RESEARCH ARTICLE

Effects of alfalfa and alfalfa-grass mixtures with nitrogen fertilization on dry matter yield and forage nutritive value

Iryna McDonald1https://orcid.org/0000-0002-4515-3305, Rudra Baral1,*https://orcid.org/0000-0002-9018-9470, Doohong Min1https://orcid.org/0000-0002-9404-5175
Author Information & Copyright
1Department of Agronomy, Kansas State University, Manhattan, KS 66506, USA
*Corresponding author: Rudra Baral, Department of Agronomy, Kansas State University, Manhattan, KS 66506, USA. Tel: +1-785-532-2292, E-mail: rudrabaral@ksu.edu

© Copyright 2021 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Oct 07, 2020; Revised: Dec 12, 2020; Accepted: Dec 21, 2020

Published Online: Mar 31, 2021

Abstract

Alfalfa (Medicago sativa L.) is an important forage legume grown in Kansas, USA and its productivity with cool-season grasses however is unknown. The objective of this study was to determine the dry matter yield (DMY) and forage nutritive value of alfalfa-grass mixtures compared to those of alfalfa and grasses grown in monoculture with and without nitrogen fertilization. Three different alfalfa varieties were planted (reduced-lignin alfalfa, Roundup Ready, and conventional alfalfa) and two kinds of cool-season grasses (smooth brome, Bromus inermis Leyss, and tall fescue, Festuca arundinacea Schreb) were planted as a monoculture or in alfalfa-grass mixtures. Nitrogen fertilizer (urea) was applied at green-up at a rate of 56 kg/ha and after the second cutting at a rate of 56 kg/ha in 2016 and 2017, respectively. and control treatments received no nitrogen. DMY was significantly higher in monoculture alfalfa and alfalfa-grass mixtures than in grass monocultures. Between alfalfa monoculture and alfalfa-grass mixtures, no significant differences in DMY were found. For all treatments, nitrogen application significantly increased DMY compared to the control. In 2016 and 2017, the low-lignin alfalfa monoculture had the lowest acid detergent fiber (ADF) and the grass monocultures had the highest ADF. In 2016 and 2017, neutral detergent fiber (NDF) in smooth bromegrass and tall fescue was higher than in other species treatments. A low-lignin alfalfa monoculture had significantly lower NDF concentration compared to alfalfa-grass mixtures. When averaged over 2016 and 2017, relative feed value (RFV) was highest in low-lignin alfalfa and lowest in the grass monocultures. In both years, nitrogen fertilizer application did not affect nutritive values.

Keywords: Dry matter yield; Forage nutritive value; Alfalfa; Tall fescue; Smooth bromegrass; Nitrogen application

INTRODUCTION

Alfalfa (Medicago sativa L.) is a perennial legume that is important in forage production worldwide due to its high forage nutritive value and dry matter yield (DMY) [13]. Legumes benefit grass pastures not only by transferring fixed nitrogen to the grasses [46] but also by providing biomass to the total yield of a pasture. This is important during the middle of the summer when grasses grow better with alfalfa and can provide additional forage to animals [7,8].

Farmers often plant grasses in pastures, because they can control weeds and improve grazing conditions [9,10]. Also, planting legumes with grasses helps improve forage nutritive value and increases DMY of pastures compared to pastures with pure grasses [11]. Because legume crops can provide nitrogen to the grasses, Woodfield and Clark [12] recommended that farmers use a new strategy in farming and plant legumes with grasses. Legume-grass mixtures are not as easy to manage as monoculture grasslands, because the legumes and grasses compete for water, nutrients, and light [11,1315]. However, much research has shown that the potential advantages of legume-grass mixtures can outweigh the disadvantages. It would be of interest to list a few of the agronomic or animal nutrition advantages to having important cool-season forage grasses in mixture with alfalfa.

Tracy et al. [16] showed that yields of pastures with grasses mixed with alfalfa were almost identical to yields of pastures with only grasses that were fertilized with urea nitrogen. Sengul [17] found that DMY of legume-grass mixtures, both with and without fertilizer, was higher than DMY of grass monocultures. Planting legumes with grasses can supply the grasses with > 80% of the nitrogen that they need through nitrogen fixation [4,18]. Grasses in the spring react positively to available nitrogen in the soil, and usually, nitrogen in fertilizer or manure is applied to grasses in the spring. Legumes fix little nitrogen in early spring, because of low temperatures [19]. Therefore, it is recommended that nitrogen fertilizer be applied to legume-grass mixtures in the early spring, which increases the biomass of grasses at that time [2022].

The importance of forage nutritive value cannot be over-emphasized as far as the sustainability and profitability of any farm are concerned. It is essential for sufficient animal nutrition, so the animals can have high gain rates, ample production of milk, and efficient production. Immense variations in the quality of forage within and among forage crops occur, just as is the case for variations in nutritional needs within and among animal classes and species [23]. Forage nutritive value is directly related to the content and availability of essential nutrients for the consuming animals and is defined by three processes, which are the capacity of the animal to consume, assimilate (availability), and digest (digestibility) the essential nutrients in the feed [24]. Various factors affect the nutritive value of forage. In the same or different years, similar plants can have varying nutritive value, even when each has been planted in the same field. Grass-legume mixtures are acknowledged to have higher crude protein concentrations, as well as lower fiber concentrations, compared to pure grass stands [23]. Brown et al. [25] observed that in grass-mixtures crude protein increases, as well as DMY. Forage nutritive value is affected by fertilization, where the fertilization of grasses using nitrogen usually results in a substantial increase in yield, while also generally increasing crude protein levels.

Little research on legume-grass mixtures has been done in Kansas, USA since late 1970s. Therefore, the objectives of this study were to evaluate the productivity of alfalfa-grass mixtures compared alfalfa in monoculture and grasses in monoculture and to assess the effect of nitrogen fertilizer on forage DMY and nutritive value of the monocultures of alfalfa and grasses and alfalfa-grass mixtures.

MATERIALS AND METHODS

Study site

The experiment was conducted at the Ashland Bottoms Research Farm of Kansas State University (39°14´ N, 96°64´W) between April 2015 and December 2017. The soil type at the study site was a Belvue silt loam [26]. A soil test, done at the Soil Testing Laboratory at Kansas State University, showed that the study field had a pH of 6.96 and an organic matter content of 2%. Weather data were collected from an automated Kansas Mesonet Station located at the Ashland Bottoms Research Farm (Table 1).

Table 1. Rainfall and maximum and minimum temperatures at Ashland Bottoms Research Farm, Kansas State University during the study period
Climate characteristics Month
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
2015
 Monthly rain (mm) 67.57 218.68 107.2 128 81 104.9 15.49 110.5 82.8
 Mean Max. temp. (°C) 20.6 22.95 31.08 31.93 30.4 29.68 22.42 16.22 9.36
 Mean Min. temp. (°C) 7.26 12.25 18.64 20.78 17.23 16.96 7.62 2.87 −1.96
2016
 Monthly rain (mm) 12.7 10.16 11.17 214.6 177.3 39.36 155 185.7 105.7 70.36 7.62 21.08
 Mean Max. temp. (°C) 4.5 11.8 18.2 21.32 23.73 33.45 32.35 30.37 28.45 24.26 17.39 5.6
 Mean Min. temp. (°C) −6.1 −2.97 2.37 7.66 11.26 19.48 21.24 19.61 16.02 9.42 3.53 −7.14
2017
 Monthly rain (mm) 24.89 11.94 106.9 126.8 96.76 71.63 33.78 154.7 20.58 92.96 2.28 2.79
 Mean Max. temp. (°C) 6.44 13.42 15.45 19.42 24.67 30.96 33.37 28.55 29.11 21.23 14.14 6.36
 Mean Min. temp. (°C) −5.13 −1.05 2.63 7.58 11.15 17.2 20.5 15.98 15.01 7.22 0.7 −5.38
30 years (1987–2017) average
 Monthly rain (mm) 16.54 27.78 57.45 83.94 121.42 136.32 112.26 108.20 83.00 61.51 38.63 27.28
 Mean Max. temp. (°C) 5.45 8.29 14.68 20.34 25.24 30.58 33.31 32.50 28.06 21.55 13.39 6.62
 Mean Min. temp. (°C) −7.27 −5.44 −0.16 5.81 11.61 17.14 19.87 18.57 13.21 6.50 −0.18 −5.78
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Three different alfalfa varieties were planted: ‘Hi-Gest 360 LL,’ a reduced-lignin variety; ‘RR Tonica,’ a Roundup Ready variety, and ‘Gunner,’ a conventional alfalfa variety. Two kinds of cool-season grasses, smooth bromegrass and tall fescue were planted as monoculture crops and in the legume-grass mixtures as binary and trio mixtures. This resulted in 14 different treatments (Table 2). All 14 treatments then were treated with nitrogen to provide 28 treatments in total: 14 with nitrogen and 14 without nitrogen. The treatments without nitrogen were called the controls. Nitrogen was applied as urea at a rate of 56 kg/ha after planting on April 24, 2015 and at the green up and after the second cutting in 2016 and 2017. In monocultures, the seeding rates were as follows: alfalfa variety, 20.18 kg/ha; tall fescue, 13.45 kg/ha; and smooth bromegrass, 16.81 kg/ha. In binary mixtures, seeding rates were as follows: alfalfa variety, 11.21 kg/ha; tall fescue, 8.97 kg/ha, and smooth bromegrass, 11.21 kg/ha. In trio mixtures, an alfalfa variety, smooth bromegrass, and tall fescue all had a seeding rate of 7.85 kg/ha. During the establishment year, the favorable growing conditions have led to a high weed population, and due to this the first cut in 2015 was controlled by mowing the study field completely, and after that, the weed population was controlled by hand-weeding.

Table 2. Composition of alfalfa and grasses as monocultures and legume-grass mixtures
Treatment Species Abbreviation
1 Gunner conventional alfalfa A
2 Low-lignin Hi-Gest 360 LL alfalfa LLA
3 Roundup ready RR tonica alfalfa RRA
4 Smooth bromegrass SB
5 Tall fescue TF
6 Gunner conventional alfalfa + smooth bromegrass A + SB
7 Gunner conventional alfalfa + tall fescue A + TF
8 Low-lignin Hi-Gest 360 LL alfalfa + smooth bromegrass LLA + SB
9 Low-lignin Hi-Gest 360 LL alfalfa + tall fescue LLA + TF
10 Roundup ready RR tonica alfalfa + smooth bromegrass RRA + SB
11 Roundup ready RR tonica alfalfa + tall fescue RRA + TF
12 Gunner conventional alfalfa + smooth bromegrass + tall fescue A + SB + TF
13 Low-lignin Hi-Gest 360 LL alfalfa + smooth bromegrass + tall fescue LLA + SB + TF
14 Roundup ready RR Tonica alfalfa + smooth bromegrass + tall fescue RRA + SB + TF
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The experimental design was a split-plot (with or without nitrogen) in a randomized complete block design with four replications. There were 28 treatments of two nitrogen application rates as whole plots and mixture treatments as subplots. Nitrogen was applied as urea at a rate of 56 kg/ha after planting on April 24, 2015 and 56 kg/ha at the green up and 56 kg/ha after the second cutting in 2016 and 2017. Plot size was 1 meter wide and 6 meters long, for a total of 112 plots. In 2016, plots were harvested on 19 May, 22 June, 27 July, and 3 September; in 2017, they were harvested on 24 May, 26 June, 28 July, and 1 September. In both years, plots were harvested when alfalfa reached 1/10th bloom stage.

Because smooth bromegrass and tall fescue are cool-season grasses, they recovered poorly from the second cut in 2016 and first cut in 2017, and this led to slow regrowth and DMY. Therefore, the plots with pure grasses were not harvested in the third cut in 2016 and second cut in 2017.

Dry matter yield

Sub-samples were collected, placed into paper bags, and put in a cooler; bags were taken out of the cooler, weighed, and placed in a custom-built forage dryer made by the Agronomy Farm Shop at Kansas State University at 60°C for 72 h. After drying, samples were weighted again and the dry matter per unit area was calculated, Then, the calculated dry matter per unit area was converted to DMY per hectare (Mg/ha).

Forage nutritive value

The dry samples were ground in a Wiley mill with a 0.04 inches screen. The total nitrogen in the samples was determined by the Soil Testing Laboratory at Kansas State University. The laboratory digests the samples using a sulfuric peroxide acid digestion, and then the digest is analyzed for nitrogen by a colorimetric procedure using the Rapid Flow Analyzer (Model RFA-300) and RFA Methodology No. A303-S072 from Alpkem (Clackamas, OR, USA). Crude protein (CP) in percent was calculated by multiplying the total nitrogen (%) by 6.25. Following the protocol of ANKOM Technology (Macedon, NY, USA), the acid detergent fiber (ADF) and neutral detergent fiber (NDF) were analyzed using ANKOM method.

Relative feed value (RFV) is calculated from digestible dry matter digestibility (DMD) and dry matter intake (DMI) using ADF (%) and NDF (%), respectively [27]. Using the equations of Rohweder et al. [27], the RFV of legume-grass mixtures and monocultures was calculated as follows:

DMD ( % ) = 88.9 ( 0.779 × ADF ) DMI ( % ) = 120 / ( NDF ) RFV= ( DMD × DMI ) / 1.29
Statistical analysis

Differences in DMY and forage nutritive value were evaluated by ANOVA using PROC GLIMMIX (SAS, 2011). The ANOVA for these variables was conducted for each year, and for each cut, with 14 combinations of varieties as a fixed effect. Block was a random effect, and all data were checked for normality and homogeneity of variance. Mean separation was performed using the Bonferroni correction [28,29]. All statistical comparisons were made at the significance level of α = 0.05.

RESULTS AND DISCUSSION

Dry matter yield

The means and total of DMY are shown in Table 3. There was no interaction between species mixture treatments and nitrogen fertilizer application in all cuts of both years for DMY.

Table 3. Mean and total dry matter yield (Mg/ha) in 2016 and 2017
Treatments 2016 2017
Cut 1 Cut 2 Cut 3 Cut 4 Total Cut1 Cut 2 Cut 3 Cut 4 Total
N 0 4.8b1) 4.7b 4.8a 3.7b 18.0b 5.0a 5.3a 4.7b 3.9a 18.9b
N 112 5.3a 5.2a 4.8a 3.9a 19.2a 5.5a 5.3a 5.0a 4.0a 19.8a
A 5.3a 5.1a 4.9a 3.5ab 18.8a 5.1ab 5.2a 4.8ab 4.1ab 19.2a
LLA 4.9a 5.3a 4.7a 3.9ab 18.8a 5.1ab 5.0a 4.8ab 4.1ab 19.0a
RRA 5.0a 5.7a 5.1a 3.7ab 19.5a 4.5ab 5.4a 5.0a 4.2a 19.1a
SB 2.5b 1.3b N/A 4.6a 8.4b 3.5b N/A 3.9b 2.8c 10.2b
TF 2.3b 1.9b N/A 4.3ab 8.5b 4.4ab N/A 4.5ab 3.4bc 12.3b
A + SB 5.5a 5.5a 5.0a 3.6ab 19.6a 5.6ab 5.5a 5.1a 4.2a 20.4a
A + TF 5.7a 5.5a 4.8a 3.7ab 19.7a 5.6ab 5.4a 5.0a 4.2a 20.2a
LLA + SB 5.4a 5.3a 4.6a 3.8ab 19.1a 5.8a 4.8a 4.9ab 4.3a 19.8a
LLA + TF 5.7a 5.6a 4.9a 3.9ab 20.1a 5.5ab 5.2a 5.0a 4.2a 19.9a
RRA + SB 5.9a 5.5a 4.4a 3.6ab 19.5a 5.7a 5.7a 5.0a 3.8ab 20.2a
RRA + TF 5.9a 5.6a 4.7a 3.3b 19.5a 5.6ab 5.2a 4.6ab 4.0ab 19.4a
A + SB + TF 6.0a 5.4a 5.1a 3.8ab 20.3a 5.9a 5.2a 5.2a 4.0ab 20.3a
LLA + SB + TF 5.8a 5.4a 4.7a 3.6ab 19.5a 5.3ab 5.2a 4.7ab 3.8ab 19.0a
RRA + SB + TF 5.5a 5.6a 4.6a 3.6ab 19.3a 5.7a 5.3a 4.7ab 4.2a 19.9a
p-value
 N 0.0016 0.0347 0.8406 0.0084 < .0001 0.051 0.8537 0.0495 0.145 < .0001
 T < .0001 < .0001 0.7919 0.0315 < .0001 0.0057 0.2185 0.0075 < .0001 < .0001

1) The LSMEANS with same letter are not significantly different at p-value of 0.05 level.

N0, control without fertilizer; N112, nitrogen application 112 kg/ha; A, gunner conventional alfalfa; LLA, low-lignin Hi-Gest 360 LL alfalfa; RRA, roundup ready RR tonica alfalfa; SB, smooth bromegrass; N/A, not applicable; TF, tall fescue; N, nitrogen fertilizer; T, treatment.

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The DMY of the first cut in 2016 was significantly higher in alfalfa monocultures and alfalfa-grass mixtures than in the monocultures of two cool-season grasses. No difference in DMY was found in three different alfalfa varieties, two cool-season grasses monocultures, and alfalfa-grass mixtures, respectively. DMY with nitrogen application was significantly higher than the control (DMY with N application: 5.3 Mg/ha and control: 4.8 Mg/ha) indicating that nitrogen fertilization appears to be a desirable practice in increasing DMY in this study. The DMY of the second cut in 2016 was significantly higher in alfalfa monoculture and legume-grass mixtures than in monocultures of two grasses. The yield difference between monoculture grasses and other species treatments was greater in the second cutting than the first cutting. This might be due to increasing air temperature and a lot lower precipitation after first cutting until second cutting. The DMY with nitrogen application was significantly higher than without (DMY with N application: 5.2 Mg/ha and without 4.7 Mg/ha).

In third cut in 2016, there was no statistical difference in DMY across treatments and fertilizer applications. The DMY of the fourth cut 2016 was significantly higher in the smooth bromegrass plots than in RRA + TF. No difference in DMY was found in different alfalfa varieties and two cool-season grasses. Also, the DMY with nitrogen application treatment was significantly greater than that of the control.

The DMY of smooth bromegrass in the first cut in 2017 was significantly lower than the following combinations: Roundup Ready alfalfa with smooth bromegrass; Gunner alfalfa with smooth bromegrass and tall fescue; and Roundup Ready alfalfa with smooth bromegrass and tall fescue (Table 3). No significant differences were found in DMY among forage species due to nitrogen application. In general, in the third cut of 2017, the DMY of most combinations was higher than that of the smooth bromegrass. No DMY difference was found in alfalfa monocultures and alfalfa-grass mixtures. The DMY was significantly higher with nitrogen fertilizer application than control. Similar results were found in the fourth cut in 2017 where DMY was significantly higher in alfalfa monoculture varieties and legume-grass mixtures than in smooth bromegrass. Unlike the fourth cutting in 2016, there was no significant difference in DMY with nitrogen application in the fourth cutting in 2017.

The possibility of legume-grass mixtures in improving forage quality and increasing DMY over grass monocultures has been studied by many people [8,30,31]. In general, results show that legume-grass mixtures provide higher DMY compared to grass monoculture stands [11]. In both years of this study (2016 and 2017), DMY of alfalfa monocultures and alfalfa-grass mixtures were significantly higher than DMY of monoculture grasses. Unexpectedly, the DMY of alfalfa in monocultures was not significantly different from that of alfalfa in mixtures with grasses. But the alfalfa grass binary mixture has several advantages such as higher total forage yield obtained in the establishment year, faster drying, more digestible fiber, less winter kill, less weed pressure and improved seasonal forage distribution than alfalfa and grass monoculture [32,33]. The monocultures of smooth bromegrass and tall fescue had the lowest biomass of all treatment (Table 3). Sleugh et al. [8], Mortenson et al. [34], and Rao et al. [35] observed that planting grasses with legumes increases yield and availability of nitrogen in the soil. Our results do agree with those of Kopp et al. [7], who found that applying nitrogen fertilizer at a rate of 68 to 110 kg/ha improved forage production by 64%. Nitrogen application increased the total yield but did not improve the yield significantly in each treatment of our study. In both years, the total yield was significantly higher with nitrogen application than without (with nitrogen in 2016 DMY: 19.2 Mg/ha, in 2017 DMY: 19.8 Mg/ha; without in 2016 DMY: 18.0 Mg/ha, in 2017 DMY: 18.9 Mg/ha) (Table 3).

Forage nutritive values
Crude protein

The means CP concentration of each cut in 2016 and 2017 are shown in Table 4 where CP was found from 65 to 199 g/kg dry matter (DM). In all cuts of both years, there was no interaction between species treatments and fertilizer applications for CP.

Table 4. Mean CP concentration (g/kg DM) in 2016 and 2017
Treatments 2016 2017
Cut 1 Cut 2 Cut 3 Cut 4 Average Cut1 Cut 2 Cut 3 Cut 4 Average
N 0 170a1) 127a 126a 140a 141a 137a 163a 143a 141a 145a
N 112 175a 127a 130a 131a 141a 136a 161a 146a 142a 145a
A 182a 129a 151a 157ab 155a 145a 163a 147a 139a 148ab
LLA 199a 131a 151a 167a 162a 147a 158a 139a 148a 148ab
RRA 187a 135a 141a 148ab 153a 147a 171a 142a 144a 151ab
SB 137bc 120a N/A 65c 107b 119ab N/A 136a 133a 130bc
TF 132c 111a N/A 77c 107b 107b N/A 132a 135a 125c
A + SB 162abc 129a 149a 140ab 145a 134ab 153a 144a 142a 143abc
A + TF 173ab 115a 148a 136ab 143a 145ab 152a 150a 149a 149ab
LLA + SB 172ab 131a 155a 126b 146a 137ab 164a 148a 129a 145abc
LLA + TF 184a 111a 147a 154ab 149a 137ab 171a 155a 146a 152a
RRA + SB 168ab 138a 153a 154ab 153a 137ab 161a 142a 154a 149ab
RRA + TF 182a 132a 144a 156ab 154a 149a 163a 152a 138a 151ab
A + SB + TF 180a 133a 148a 130ab 148a 135ab 160a 143a 140a 145abc
LLA + SB + TF 182a 137a 159a 139ab 154a 136ab 161a 151a 145a 148ab
RRA + SB + TF 175a 126a 147a 151ab 150a 138ab 165a 141a 140a 146abc
p-value
 N 0.1601 0.9075 0.161 0.1486 0.8494 0.5725 0.6211 0.3376 0.8758 0.8709
 T < .0001 0.3195 < .0001 < .0001 < .0001 0.0075 0.535 0.6692 0.203 0.0006

1) The LSMEANS with same letter are not significantly different at p-value of 0.05 level.

CP, crude protein; DM, dry matter; N0, control without fertilizer; N112, nitrogen application 112 kg/ha; A, gunner conventional alfalfa; LLA, low-lignin Hi-Gest 360 LL alfalfa; RRA, roundup ready RR tonica alfalfa; SB, smooth bromegrass; N/A, not applicable; TF, tall fescue; N, nitrogen fertilizer; T, treatment.

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The CP concentration in the first cut in 2016 was significantly lower in grasses monoculture than in alfalfa monoculture and alfalfa-grass mixtures except the mixture of conventional Gunner alfalfa with smooth bromegrass. In the second cut and third cut of 2016 no significant differences in CP concentration among treatments was found. When we harvested the smooth bromegrass and tall fescue in the fourth cut in 2016, we found the significantly lower CP concentration in both grasses. The low-lignin alfalfa monoculture had a significantly higher CP concentration than mixture of low-lignin alfalfa with smooth brome grass. In the first cut of 2017, the tall fescue monoculture had a significantly lower (107 g/kg DM) CP concentration than alfalfa monocultures and alfalfa-grass mixture of roundup ready Tonica alfalfa with tall fescue (149 g/kg DM). In the second, third and fourth cuts of 2017, no differences in CP concentration were found, because nitrogen was applied after the second cut, and grasses monoculture did not harvest due to poor regrowth. However, it has been shown that planting alfalfa with grasses in mixtures increases crude protein compared to crude protein in grass monocultures [25], and we agree with this statement, on average, the CP concentration of alfalfa monocultures and alfalfa-grass mixture treatments was significantly higher than monoculture cool-season-grasses.

Overall, in 2016 and 2017 with the same fertilizer treatment, there were no significant differences in CP with nitrogen fertilizer treatment. On average, the CP was higher in 2017 than in 2016 (CP 80–120 g/kg DM in 2016; CP 125–152 g/kg DM in 2017). In both years with nitrogen application, there was no difference in CP between the grasses and other treatments. No significant differences in crude protein were observed between the mixtures and the alfalfa monocultures. Schmidt (1993) saw that change in forage nutritive value could be due to temperature and precipitation variance. In our study, 2017 was much dryer than 2016, and the dryness might have led to an increase in the crude protein in the forage treatments.

Acid detergent fiber

The concentration of ADF was significantly higher in smooth bromegrass than in alfalfa monocultures in the first cut in 2016, but there were no differences in ADF among the legume-grass mixtures and alfalfa monocultures (Table 5). In the second cut in 2016, the low-lignin alfalfa variety had a significantly lower ADF content than mixtures alfalfa (A) + smooth brome (SB) and A + tall fescue (TF) (364, 418, and 431 g/kg DM). Smooth bromegrass and tall fescue were not harvested during the third cut in 2016. No differences in ADF concentration were observed for species treatments in the third cut. In the fourth cut of 2016, ADF content of low lignin alfalfa was significantly lower than grass monocultures and conventional alfalfa with grass mixtures. Nitrogen application had no significant effect on ADF concentration in all cuts.

Table 5. Mean ADF concentration (g/kg DM) in 2016 and 2017
Treatments 2016 2017
Cut 1 Cut 2 Cut 3 Cut 4 Average Cut1 Cut 2 Cut 3 Cut 4 Average
N 0 347a* 406a 392a 389a 383a 362a 339a 365a 350a 355a
N 112 34.7a 405a 381a 395a 382a 362a 340a 360a 350a 354a
A 323d 404abc 398a 380bc 376ab 359a 318ab 357ab 354ab 347abc
LLA 332bcd 364c 381a 352c 357b 355a 330ab 335b 312c 333c
RRA 329cd 410abc 398a 386abc 379ab 347a 309b 377a 357ab 347abc
SB 367a 390abc N/A 401ab 386a 364a N/A 371ab 371a 369a
TF 357abc 379bc N/A 398ab 378ab 360a N/A 363ab 362ab 361a
A + SB 345abcd 418ab 390a 395ab 387ab 374a 347ab 375ab 352ab 362a
A + TF 349abcd 431a 374a 404ab 390a 353a 354ab 372ab 353ab 358ab
LLA + SB 350abcd 413abc 374a 379bc 379ab 364a 343ab 345ab 343abc 349abc
LLA + TF 343abcd 407abc 373a 378bc 375ab 357a 326ab 339ab 330bc 338bc
RRA + SB 348abcd 407abc 401a 401ab 389a 371a 365a 366ab 342abc 361a
RRA + TF 344abcd 402abc 378a 401ab 381ab 370a 338ab 370ab 361ab 360a
A +SB + TF 364ab 428ab 388a 417a 400a 376a 361ab 369ab 356ab 365a
LLA + SB + TF 350abcd 402abc 389a 386abc 382ab 357a 334ab 358ab 340abc 347abc
RRA + SB + TF 354abcd 417abc 392a 409ab 393a 358a 350ab 376ab 362ab 361a
p-value
 N 0.9838 0.8416 0.1168 0.1327 0.5583 0.863 0.6824 0.286 0.9835 0.6704
 T 0.0003 0.0018 0.8074 < .0001 0.0005 0.5159 0.0212 0.0032 < .0001 < .0001

The LSMEANS with same letter are not significantly different at p-value of 0.05 level.

ADF, acid detergent fiber; DM, dry matter; N0, control without fertilizer; N112, nitrogen application 112 kg/ha; A, gunner conventional alfalfa; LLA, low-lignin Hi-Gest 360 LL alfalfa; RRA, roundup ready RR tonica alfalfa; SB, smooth bromegrass; N/A, not applicable; TF, tall fescue; N, nitrogen fertilizer; T, treatment.

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In the first cut in 2017, no significant differences in ADF concentration among the alfalfa monocultures, grasses, and legume-grass mixtures were observed. In the second cut of 2017, there were no significant differences among treatments, except the Roundup Ready alfalfa had a significantly lower ADF than a mixture of Roundup Ready alfalfa with smooth bromegrass (RRA: 309 g/kg DM, RRA + SB: 365 g/kg DM). In the third cut in 2017, the reduced-lignin alfalfa variety had significantly lower ADF than the Roundup Ready alfalfa (LLA: 335 g/kg DM, RRA: 377 g/kg DM). In the fourth cut of 2017, the low-lignin alfalfa had the lowest ADF (312 g/kg DM), and smooth bromegrass had significantly (p < 0.05) the highest ADF (371 g/kg DM). Nitrogen fertilizer application did not affect ADF concentration in all cuts.

Results in our study showed that, on average, ADF was significantly lower in the low-lignin alfalfa in both years than ADF in pure grasses and alfalfa-grass mixtures. The ADF concentration is related to the cell wall portion of the plants, which is made up of lignin and cellulose. Digestibility is dependent upon ADF; as ADF concentration increases, the digestibility decreases [36]. Our results also suggest that mixed cropping of alfalfa with other grasses or species may not only increase forage yield compared to grasses in monoculture but also decrease the risk of bloating [37].

Neutral detergent fiber

The NDF concentration in the first cut of 2016 was significantly lower in alfalfa monocultures compared to grasses in monoculture (Table 6). The low-lignin alfalfa variety had the lowest NDF (LLA: 460 g/kg DM, SB: 614 g/kg DM, TF: 574 g/kg DM). No difference in NDF was found in alfalfa-grass mixtures. Nitrogen fertilizer application significantly increased the NDF. With nitrogen application, the NDF was 534 g/kg DM, and without nitrogen NDF it was 513 g/kg DM. In the second cut of 2016, tall fescue had a significantly higher NDF than alfalfa in monocultures and alfalfa-grass mixtures. No significant differences were observed due to nitrogen application. In the third cut in 2016, the NDF was significantly lower in the low-lignin alfalfa monoculture than in these mixtures: A + SB, A + TF, RRA + SB, A + SB + TF. Nitrogen application had a significant effect on NDF concentration; the mean of NDF was significantly lower with nitrogen than without nitrogen (with nitrogen application NDF: 484 g/kg DM, without NDF: 496 g/kg DM). In the fourth cut of 2016, NDF was significantly higher in grass monocultures than in the other treatment (SB: 588 g/kg DM, TF: 585 g/kg DM). The low-lignin alfalfa had a significantly lower NDF than the grasses and mixtures, except for the mixture low-lignin alfalfa with tall fescue (LLA: 465 g/kg DM, LLA + TF: 487 g/kg DM). With nitrogen application, NDF was significantly higher than without nitrogen application.

Table 6. Mean NDF concentration (g/kg DM) in 2016 and 2017
Treatments 2016 2017
Cut 1 Cut 2 Cut 3 Cut 4 Average Cut1 Cut 2 Cut 3 Cut 4 Average
N 0 513b1) 518a 496a 508a 512b 475a 411a 465a 467a 458a
N 112 534a 522a 486b 515a 518a 482a 406a 464a 476a 462a
A 470ef 503b 476ab 491bc 486de 458c 396a 432b 459bc 436cde
LLA 460f 489b 441b 466c 465e 443c 398a 422b 412d 419e
RRA 470ef 513b 487ab 493bc 491cde 450c 388a 481ab 472bc 448bcde
SB 614a 548ab N/A 601a 588a 534ab N/A 520a 521a 525a
TF 574ab 573a N/A 608a 585a 550a N/A 518a 523a 530a
A + SB 528bcde 512b 508a 501bc 513cd 487bc 416a 474ab 483abc 465bc
A + TF 516bcdef 549ab 500a 517bc 520b 471c 424a 463ab 480abc 460bcd
LLA + SB 526bcdef 504b 481ab 506bc 504bcd 445c 395a 441b 439cd 430de
LLA + TF 500cdef 505b 473ab 471bc 487de 457c 400a 455ab 445bcd 440bcde
RRA + SB 536bcd 520ab 497a 519bc 518bc 484bc 415a 459ab 460bc 454bcd
RRA + TF 519bcdef 536ab 486ab 476bc 504bcd 469c 404a 437b 481abc 448bcde
A + SB + TF 546bc 514b 526a 520b 527b 484bc 420a 478ab 487ab 467b
LLA + SB + TF 526bcdef 498b 493ab 492bc 502bcd 481bc 420a 459ab 456bcd 454bcd
RRA + SB + TF 539bc 518ab 510ab 503bc 518bc 486bc 425a 467ab 482abc 465bc
p-value
 N 0.000 0.535 0.044 0.159 0.022 0.410 0.303 0.857 0.186 0.219
 T < 0.000 < .000 0.000 < .000 < .0001 < .000 0.042 < .000 < .000 < .0001

1) The LSMEANS with same letter are not significantly different at p-value of 0.05 level.

NDF, neutral detergent fiber; DM, dry matter; N0, control without fertilizer; N112, nitrogen application 112 kg/ha; A, gunner conventional alfalfa; LLA, low-lignin Hi-Gest 360 LL alfalfa; RRA, roundup ready RR tonica alfalfa; SB, smooth bromegrass; N/A, not applicable; TF, tall fescue; N, nitrogen fertilizer; T, treatment.

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In first cut in 2017, tall fescue had a significantly higher NDF concentration than the other treatments. There were no significant differences due to nitrogen application in all cuts. In the second cut in 2017, no significant differences were observed in NDF among combinations or due to nitrogen application. In the third cut in 2017, the low-lignin alfalfa variety, the conventional variety, and two mixtures (low-lignin alfalfa with smooth bromegrass and roundup ready alfalfa with tall fescue) had a significantly lower NDF concentration than smooth bromegrass and tall fescue. In the fourth cut, low-lignin alfalfa had the lowest NDF and grasses had the highest NDF (LLA: 413 g/kg DM, SB: 522 g/kg DM, TF: 523 g/kg DM). Nitrogen fertilizer application did not affect NDF.

Based on our average results for 2016 and 2017, the low-lignin alfalfa had the lowest NDF compared to mixtures and pure grasses. When the NDF concentration increases, DMI by animal decreases [38]. A higher fiber concentration usually occurs in grasses than in leguminous plants [3941]. Planting grasses as a mixture with alfalfa will improve forage nutritive value by reducing the fiber content. Our results showed that the low-lignin alfalfa monoculture had significantly lower ADF and NDF. Also, some mixtures with low-lignin alfalfa had a relatively lower fiber content, which can be a benefit to the producer.

Relative feed value

There was no interaction between species treatments and nitrogen fertilizer application in all cuts of both years for RFV (Table 7).

Table 7. Mean RFV indexes in 2016 and 2017
Treatments 2016 2017
Cut 1 Cut 2 Cut 3 Cut 4 Average Cut1 Cut 2 Cut 3 Cut 4 Average
N 0 109b1) 104a 110b 108a 108a 120a 142a 122a 124a 126a
N 112 113a 103a 115a 106a 107a 118a 143a 123a 121a 125a
A 126ab 107ab 114ab 113abc 115ab 125ab 151a 133a 124bcd 133ab
LLA 129a 115a 125a 123a 123a 129a 148a 139a 146a 140a
RRA 125abc 104ab 111ab 112abc 113bc 129a 156a 115ab 121bcde 130abcd
SB 91e 100ab N/A 89d 93f 106bc N/A 108b 108e 107e
TF 99de 96b N/A 89d 95ef 103c N/A 109b 108e 107e
A + SB 110bcd 102ab 108ab 108bc 107bcd 115abc 139a 117ab 119cde 122cd
A + TF 112abcd 94b 112ab 104c 105cd 122abc 135a 121ab 119bcde 124bcd
LLA + SB 109dc 105ab 116ab 110abc 110bcd 127a 147a 131ab 132bc 134ab
LLA + TF 116abcd 106ab 118ab 118ab 114ab 124ab 147a 128ab 133ab 133abc
RRA + SB 108de 103ab 108ab 104c 106cd 116abc 136a 123ab 127bcd 125bcd
RRA + TF 112abcd 101ab 114ab 113abc 110bcd 120abc 145a 129ab 118cde 128bcd
A + SB + TF 103de 101ab 105b 101dc 102cd 116abc 135a 118ab 117de 121d
LLA + SB + TF 109dc 108ab 111ab 112abc 110bcd 118abc 139a 125ab 128bcd 127bcd
RRA + SB + TF 106de 103ab 107ab 106bc 105cd 117abc 135a 119ab 117de 122cd
p-value
 N 0.003 0.689 0.033 0.0722 0.168 0.535 0.579 0.552 0.396 0.635
 T 0.000 0.003 0.044 < .0001 < .0001 < .0001 0.021 0.000 < .0001 < .0001

1) The LSMEANS with same letter are not significantly different at p-value of 0.05 level.

RFV, relative feed value; N0, control without fertilizer; N112, nitrogen application 112 kg/ha; A, gunner conventional alfalfa; LLA, low-lignin Hi-Gest 360 LL alfalfa; RRA, roundup ready RR tonica alfalfa; SB, smooth bromegrass; N/A, not applicable; TF, tall fescue; N, nitrogen fertilizer; T, treatment.

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The RFV in the first cut in 2016 was significantly higher in low-lignin alfalfa (LLA: 129) than in grasses (SB: 91, TF: 99), and alfalfa-grass mixtures (A + SB:110, RRA + SB:108, LLA + SB:109, A + SB + TF: 103, RRA + SB + TF: 106, LLA + SB + TF:109). The RFV with nitrogen application was significantly higher than in the control (113, 109). In the second cut in 2016, the RFV of reduced-lignin alfalfa was significantly higher than the RFV of tall fescue and a mixture of conventional alfalfa with tall fescue (LLA: 115, TF: 96, A + TF: 94). In the third cut in 2016, reduced-lignin alfalfa had a significantly higher RFV value than that of the mixtures of conventional alfalfa with smooth bromegrass and tall fescue (LLA: 125, A + SB + TF: 108). Also, nitrogen application had a significant effect on RFV; RFV was higher with nitrogen than without nitrogen (with nitrogen: 115, without 110). In the fourth cut in 2016, the highest RFV was in the low-lignin alfalfa (123) compared to other treatments. The smooth bromegrass and tall fescue had significantly lower RFV than the other treatments (SB: 89, TF: 89). No significant differences were observed in RFV values due to nitrogen application.

The RFV values in first cut in 2017 were significantly (p < 0.05) higher in low-lignin alfalfa, conventional alfalfa, and the mixture of reduced-lignin alfalfa with smooth bromegrass than monocultures of tall fescue and smooth bromegrass (LLA: 129, RRA: 129, LLA + SB: 127, SB: 106, TF: 103). No significant differences in RFV values were observed due to nitrogen fertilizer application.

In the second cut in 2017, no significant difference in RFV values among the treatments and nitrogen application was observed, this was probably due to the smaller portion in alfalfa-grass mixtures grasses, and alfalfa had a larger proportion in the mixtures. In the third cut in 2017, the RFV values were significantly higher in the reduced-lignin alfalfa and the conventional variety of alfalfa than in the smooth bromegrass and tall fescue (A: 133, LLA: 139, SB: 108, TF: 109). In 2017 in the fourth cut, the highest RFV value was in the low-lignin alfalfa and low-lignin alfalfa with tall fescue (LLA: 146, LLA + TF: 133). On average in 2016 and 2017, RFV was highest in LLA and lowest in monocultures of smooth bromegrass and tall fescue. The average RFV in 2016 was 123; in 2017, it was140. The RFV of SB in 2016 was 93 and for TF it was 95. In 2017, the RFV of SB was 107 and the RFV of TF was 107. In both years, there were no significant differences due to fertilizer application.

CONCLUSION

The study evaluated the DMY and forage nutritive value of alfalfa-grass mixtures as compared to alfalfa monocultures and grass monocultures for forage production. It also assessed the effect of nitrogen fertilization on dry matter production and forage nutritive value of the different perennial forage species grown in monoculture, in binary mixture, and in trio mixture. According to our study, the forage yields and nutritive value of alfalfa-grass mixtures were higher than monocultures of smooth bromegrass and tall fescue and equal to alfalfa monocultures. All treatments had higher DMY with nitrogen fertilizer application than without nitrogen application. The DMY of the low-lignin alfalfa variety was not significantly different from the DMY of the other two alfalfa varieties (conventional and roundup ready). The lowest forage nutritive value and yield was observed in monocultures of smooth bromegrass and tall fescue. Based on two production year data in 2016 and 2017, the results indicate that planting two most common grasses in Kansas such as smooth bromegrass and tall fescue with legume species such as alfalfa appears to be more desirable practice than monoculture grasses. Also, alfalfa varieties did not affect the DMY indicating that variety selection of alfalfa doesn’t appear to affect when alfalfa is being planted with cool-season grasses.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

This research was funded by the Department of Agronomy, Kansas State University.

Acknowledgements

Not applicable.

Availability of data and material

Upon reasonable request, the datasets of this study can be available from the corresponding author.

Authors’ contributions

Conceptualization: Min D.

Data curation: McDonald I.

Formal analysis: McDonald I, Baral R.

Methodology: McDonald I, Min D.

Software: Baral R.

Validation: Min D, Baral R.

Investigation: McDonald I.

Writing - original draft: McDonald I.

Writing - review & editing: Baral R, Min D.

Ethics approval and consent to participate

This article does not require IRB/IACUC approval because there are no human and animal participants.

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