lower energy density (52% less RFQ) relative to the alfalfa monoculture. The ADL concentration of HiGest was 98.8%, 86.7%, and 99.2% (avg. 94.9%) of that of Grazeland alfalfa (check cultivar) for the stage 1, stage 2, and stage 3, respectively. In agreement with the current study, others, [
23,
24], also reported that low lignin alfalfa decreased lignin concentrations ranging from 4 to 12% compared to control cultivars. The ADL concentrations of HiGest-HBG were 106.3%, 93.7%, and 90.9% of (avg. 96.7%) that of Grazeland-HBG for the stage 1, stage 2, and stage 3, respectively (
Table 5). The ADL concentrations for reference cultivars (cv. Grazeland) were comparable to previous findings by others [
25,
26]. Likewise, the cultivar description by Alforex (2021) stated that the whole plant lignin of Hi-Gest
® 360 alfalfa is lower (by 7–10%) than non-selected elite commercial cultivar, which was supported by the results in the current study.
As a plant grows, the deposition of lignin is necessary to provide the strength and rigidity for a plant to stand upright [
3,
4]. Reductions in lignin are generally associated with negative impacts on plant growth, development, lodging tolerance, and/or productivity [
27]. However, as noted in the previous section in the current study, HiGest did not differ in lodging resistance relative to Grazeland in monocultures or in binary mixtures with HBG. The HiGest-HBG at stage 3 did not differ (
p > 0.05) from Grazeland-HBG at stage 2 in eight of nine measured nutrient profiles parameters (data not shown). However, HiGest-HBG at stage 3 was greater in Ca (
p = 0.014; 1.7 vs. 1.6% DM) relative Grazeland-HBG at stage 2 (data not shown). Thus, delaying HiGest alfalfa harvest increased forage yield and maintained quality. Alfalfa monocultures had lower (
p < 0.05) ADF, NDF, and NDFD
48h, but had higher CP, TDN, ADL, and RFQ than binary mixtures (Grazeland-HBG and HiGest-HBG), as was mostly expected. The difference in NDF concentration between alfalfa and alfalfa-HBG (42.7 vs. 65.3% DM) can be accounted for by the difference between NDF and ADF of these two mixtures, which is primarily hemicellulose (9.6 vs. 27.2%, data not shown), which was in agreement with the findings of Elizalde et al. [
28]. Likewise, Hoffman et al. [
29] also reported higher NDF in grasses (i.e., timothy, orchardgrass, perennial ryegrass, quackgrass, and bromegrass) compared with legumes (i.e., alfalfa, red clover, and birdsfoot trefoil).
Also seen in the current study, averaged by two sites, an increase of 10% NDFD
48h for HiGest compared with Grazeland was observed, which concurred with Guo et al. [
4] who observed an increase of eight percent NDFD for one of these transgenic reduced lignin lines compared with its isogenic counterpart. In the current study, in both HiGest and HiGest-HBG binary mixtures, the ADL concentration of the forages was relatively lower and consistent up to stage 2 and increased rapidly thereafter (
Table 5). Furthermore, for Grazeland monoculture or Grazeland-HBG binary mixtures, ADL concentration gradually increased as plant maturity advanced. However, an opposite pattern (decreased) with lignin was observed on CP, TDN, and RFQ, as well as on P. In agreement with the current study, Hall et al. [
30] and Yu et al. [
7] also reported declines in CP concentrations with advancing morphological development across multiple harvests. The greater reduction in RFQ (from 142.3 to 118; by 20.5%) from stage 2 to stage 3 for HiGest likely suggests that the ideal harvest stage for Hi-Gest for hay is stage 2.
Alfalfa is generally harvested at the 40% bloom stage of maturity in western Canada, balancing yield and nutrient quality [
31]. Crude protein concentrations differed among cutting stages at both sites (
Table 4 and
Table 5). Across sites, CP concentrations for both alfalfa cultivars ranged from 18.9 to 23.5% DM and were comparable to previously reported values by others [
26,
30,
32]. Concurring with the current study, previous studies examining reduced lignin alfalfa experimental lines have, also, found similar CP concentrations for LL alfalfa compared to reference alfalfa cultivars [
2,
33,
34]. To the best of our knowledge, this is the first study comparing forage accumulation between low-lignin HiGest and reference alfalfa cultivars under diverse cutting schedules in monocultures and binary mixtures in western Canada; therefore, no data for comparison was available. Overall, the reduction in ADL was present across both sites. Significant differences (
p < 0.05) in ADL were observed between stage 1 and stage 3, or between stage 2 and stage 3.
According to Van Soest [
35], legumes tend to have lower ADF and NDF concentrations compared to grass species. As plants mature over the growing season, the nutritive value of both annual and perennial forages declines [
36] as a result of a simultaneous decrease in CP and increase in NDF concentrations and decrease in fiber digestibility. This decline in nutritive value of forages was evident in the current study.
However, Ca concentrations of the binary mixtures in Saskatoon averaged 0.47% greater compared to those in Lanigan (0.27% DM) which was likely, as we speculated, due to higher proportions of alfalfa at the Saskatoon site since alfalfa has greater Ca concentrations compared to grasses [
37].
3.4. Relationship between Forage Lignin and Yield and Nutrient Profiles
Pearson correlation analysis between the DMY and ADL concentration of forage pooled at both sites is presented in
Table 7. Expectedly, ADL concentration was moderately and positively correlated with DMY in both Grazeland (
r = 0.49;
p < 0.001) and HiGest (
r = 0.42;
p < 0.001) alfalfa. For HiGest, ADL concentration moderately correlated with ADF (
r = 0.47;
p < 0.001), NDF (
r = 0.43;
p < 0.01), RFQ (
r = −0.52;
p < 0.001), NDFD
48h (
r = −0.51;
p < 0.001), and TDN (
r = −0.47;
p < 0.001). It can be assumed that the lower value of ADL in HiGest, the magnitude of
r value between ADL concentration and DMY as well as nutrient profiles was lower in HiGest relative to Grazeland excluding the
r value between NDFD
48h and ADL, which was numerically greater in HiGest. A moderate correlation was also observed between ADL and DMY in both Grazeland-HBG (
r = 0.57;
p < 0.001) and HiGest-HBG (
r = 0.43;
p < 0.01). However, very weak or no correlation was also observed between ADL and nutrient profiles in both Grazeland-HBG and HiGest-HBG. Changes in lignin concentration account for most of the improvements in digestibility rather than the effects of lignin composition/structure as strong negative correlations were reported for lignin concentrations with digestibility [
39,
40,
41]. Summarizing all four forages on both sites through all years, each percentage unit increase in lignin concentration, the main factor hindering cell wall digestion, decreased cell wall in vitro NDFD
48h degradability by 3.8 percentage units (NDFD
48h, % of NDF = 65.80 − 3.766 × ADL, % of DM;
r2 = 0.38, n = 192,
p < 0.001; data not shown). In a similar fashion with the current study, others, [
42], have also documented that the lignin concentration predicted NDF in degradability with reasonable accuracy. Likewise, a 1-unit increase in forage NDF digestibility is associated with 0.17 and 0.25 kg d
−1 increases in DMI and milk production, respectively [
43].
Furthermore, in perennial ryegrass (
Lolium perenne L.), a 5 to 6% increase in digestibility was estimated to increase milk production by up to 27% [
44]. Thus, each percentage unit increase in lignin concentration in forage cell walls can severely constrain DMI and milk production. Moreover, several studies have confirmed the positive effect of feeding forage with increased NDF digestibility on DMI and productivity of dairy cattle [
45]. It is well known that forage quality may be affected by cultivar [
46], soil type, climatic conditions [
47], as well as harvest time or maturity stage [
7,
48] among other factors. As plants mature, leaf proportions decrease, stem proportions increase, stem cell wall concentrations increase, and whole plant nutritive value decreases [
49,
50,
51].
Hall et al. [
30] concluded that the selection for greater nutritive value did not inadvertently result in the selection for delayed morphological development. As the lodging tolerance score indicated in the case of Hi-Gest
® 360 alfalfa, our results demonstrated the genetic selection for lower ADL concentrations and greater NDFD had essentially no difference in morphological development than the non-LL cultivar. As expected, the results of the current study indicate that binary mixtures had decreased energy density (less lignin, CP and higher ADF, NDF) compared to alfalfa monocultures.
Guo et al. [
4] examined the lignin concentration and NDFD of six independent transgenic alfalfa lines with reduced lignin concentration compared with control lines and reported a range from 13 to 29% in lignin concentration, which was in agreement with the results of the current study. Furthermore, they observed an increase of 8% in vitro NDFD for one of these reduced lignin lines compared with its isogenic counterpart. In agreements with the latter, HiGest alfalfa showed increased NDF digestibility relative to AC Grazeland alfalfa cultivar in the current study. Small decreases in the lignin concentration of forages can be expected to improve the fiber digestibility at any plant maturity stage [
24]. Overall, the result of the current study indicated that the Hi-Gest
® 360 alfalfa could be harvested from 7 to 14 days later and still maintain slightly greater than or similar nutrient quality concentrations and similar (at the Lanigan) or lower (at the Saskatoon) forage yields as the AC Grazeland alfalfa checks harvested earlier.
3.5. Economics
The stand establishment costs for monoculture and binary treatments are presented in
Table 8. At the Lanigan site, glyphosate was applied three times (total of 3.24 L ha
−1) for a product cost of 20.74 CAD ha
−1 and an application cost of 37.05 CAD ha
−1 (12.35 CAD ha
−1 per application). Seed costs were calculated from actual seeding rates which varied by treatment and were based on germination tests and seed weight (per 100 seeds), and the actual price paid for the seed. For HiGest alfalfa, the seed was purchased for 16.41 CAD kg
−1 (7.46 CAD lb
−1) for a total cost of 147.22 CAD ha
−1. The AC Success seed was purchased for 10.89 CAD kg
−1 (4.95 CAD lb
−1). The seed cost for the HiGest-HBG binary treatment was 122.25 CAD ha
−1. AC Grazeland alfalfa seed was valued at 11.66 CAD kg
−1 (5.30 CAD lb
−1), for a total cost of 104.59 CAD ha
−1. The total seed cost for Grazeland-HBG was 111.60 CAD ha
−1. In each treatment, 11.2 kg ha
−1 of P (11-51-0) was added to improve seed flow. Phosphorus was valued at 685.47 CAD tonne
−1 based on published prices for 11-51-0 fertilizer for January to June 2017 by Alberta Agriculture and Forestry [
18]. The inclusion of fertilizer for seed flow costed 7.66 CAD ha
−1 (3.10 CAD ac
−1). Stand total establishment costs varied by treatment from a high of 269.47 CAD ha
−1 for HiGest monoculture to a low of 226.85 CAD ha
−1 for Grazeland monoculture.
At the Saskatoon site, rototilling was used for weed control on the Saskatoon plots which has been equated with cultivating (
Table 8). The suggested custom rate for cultivating is 22.48 CAD ha
−1 in the 2016–2017 Farm Machinery Custom and Rental Rate Guide [
17]. Seed costs for Saskatoon varied from Lanigan due to differing seeding rates. HiGest was seeded at a 72% higher seeding rate than Grazeland resulting in higher seed costs for treatments containing HiGest. The HiGest monoculture seed cost 251.10 CAD ha
−1 (15.3 kg ha
−1 × 16.41 CAD kg
−1) at Saskatoon. The AC Grazeland seed cost 103.77 CAD ha
−1 (8.9 kg ha
−1 × 11.66 CAD kg
−1). The HiGest-HBG treatment had 282.10 CAD ha
−1 seed costs and Grazeland-HBG was 207.03 CAD ha
−1. There was no fertilizer or herbicide applied on the Saskatoon plots. At the Saskatoon site, the Grazeland monoculture and binary treatments had 45% and 21% lower establishment costs, respectively. The difference is due to the higher seeding rates and seed prices for HiGest. Establishment costs for the Saskatoon treatment plots from lowest to highest were: Grazeland (183.06 CAD ha
−1), HBG-Grazeland (286.32 CAD ha
−1), HiGest (330.39 CAD ha
−1), and HBG-HiGest (361.39 CAD ha
−1).