Unlocking the Potential of Indian Pasture Legumes: Nutrition, Mineral, Gas and Methane Production Insights for Use in Semiarid Regions

This study evaluated five annual and eleven perennial Indian pasture legumes species for their nutritive value, dry matter, mineral contents and in vitro fermentation parameters, aiming to boost their potential as animal feed. Legume species significantly differed (p<0.05) in organic matter, crud protein (CP), ether extracts, fibres, and protein fractions. Clitoria ternatea (CT) a perennial had higher (p<0.05) buffer soluble protein (477), while neutral detergent soluble protein was highest in annually grown Lablab purpureus (420 g/kg CP). Atylosia scarabaeoides (AS) had higher NSC (392 g/kg DM) than SC (367 g/kg DM). Rapidly degradable fraction (51.7 g/kg tCHO) was lower (p<0.05) than other fractions of carbohydrate. Total digestible nutrients, digestible energy and metabolisable energy varied and values were higher for Desmenthus virgatus and lowest for Stylosanthas seabrana (SSe). The NE values for lactation (NEL), maintenance (NEM) and gain (NEG) were also higher for DV (6.6, 7.95 and 4.28) and lowest for SSe (3.54, 4.24 and 0.58 kJ/g DM), respectively. dry matter intake, digestible DM (DDM) and relative feed value of legumes differed (p<0.05) with mean values of 2.22% body weight, 592 g/kg DM and 102%, respectively. Annual grasses like Dolichos biflorus, Macroptilium atropurpureum, Rhynchosia minima (RM) were found better with micro minerals compared to other perennials. In vitro dry matter degradability, partition factor, short chain fatty acids and microbial protein production of legumes varied (P<0.05) and mean values were 578 g/kg DM, 5.61 mg DDM/mL, 2.42 mm/g and 352 mg/g, respectively. Gas and CH4 production (mL/g and mL/g DDM) varied (p<0.05) being lowest gas from AS (55.5) and highest from CT-blue (141), while CH4 production was lower (p<0.05) from CT-white and AS (8.24 and 9.14) and higher from Arachis glabrata (AG) and Arachis hagenbackii (AH) (15.2 and 15.1). Methane in total gas was low for DV, RM and CT-w (8.99, 9.72 and 9.51%) and loss of DE and ME as CH4 varied (P<0.05) amongst the legumes with mean values of 4.62 and 7.86%, respectively. Based on these findings, each legume offers unique benefits, allowing for tailored combinations of annual and perennial legumes to optimize rumen feed efficiency.

Keywords:

1. Introduction

Inadequate availability feed and forage coupled with their low nutritive value (crop residues, grasses of pasture and grazing lands) are the prime factors for poor animal production in tropics and subtropics. For ages forage legumes had played an important role in dairy and meat production [1]. Forage legumes species usually had high feeding value as they are generally rich in protein, provides substantial amount of energy, mineral and vitamins with higher intake and digestibility [2,3]. Even in grain-based feed lot livestock production, forage legumes are essential to maintain animal health [4]. In recent many researchers have reviewed the role of forage legumes, for sustenance of mixed crop-livestock production, sustenance of pasture and grazing lands along with ecosystem services [5,6,7]. Dietary supplementation of forage legumes not only increases livestock production primarily through higher intake, nutrient contents, digestibility than cereal crops-grasses, sorghum, maize etc. [8,9] but also improves the rumen fermentation efficiency through enhanced metabolisable energy, protein ratio and ruminal bypass protein availability to animals [10], increased N retention [11] and reduced methane emission [8,12,13]. Further the introduction of forage legumes in grass production areas as grass-legume mixture could be one of the promising strategies to mitigate the GHG emissions from pasture-based livestock production [7].

As per Food and Agriculture Organization of the United Nations (FAO; www.feedipedia.org) lists around 169 legume species are being used as forage and about 20 Mha land area in under forage legume monoculture [14]. Species of Aeschynomene, Arachis, Centrosema, Desmodium, Macroptilium, and Stylosanthes offer promise for improved tropical pasture systems [15]. Alike the wider morphological genetic variability in legume species [16] there exists nutritional variability [17]. In addition to genetic differences the nutritional composition of legumes also influenced by the season and growing location. So, to have correct information on legumes level of supplementation and nutritive value, their evaluation for chemical composition has a substantial impact on the understanding of their nutritional value and also a great influence on animal nutrition [8,18]. The chemical composition (CP, NDF, ADF and lignin) and total digestible nutrients TDN, in vitro digestibility has limited application for formulating precise diet specific to animal species and their physiological stages [19,20]. To have comprehensive information on feeding value of each legume, the present study was planned to evaluate 16 annual and perennial different legume species from nine genera for protein, cell wall constituents, carbohydrate fractions, protein fractions, energy contents, minerals, intake, digestibility and in vitro fermentation pattern (gas and methane production) for their judicious use in ruminant diets.

2. Materials and methods

2.1. Experimental Sites

The study was carried out at Plant Animal relationship Division, ICAR-Indian Grassland and Fodder Research institute, Jhansi (India). Laboratory procedures and animal management for donor sheep were carried out as per Institute animal ethics committee guidelines.

2.2. Sample collection and processing of forage legumes

Annual species: Dolichos biflorus (DB), Lablab purpures (LLP), Macroptilium atropurpureum (MA), Rhynchosia minima (RM), Stylosanthas hamata (SH); Perennial species: Arachis glabrata (AG), Arachis hagenbackii (AH), Atylosia scarabaeoides (AS), Clitoria ternatea-white (CT-W), Clitoria ternatea-blue (CT-B), Centrosoma pubescene (CPb), Desmenthus virgatus (DV), Stylosanthas scabra (SSc), Stylosanthas scofield (SSco), Stylosanthas seabrana (SSe) and Stylosanthus viscosa (SV) were collected randomly from plots (30×10 m) maintained by Grassland and Silvipasture Management Division of Institute. The legumes samples were harvested after monsoon (rainy season) growth in first week of September 2016. Collected samples were dried initially under shade on cemented floor and then in hot air oven at 60°C for 2-3 consecutive days. The DM contents were 36.03, 29.26, 22.21, 17.98, 16.62, 31.14, 28.48, 32.23, 29.13, 35.57, 20.40, 30.38, 39.04, 29.77, 15.83 and 17.71% for AG, AH, CP, DB, CT-w, SSe, SH, SSc, SSco, MA, AS, DV, RM, CT-b and LLP, respectively.

2.3. Chemical analyses

Samples dry matter (DM), crude protein (CP), ether extract (EE) and ash were estimated as per methods of AOAC (1995). CP of samples was estimated as Kjeldahl N × 6.25 by digesting in sulfuric acid and digestion mixture (Consisting of sodium/ potassium sulphate and copper sulphate in 10:1 ratio) using semi auto analyser (Kel Plus Classic-DX, Pelican). The EE was determined by refluxing samples in petroleum ether using extraction apparatus. For ash estimation samples were put in tarred silica basins, desmoked and then basins were put into a muffle furnace at 600^°C temperature for 4h. Neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose and lignin (ADL) were estimated by sequential procedure modified by Van Soest et al. [21] using fiber tech (Fibra Plus FES 6, Pelican). Heat labile alpha amylase and sodium sulphite were not used in NDF solution. For lignin (ADL) estimation sample left after ADF estimation were treated with 72% H₂SO₄ followed by ashing in muffle furnace.

2.4. Carbohydrate and protein fractionation

Carbohydrate fractions were estimated according to the CNCPS [22]. This is broadly classified into 4 fractions as follows:

a): C_A: rapidly degradable CHO including sugars.
b): C_B1: intermediately degradable starch and pectin.
c): C_B2: slowly degradable cell wall.
d): C_C: unavailable/lignin bound cell wall.

Total carbohydrate (tCHO g/kg DM) was determined by subtracting CP, EE and ash contents from 1000. Structural carbohydrates (SC) were calculated as the difference between NDF and neutral detergent insoluble protein and non fiber carbohydrates were estimated as the difference between total CHO and SC [23]. Starch estimated by samples extraction with 80% ethyl alcohol to solubilize free sugars, lipids, pigments and waxes. Residue rich in starch was solubilized with perchloric acid and extract was treated with anthrone-sulphuric acid to determine glucose colorimetrically using standard glucose [24].

Protein fractions of samples were partitioned into five fractions according to Licitra et al. [25]. These are as follows:

P_A: non-protein nitrogen (NPN), the difference between total nitrogen and true CP nitrogen precipitated with sodium tungstate (0.30 M) and 0.5 M sulfuric acid.

P_B1: buffer soluble protein, the difference between true protein and buffer-insoluble protein, estimated with borate-phosphate buffer (pH 6.7 to 6.8) and freshly prepared 10% sodium azide solution.

P_B2: neutral detergent soluble protein (NDSP), buffer-insoluble protein minus ND insoluble protein.

P_B3: acid detergent soluble CP, the difference between ND insoluble protein and acid detergent insoluble CP.

P_C: indigestible.

All fractions, including CP, were analysed in triplicate and N content determined by Kjeldahl. Crude protein was determined as Kjeldahl N × 6.25 using semi auto analyzer (Kel Plus Classic-DX Pelican India).

2.5. Dry matter intake, digestibility and energy calculations

Legumes dry matter intake (DMI), digestible DM (DDM), relative feed value (RFV), total digestible nutrients (TDN) and net energy for different animal functions i.e. lactation (NEl), gain (NEg) and maintenance (NEm), were calculated using the following equations (DMI = 120/NDF; DDM =88.9-0.779×ADF; RFV =(DDM×DMI)×0.775; TDN = 104.97−(1.302×ADF); NEl =(TDN×0.0245)-0.012; NEg=TDN×0.029)-1.01; NEM = (TDN×0.029)-0.29) of Undersander et al. [26]. Digestible energy (DE, KJ/g DM; DE= TDN×0.04409) and metabolizable energy (ME, KJ/g DM) values were calculated using the equations of Fonnesbeck et al. [27] and Khalil et al. [28], respectively. Metabolizable energy was calculated as 0.821 × DE.

2.6. Estimation of minerals

For minerals estimation legumes samples were wet digested with 3:1 HNO₃: perchloric acid mixture, cooled and filtered through Whatman 42 filter paper. The aliquot was used for estimation of calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) using an atomic absorption spectrophotometer (Varian AA 240) against their standards.

2.7. Donor animals and inoculum preparation

Four male adults of Jalauni sheep with mean body weight of 36.2± 0.217 kg were used as inoculums donors. These animals were maintained on sole berseen hay diet and had free access to clean drinking water. Rumen liquor was collected in a pre-warmed thermos from each animal before feeding using a perforated tube from stomach with the help of vacuum pressure pump. Collected rumen liquor was filtered through four layers of muslin cloth and thus strained rumen liquor obtained from different animals was mixed, kept at 39°C in water bath, gassed with CO₂ till used for mixing with incubating buffer media.

2.8. In vitro incubations

In vitro gas production was determined according to the pressure transducer technique of Theodorou et al. [29]. Ruminal fluid was collected in pre warmed thermos using a stomach tube before feeding from adult male Jalauni sheep maintained on berseem hay diet. Collected rumen fluid was filtered through a double layer of cheese cloth and bubbled with CO₂. The incubation medium (1L) was prepared by sequential mixing of buffer solution, macro-mineral solution, micro-mineral solution and resazurin solution (Menke and Steingass 1988). Incubation medium was fluxed with CO₂ till the pink colour turned colourless and then 250 mL rumen liquor was added to attain incubation medium: rumen liquor ratio of 80:20. Samples (0.5g) of air dry foliages were weighed into three 100 mL serum bottles. Three serum bottles without substrate/sample were used as blanks. Sample and control serum bottles were initially gassed briefly with CO₂ before adding 50 mL of inocuolum medium. Bottles were continuously fluxed with CO₂ and then sealed with aluminium crimps. Before incubation, the gas pressure transducer was used to adjust head space gas pressure in each bottle to adjust the zero reading on the LED display and then incubated at 39°C for 24 h to estimate the total gas production.

2.9. Methane measurements

Methane in total gas measured at 24h from three bottles incubated for each of the tree leaves was analysed by gas chromatography (Nucon 5765 Microprocessor controlled gas chromatograph, Okhla, New Delhi, India) equipped with a stainless-steel column packed with Porapak-Q and a Flame Ionization Detector. One ml gas sampled using a Hamilton syringe from total gas produced was injected manually (pull and push method of sample injection) into the GC, which was calibrated with standard CH₄ and CO₂. Methane was also measured from blank bottles incubated for 24h and used for correction of CH₄ produced from the inoculum. Methane measured was related to total gas to estimate its concentration [30] and converted to energy and mass values using 39.54 kJ/l CH₄ and 0.716 mg/mL CH₄ factors, respectively [31]. Short chain fatty acids (SCFA) were calculated using 24h gas production [32], while the partition factor (PF) and microbial mass (MBM) were estimated as described in previously described method [33].

2.10. Statistical analysis

The means of nutritional and gas fermentation parameters of legumes were compared using one way ANOVA of SPPS (version 16) with legumes as fixed factor and parameters as dependent variables. Post hoc multi comparison was performed using Duncan Multiple Range test to differentiate the means at p<0.05 level.

3. Results

3.1. Chemical composition

Legumes differed (p<0.05) in OM, CP, EE, NDF, ADF, cellulose and lignin contents and their mean values were 906, 125, 33.4, 549, 382, 281 and 93.9 g/kg DM, respectively (Table 1). The protein contents were > 150 g/kg DM in CP, CT-b and LLP and lower < 100 g/kg DM in DB, SSe, SH, SSc and SSco (87.9, 80.4, 93.5, 92.2 and 94.8 g/kg DM). The EE contends were higher in CT-w, CT-b and DV (48.3, 46.6 and 63.8 g/kg DM), while lignin contents were lower in LLP (62.7) and AH (68.1) against highest in SSe (134) and CT-w (124 g/kg DM). Stylosanthes species had higher ADF (401 to 508) and cellulose (297 to 365 g/kg DM) contents except (DB) than other evaluated legumes.

3.2. Protein and carbohydrate fractions

Contents of tCHO, SC and NSC varied (p<0.05) in legumes and their mean values were 747, 484 and 263 g/kg DM, respectively. Amongst the evaluated legumes AS is the legume which had higher NSC than SC (392 vs 367 g/kg DM Table 3). SC contents were highest in DB and lowest in AS (684 vs 367 g/kg DM). Carbohydrate fractions C_A, C_B1, C_B2 and C_C differed (p<0.05) in legumes and ranged between 143-589, 33.5-84.7, 4.30-551 and 221-428 g/kg tCHO, respectively. Rapidly degradable carbohydrate fraction C_B1 (51.7) was lower (p<0.05) than C_A (405), C_B2 (242) and C_C (302 g/kg tCHO), respectively.

Legumes protein fractions P_A, P_B1, P_B2, P_B3 and P_C differed (p<0.05 Table 2) and had mean values were 226, 295, 209, 167 and 102 g/kg CP, respectively. Legumes had highest (295) accumulation of rapidly degradable protein fraction (P_B1) against lowest (1023 g/kg CP) of lignin bound protein fraction (Pc). Clitoria species (CT-w and CT-b) and MA had higher P_B1 fraction (437, 477 and 430) and lowest P_C fraction (52.9, 71.6 and 75.2 g/kg CP).

Table 2. Carbohydrates (g/kg DM), its fractions (g/kg tCHO) and protein fractions (g/kg CP) of range legumes.

Legumes	Carbohydrate and its fractions							Protein fractions
Legumes	tCHO	SC	NSC	C_A	C_B1	C_B2	C_C	P_A	P_B1	P_B2	P_B3	P_C
AG	730^e	424^bc	306^ghi	470^f	71^g	127^bc	315^gh	223^de	394^fg	161^cd	116^cd	98.8^d
AH	739^f	430^bcd	309^ghi	439^ef	57^e	282^gh	221^a	228^de	374^ef	224^e	92.8^bc	81.5^c
CP	705^c	457^cde	248^def	459^ef	48^cd	153^cd	349^def	214^cd	185^b	305^g	202^ef	93.7^d
DB	781ⁱ	683^h	976^a	143^a	52^d	551^j	253^a	324^h	123^a	300^g	118^cd	134^fg
CT-w	725^de	500^f	225^cd	429^ef	37^a	121^bc	428^g	211^c	437^h	91.5^b	208f	52.9^a
SSe	823^L	561^g	261^ef	358^c	43^b	208^def	391^fg	184^b	352^e	172^d	178^e	113^e
SH	802^k	589^g	213^bc	278^b	61^e	365ⁱ	297^bc	277^g	232^c	346^h	60.8^a	83.7^c
SSc	780^j	483^ef	296^gh	406^de	37^a	332^hi	224^a	236^ef	182^b	292^g	139^d	150^h
SSco	773ⁱ	454^cde	319^hi	431^ef	58^e	251^efg	260^ab	238^ef	194^b	179^d	246^g	142^gh
SV	797^k	464^de	333ⁱ	414^de	65^f	277^fgh	243^a	214^cd	265^d	138^c	254^g	129^f
MA	689^b	415^b	274^fg	478^f	85^h	70^b	367^ef	165^a	430^h	252^f	78.3^ab	75.2^cd
AS	759^h	367^a	392ⁱ	589^g	43^b	4.3^a	364^def	250^f	205^bc	99.1^b	330^h	116^e
DV	721^d	458^cde	263^ef	410^de	49^cd	296^gh	245^a	215^cd	281^d	310^g	110^cd	82.9^c
RM	749^g	516^f	233^cd	375^cd	45^bc	254^efg	326^cde	210^c	418^gh	25.2^a	245^g	1015^d
CT-b	708^c	453^cde	254^def	450^ef	42^b	187^cde	321^cd	205^c	477ⁱ	33.8^a	213^f	71.6^b
LLP	676^a	484^ef	191^b	356^b	33^a	388ⁱ	223^a	215^cd	177^b	420ⁱ	76.8^ab	111^e
Mean	747	484	263	405	52	242	302	226	295	209	167	102
SEM	0.343	2.74	2.77	14.03	1.96	19.5	9.50	5.30	16.4	16.4	11.3	3.90
Significance	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001

NSC, non-structural carbohydrates; P_A, non-protein nitrogen; P_B1, buffer soluble protein; P_B2, neutral detergent soluble protein; P_B3, acid detergent soluble protein; P_C, indigestible protein; SEM, standard error of means; SC, structural carbohydrates; tCHO, total carbohydrates.

3.3. Energy, energy efficiency, intake, digestibility and relative feed value of legumes

The TDN, DE and ME contents of legumes varied (p<0.05) and their values were higher for DV (703, 12.9 and 10.6) and lowest for SSe (398, 7.28 and 5.99 kJ/g DM Table 3). Similarly, the NE values of legumes for lactation (NE_L), maintenance (NE_M) and gain (NE_G) were higher for DV (6.66, 7.94 and 4.28) and lowest for SSe (3.53, 4.24 and 0.582 kJ/g DM), respectively. DMI, DDM and RFV of evaluated legumes differed (p<0.05) with mean values of 2.22 % body weight, 592 g/kg DM and 102%, respectively.

Table 3. Energy value of range legumes.

Legumes	TDN	DE	ME	NE_L	NE_M	NE_G	DMI	DDM	RFV
AG	553^de	10.1^e	8.32^d	5.16^f	6.16^e	2.45^de	2.51^g	592^de	114.94^ef
AH	604^f	11.1^f	9.11^e	5.66^g	6.74^f	3.08^f	2.51^g	623^f	121.26^fg
CP	555^e	10.2^e	8.36^d	5.16^ef	6.16^e	2.50^e	2.19^def	593^e	100.45^cd
DB	511^ab	9.36^bc	9.69^bc	4.70^be	5.62^bc	1.96^bc	1.64^a	567^bc	72.20^a
CT-w	534^cde	9.82^cde	8.03^cd	4.95^cdef	5.91^cde	2.25^cde	2.12^cd	581^cde	95.48^c
SSe	398^a	7.28^a	5.99^a	3.54^a	4.24^a	0.58^a	1.99^bc	499^a	76.80^a
SH	525^c	9.65^cd	7.90^c	4.87^cde	5.78^c	2.12^c	1.93^b	575^c	86.16^b
SSc	498^b	9.150^b	7.48^b	4.58^b	5.49^b	1.83^b	2.20^def	559^b	95.65^c
SSco	528^cd	9.69^cde	7.94^cd	4.87^cde	5.82^cd	2.16^cd	2.27^ef	577^cd	101.46^cd
SV	520^ab	9.52^bc	7.82^bc	4.78^f	5.74^c	2.08^bc	2.20^def	572^bc	97.68^c
MA	552^de	10.1^de	8.32^d	5.12^bc	6.12^de	2.45^de	2.50^g	591^de	114.36^e
AS	605^f	11.1^f	9.11^e	5.66^g	6.78^f	3.08^f	2.73^h	623^f	131.73^h
DV	703^g	12.9^g	10.6^f	6.66^h	7.95^g	4.28^g	2.31^f	682^g	122.22^g
RM	611^f	11.2^f	9.19^e	5.74^e	6.82^f	3.16^f	2.02^be	627^f	97.92^c
CT-b	534^cde	9.77^cde	8.03^cd	4.95^b	5.91^cde	2.25^cde	2.21^def	580^cde	99.45^cd
LLP	613^f	11.2^f	9.23^e	5.74^e	6.86 ^f	3.20^f	2.16^de	627^f	104.94^d
Mean	553	10.15	8.40	5.12	6.12	2.47	2.22	592	102.04
SEM	2.04	0.029	0.029	0.021	0.025	0.025	0.011	1.22	0.548
Significance	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001

DM, dry matter; EE, ether extract; Lignin(sa), lignin solubilized with sulphuric acid; fibre expressed inclusive residual ash; NE_L, net energy for lactation; NE_M, net energy for maintenance; NE_G, net energy for growth; NSC, non-structural carbohydrates; OM, organic matter; SEM, standard error of means; TDN, total digestible nutrients.

3.4. Minerals

Micro minerals (Cu, Zn, Fe and Mn) of legumes varied (p<0.05) in range of 11.11-76.74, 20.77-65.85, 31.53-1288.04 and 18.61-70.51 ppm, respectively (Table 4). Ca and Mg varied from 0.55-2.77 and 0.25-0.88% with mean values of 1.26 and 0.42%, respectively.

3.5. Fermentation pattern

Fermentation parameters (DMD, ME, PF, SCFA and MBP) of the legumes incubated in sheep inoculums varied (p<0.05 Table 5) and their mean values were 578 g/kg DM, 5.88 kJ/g DM, 5.61mL/mg DM, 2.42 mm/g and 351.65 mg/g, respectively. Microbial protein production efficiency varied (p<0.05) from 0.32 for DV to 0.68 for CT-w.

3.6. Gas, methane and loss of energy as methane

In vitro gas and methane production (mL/g and mL/g DDM) of legumes varied (p<0.05 Table 6). Gas production (mL/g) was lowest from AS (55.5) and highest from CT-b (141), while CH₄ production was lower (p<0.05) from CT-w and AS (8.24 and 9.14) and higher from AG and AH (15.2 and 15.1). Methane in total gas varied (p<0.05) and was low for DV, RM and CT-w (8.99, 9.72 and 9.51%) and higher for MA, AG and AH (14.1, 14.0 and 13.9%), respectively. The loss of DE and ME as CH₄ varied (p<0.05) amongst the legumes with mean values of 4.62 and 7.86%, respectively.

4. Discussion

4.1. Chemical composition

Information on the chemical composition of forages has significant impact on the understanding of their nutritive value and animal production [8,18]. Legumes evaluated in present study except DB and Stylosanthes species had CP contents above 110 g/kg DM recommended to fulfil the protein requirement of growing cattle. Crude protein and NDF contents of Arachis, Centro, Stylo and Siratro legumes in range of 129-191 and 452-592 g/kg DM [34] were within range of our values baring Stylosanthes species and AS. The earlier reported CP contents of C. pubescens (221) and S. guyyanensis (179 g/kg DM) reported were higher than our values (172 and 80.4-105 g/kg DM), respectively [17]. The CP, EE, NDF, AFD, cellulose and lignin contents of C. pubesecens were found similar to earlier reports [17,35]. Whereas S. gyuyanensis was in lower contents than Stylosanthes except lignin. Further the EE contents of C. pubescens and S. guyanensis (24.0 and 47.0 g/kg DM) of these workers were similar to values of CPb (25.0) and SSc (43.2 g/kg DM), respectively. The OM, NDF and ADF contents in C. pubescens, S. hamata, and S. scabra was recorded as same by Musco et al. [36]. However, the lignin contents of C. pubescens (167) and S. scabra (187) were higher than our values (103 and 72.9 g/kg DM), respectively. The NDF and ADF contents of C. pubescens, Macroptelium bracteatum and M. gracile was at par with previous study [37]. Similarly, the range of CP, EE, OM, NDF, ADF, cellulose and lignin contents in Macroptilium species, Rhynchosia minima, S. humilis, Clitoria ternatea were found in coherence with other studies [38,39]. In contrast, significant differences in DM contents (154-253 g/kg DM) of 13 legumes were also recorded previously [40]. The CP content in 24 accessions of Arachis species was ranged from 14.7 to 22.55 [41] that was like our results whereas in a study it was recorded even at higher range (184-250 g/kg DM) [42]. Whereases OM and lignin contents (873-919) and 63-82 g/kg DM) were like values presented in present study. The CP (73-129), EE (15-36) and ADF (373-424) except NDF (416-510 g/kg DM) of ten Stylosanthes guyanensis varieties recorded by Li et al. [43] were within the range of our observed values of Stylosantehs species. Furthermore, the value chemical composition recorded in Arachis hypogeal, C. pubescens, Clitoria ternatea, M. atropurpureus and S. guyanensis were found similar of earlier studies [44,45].

4.2. Protein and carbohydrate fractions

Feeds protein fractions composition reflect rumen degradation rates that estimate the dietary nitrogen efficiency. Thus, the utilization level of the nitrogenous fraction is important at the evaluation of feeds and at the specification of nutritional requirements of ruminants [46]. Protein fractions (P_B1, P_B2, P_B3 and P_C) differed (p<0.05) across legumes may be attributed to differences in concentrations of CP and lignin. About 50‒150 g/kg CP of total forage N is bound to lignin, or rather, is unavailable to ruminal microorganisms [47] and our legumes values for P_C lies within this range (52.9 to 150 g/kg CP). The P_C fraction of Arachis pinto ten accessions in range of 178-276 g/kg CP [42] (Ferreira et al. 2012) was higher than our Arachis species values. Due to good source of protein the annual Lablab purpures is predominantly cultivated in dry and semi-dry tropical regions [48].

Carbohydrates constitute the main energy source of plants (500-800 g/kg DM) and play an important role in animal nutrition as a prime source of energy for rumen microorganisms [47]. Carbohydrate accumulation in fodder crops is influenced by several factors like plant species, variety, growth stage and environmental conditions during growth (Buxton and Fales 1994). Mlay et al. [49] reported mean tCHO content of 746 g/kg DM for Macropttlium atropurpurus legume which was higher than our values (689 g/kg DM). The higher tCHO contents for blue (800) and white (830 g/kg DM) varieties of Clitoria ternatea than our CT-w (725) and CT-b (708 g/kg DM) values was reported earlier [38]. The tCHO (626-701 g/kg DM) for ten Arachis pinto accessions found marginally lower than our AG and AH values [42]. Higher SC in DB, SSe, SH and RM may be due to their higher NDF contents recorded in the present study. The legumes AG, AH, CPb, MA, CT-b and AS had higher contents of C_A fraction (> 44 g/kg tCHO) and feeds higher in this fraction are considered good energy sources to stimulate rumen microorganism growth [50] and the synchronism between the protein and carbohydrate digestion rates, having an important effect on the end products of fermentation and on animal production [51]. Low contents of unavailable carbohydrate fraction (C_C) in AH, DB, SSe, DV, SV and LLP may be due to their lower lignin contents. This indigestible fraction with C_B2 usually affects animal intake by the rumen fill, which can reduce animal performance [52]. In our study we recorded that DB, DV, RM, SH and LLP legumes with higher hemicelluloses contents (315, 253, 258, 221 and 220 g/kg DM) had higher C_B2 fraction contents (551, 296, 254, 365 and 388 g/kg tCHO) which is more slowly degraded in the rumen, impacting microbial synthesis and animal performance [53]. Higher hemicellulose concentrations result in higher concentrations of C_B2 fraction. Carvalho et al. [50] reported that NDF concentration influences carbohydrate fraction C_B2 and forages high in NDF concentration usually have higher values of C_B2. Our results partially agree with these observations that most of the legumes with higher NDF contents had higher C_B2 fraction value. Additionally, annual forage RM rich in flavonoids and found suitable for long-term rangelands improvement [54,55].

4.3. Energy, energy efficiency, intake, digestibility and relative feed value of legumes

Feed or fodder nutritive value is a function of its dry matter intake and its ability to provide the nutrients in the right proportion required by animals for different physiological functions [56]. Greater calculated DMI value for AG, AH, MA and DV (2.51, 2.51, 2.50 and 2.73%) than other legumes (1.64-2.31%) may be due to lower NDF contents. The NDF of forages has been negatively correlated with DMI which is always not consistent, although the NDF is positively related with resistance to comminution [57]. Lowest and highest DMD of SSe and DV legumes may be due to their highest (501) and lowest (266 g/kg DM) ADF contents, respectively.

Musco et al. [36] reported higher NE_L values for C. pubescens, S. hamata and S. scabra (9.53, 6.16 and 5.49 kJ/g DM) than our recorded values (5.16, 4.88 and 4.58 kJ/g DM), respectively. Similarly, ME values of C. pubescens were higher (6.53) than our values (5.78 kJ/g DM) [58]. The ME contents (9.44-10.36 kJ/g DM) of 13 legumes were higher than ME values observed in present study [40]. The TDN consists of digestible nutrients that are available for livestock and is primarily related to forage ADF contents. With increase in ADF there is decline in TDN resulting unavailability of forage nutrients to animals [59]. The relatively higher values for TDN (634 g/kg DM), GE (17.2), DE (11.5) and ME (9.44 kJ/g DM) were recorded than values of MA in present study [49].

The IVDMD of C. pubescens and S. guyanensis (530 and 570 g/kg DM) reported earlier was like our values of CPb (513) and SSe and SV (578 and 580 g/kg DM) but lower to SH (641, SSc (631) and SSco (646 g/kg DM), respectively [17]. The OMD of C. pubescens (478), S. hamata (609) and S. scabra (593) were relatively higher than our values (457, 447 and 471 g/kg DM), respectively. Tona et al. [35] reported higher ME and OMD for C. pubescens than our values. The DMD and OMD (614-712 and 586-688 g/kg DM) for five forage legumes [37] were more than range values (395-663 and 375-526 g/kg DM) of legumes evaluated in present study. Similarly, the mean OMD and DMD (656 and 684 g/kg DM) were higher than average OMD and DMD values of tested legumes. The higher values for OMD (642-739 g/kg DM) in legume forage than our values are mentioned earlier[40]. Fernandes et al. [60] recorded DMD of ten Arachis species over three years in range of 501-632 g/kg DM. The values of IVOMD between 600 to 740 g/kg DM for 24 accessions of Arachis pinto substantiate our DMD values of Arachis species [41]. Highest and lowest TDN values for DV and SSe in present study may be attributed to differences in their digestibility and lower and higher ADF contents as ADF increased TDN values decreases.

The NDF and ADF contents are negatively associated with OMD and ME values of legumes [40]. Intake an important part of forages nutritive value is partially related to cell wall content and bulkiness of forages. Lowest value of DMI for DB (1.64) versus highest of AS (2.73%) may be attributed to their maximum (730) and minimum NDF contents (440 g/kg DM) as the NDF contents are negatively associated with DMI [47].

The relative feed value (RFV) of ten varieties of S. guyanensis in range of 102.8-130.5% [43] were higher than values determined for Stylosanthes species (70.80-101.46%). The values of IVDMD and TDN of A. hypogeaea (78.86 and 71.44), C. pubescens (59.89 and 51.30), Clitoria ternatea (74.15 and 60.20) M. atropurpureus (66.51 and 56.15) and S. guyanensis (66.22% and 54.43%) which were higher than our values for these legumes [45]. The DMD of Arachis species (AG and AH) was higher (650 and 663 g/kg DM) except LLP than other legume species is similar to previous observations [61] where reported that DMD of Arachis species in general is greater than other tropical legumes.

4.4. Minerals

The Ca contents (0.50-0.80%) of legumes studied here were found relatively lower than earlier reported [17], while Mg contents (0.32-0.63%) were like our values of legumes. Similar trend of Ca and Mg contents were reported in other study [62]. Legumes Fe, Cu, Mn and Zn contents in range of 441-494, 2.42-7.14, 55.2-61.3 and 34.8-65.3 ppm [62] were more or less similar to our micro mineral values. In contrast, the relatively lower Ca (1.01-1.62%) and higher Mg (0.92-1.96%) of different Arachis pinto cultivars under humid and sub-humid environment were reported than our values [63]. Further the Cu, Fe, Zn and Mn reported for this legume were inconsistent to our values. Macro (Ca and Mg) and micro minerals (Cu. Zn, Fe and Mn) of white and blue varieties of Clitoria ternatea reported earlier [38] were more or less similar to our CT-w and CT-b values. Fernandes et al. [60] reported Ca and Mg values in range of 1.4-2.47% and 0.08-0.59% for ten Arachis species over three years were similar to our Arachis species values. Macro (Ca and Mg) and micro minerals (Cu, Fe, Zn and Mn) of four herbaceous legumes and two browse legumes reported by [63] were in partial agreement to our mineral values. The variation in mineral contents of legume species may be due to differences in soil mineral contents, growth stage, fertilizer application and environment conditions [64,65].

4.5. Fermentation pattern

The 1.15 µmole SCFA content for C. pubescens legume at 24h of fermentation was reported earlier [35]. The partition factor in range of 3.07-4.94 mg/mL reported earlier [36] was lower than PF values (3.45-7.08) of legumes evaluated in present study. The reason for higher PF values for evaluated legumes may be ascribed to less gas production due to lower DMD.

Partition factor an indicator of fermentation efficiency of a feedstuff is expressed as volume of gas (mL) produced per mg of substrate degraded. Legumes mean PF values were higher (5.61) than the theoretically possible maximum value (4.14) [33]. Higher PF values for CT-w (7.05) could not translate to higher microbial mass probably due to their less gas production. Higher SCFA for DV, LLP, SSc and RM may be due to their higher gas production as reported. For DV legume microbial protein production was lowest (143 mg/g) and methane production was highest (26.45 mL/g DDM) which is consistent with previous observations [66]. Greater PF values for CT-w did not translate to greater microbial mass as noted in previous study [67]. We expected with higher PF to translate to greater microbial mass as PF is the measure of efficiency of microbial production. We can only speculate the reason for this observation, but higher SCFA value noted for DV consistent with previous report that microbial mass and SCFA are inversely related [68]. It may be that DV partitioned more energy into SCFA versus microbial mass production, while in case of LLP energy partition was well distributed between SCFA and microbial mass production.

4.6. Gas, methane production and loss of energy as methane

One of the options to improve the feed efficiency through efficient rumen fermentation is that dry matter conversion to methane is less and utilization by animal is more. Methane an end-product of rumen fermentation causes 2-12% loss of dietary energy [69]. This loss of feed energy as methane varies with its quality [70,71,72,73] and animal species. Methane % of gas between 15.9 to 18.4%) from 13 legumes [40] was higher than our recorded values for 16 legumes (9.0 to 14.1%). Our values for proportion of methane in total gas except for DV, RM and CT-b were within the value (12.3-15.9%) reported earlier [62]. Lopez et al. [74] categorised methane reduction potential as low potential (% of CH₄ in gas between 11 and 14%). So, legumes evaluated in present study had low methane production potential. Furthermore, gas production was higher from AG and AH (110.25 and 108.92) than MA and CPb (90.46 and 99.14 ml/g) as recorded in present study [34]. A higher gas production for S. hamata and S. scabra (195 and 193) than C. pubescens (136 mL/g OM) were reported and these values were higher with similar gas production pattern [36]. Similarly, a report on higher gas production at 24h for C. pubescens (25.4 mL/200mg) than our values was published [58]. Like our observations [40] recorded significant difference in gas production (42.56 to 51.42 mL/200mg) from hays of 13 legumes and these values were higher than our values (55.46 to 141.26 mL/g) from 16 legumes and these differences may be due to our lower OMD values as low OM fermented for gas production. Further the methane production in range of 7.36-8.78 mL/200mg was higher than our methane production values. The differences in gas and methane production of evaluated pasture legume species may be ascribed to the variation in their chemical composition and degradation. Such variation in methane and gas production of legume species has been recorded earlier [40,75,76,77].

The proportion of methane to total gas production is an important indicator of methane emission potential of feed/fodder degradation. This ratio of methane to gas for common feeds (hays, concentrate, mixture of hays and concentrate) vary between 16-20%. Methane production from Desmodium intortum (3.67), Medicago sativa (5.90) and Vicia sativa (5.73 mL/200mg DM) legumes observed by [62] was also higher than most of our legumes but similar to Arachis species. Maccarana et al. [78] reported gas production, methane production and CH₄% of total gas in range of 72-480 mL/g DM, 7.3-77.5 mL/g and 9.4-40.61% from 390 observations of 30 studies. Relatively lower values of gas production, methane production and CH₄% total gas in present study may be due to sheep rumen liquor as values of gas and methane production and CH₄% total gas were higher when bovine rumen liquor was used for incubations [79,80]. Greater methane production may be attributed to their higher IVDMD values. In line of present findings, previous studies reported that feedstuffs with higher gas production and IVDMD tended to have higher methane production per gram DM incubated [81]. Methane emission differences within legumes may be attributed to the variability in their chemical constituents as reported earlier [82,83,84]. The differences in relative proportion of CH₄ to energy values (CH₄% of DE and % of ME) may be attributed to variations in legumes energy values, level of methane production, dry matter degradability. Variability in proportion of methane to energy of feeds and fodders has been reported earlier [62,84].

5. Conclusions

Range legumes evaluated in present study had CP more than 100 g/kg DM except for Stylosanthes species. Legumes AG, AH, DV and AS had higher energy values and relative feed values along with lower accumulation of structural carbohydrates. The CH₄% of total gas and CH₄ (% of DE and ME) were lowest for DV and RM legumes.

Author Contributions

Conceptualization, S.S., T.S., P.K. and Y.R.; methodology, S.S., T.S., P.K., M.C. and U.Y.A.; formal analysis, S.S., T.S. and P.K.; investigation, S.S., T.S. and B.K.B.; writing—original draft preparation, S.S., T.S., P.K., M.C. and Y.R; writing—review and editing, S.S, T.S, P.K., U.Y.A., M.C. and Y.R.; visualization, P.K. and U.Y.A.; supervision, S.S., B.K.B. and Y.R.; project administration, S.S. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Not applicable.

Acknowledgments

Authors are thankful to Director IGFRI for providing financial support and Head Plant Animal Relationship Division for providing laboratory and animal facilities to carry this research work.

Conflicts of interest: The authors have no conflicts of interest.

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Table 1. Chemical composition of range legumes (g/kg DM).

Legumes	OM	CP	EE	NDF	ADF	Cellulose	Lignin	Hemi cellulose
AG	873^b	123^f	20.2^ab	479^b	381^cd	2725^e	101^ef	976^a
AH	878^c	116^e	23.4^abc	478^b	342^b	273^e	68.1^ab	136^bcd
CP	905^g	172ⁱ	27.5^cd	549^cde	380^c	266^c	103^ef	169^d
DB	897^f	88^b	28.1^cd	730^h	414^ef	325^e	82.4^bcd	316^g
CT-w	919ⁱ	146^h	48.3^f	566^f	396^cde	270^c	124^gh	171^d
SSe	930^L	80^a	26.6^cd	604^g	501^g	365^g	134ⁱ	103^ab
SH	923^j	93^c	27.0^cd	624^g	403^e	297^d	99.2^e	221^e
SSc	915^h	92^c	43.2^ef	548^cde	423^f	345^f	72.9^abc	124^abc
SSco	886^d	95^c	18.5^a	529^cd	401^de	307^de	83.8^bc	129^abc
SV	927^k	105^d	24.6^bcd	545^cde	407^ef	320^g	80.7^bc	139^bcd
MA	855^a	141^g	24.9^bcd	482^b	382^cd	271^e	105^ef	99.0^a
AS	894^e	105^d	29.7^d	439^a	342^b	213^b	115^fg	98.2^a
DV	928^kl	143^gh	63.8^g	519^c	266^a	177^a	73.5^abc	253^ef
RM	934^m	143^gh	41.5^e	595^fg	337^b	229^b	1027^ef	258^f
CT-b	937ⁿ	183^j	46.6^f	544^cde	396^cde	296^d	94.6^de	147^cd
LLP	896^e	180^j	40.3^e	556^de	335^b	267^c	62.7^a	220^e
Mean	906	12.54	33.4	549	382	28.09	93.9	168
SEM	2.12	0.283	0.421	-	1.51	6.93	1.69	2.87
Significance	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001

AG, Arachis glabrata; AH, Arachis hagenbackii; AS, Atylosia scarabaeoides; CPb, Centrosoma pubescene; CT-b; Clitoria ternatea(blue) CT-w, Clitoria ternatea(white); DV, Desmenths virgatus; DB, Dolichos biflorus; LLP, Lablab purpures; MA, Macroptilium atropurpureum; RM, Rhynchosia minima; SSe, Stylosanthas seabrana; SH, Stylosanthas hamata, SSc, Stylosanthas scabra, SSco, Stylosanthas scofield; SV, Stylosanthus viscosa.ADF, acid detergent fibre expressed inclusive residual ash; CP, crude protein; EE, ether extract; Lignin(sa), lignin solubilized with sulphuric acid; NDF, neutral detergent fibre expressed inclusive residual ash; OM, organic matter; SEM, standard error of means.

Table 4. Macro (%) and micro minerals (ppm) of range legumes.

Legumes	Cu	Zn	Fe	Mn	Ca	Mg
AG	76.7^h	65.8^g	1150^cde	47.5^c	2.77^f	0.59^j
AH	60.7^g	61.1^g	737^bc	47.3^c	2.74^f	0.58^j
CP	28.7^ef	42.2^e	246^ab	31.9^ab	1.48^e	0.37^e
DB	35.7^f	31.9^bc	1271^de	70.5^ef	1.37^e	0.41^g
CT-w	24.9^cde	41.7^de	311^ab	40.8^bc	0.60^a	0.37^f
SSe	14.9^ab	35.0	215^ab	20.3^a	0.94^c	0.24^e
SH	11.1^a	26.7^ab	259^ab	77.37^f	0.80^b	0.25^ab
SSc	13.6^ab	32.0^bc	174^a	22.1^a	1.34^e	0.28^bc
SSco	16.1^abc	48.7^f	1023^cd	54.5^cd	1.47^e	0.29^cd
SV	12.4^ab	20.8^a	137^a	27.2^a	1.44^e	0.46^h
MA	21.1^bcde	30.1^bc	1573^e	61.4^e	1.17^d	0.34^ef
AS	18.5^abcd	28.5^bc	1288^de	72.3^f	0.93^c	0.36^ef
DV	27.3^def	27.1^ab	32.8^a	28.3^ab	0.93^c	0.88^k
RM	59. 6^g	25.5^ab	183^a	18.6^a	0.58^a	0.32^de
CT-b	72.0^h	35.3^cd	31.5^a	41.9^bc	0.55^a	0.53i
LLP	62.8^g	39.3^de	39.4^a	41.6^bc	0.98^c	0.44^gh
Mean	34.8	37.0	542	44.0	1.26	0.42
SEM	0.726	0.544	41.41	1.08	0.11	0.003
Significance	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001

SEM, standard error of means.

Table 5. Partition factor (PF), short chain fatty acids (SCFA), degradable dry matter and microbial protein production from fermentation of range legumes in sheep inoculum.

Legumes	ME kJ/g	DMD	PF	SCFA mm/g	MBM mg/g	EMBP mg/g
AG	6.07^cd	649^fg	6.09^cde	2.44 ^cd	421^efg	0.63 ^cd
AH	6.04^cd	663^g	6.35^cde	2.41 ^cd	445^g	0.64 ^cd
CP	5.77^bcd	513^b	5.35^bc	2.20^bcd	306 ^bc	0.60 ^bcd
DB	6.11^cd	577^cd	5.26^bc	2.54 ^cd	346 ^cde	0.56^bcd
CT-w	4.86^ab	559^cd	7.05^de	1.80^ab	392^defg	0.68^d
SSe	5.61^bc	578^cd	5.49^bc	2.41 ^cd	355 ^cdef	0.59 ^bcd
SH	5.82^bcd	641^fg	6.20^cde	2.37^cd	417 ^defg	0.63 ^cd
SSc	6.18^cd	631^efg	5.60^bcde	2.61^de	385^defg	0.59 ^bcd
SSco	5.78^bcd	646 ^fg	6.25^cde	2.42 ^cd	424 ^efg	0.63^cd
SV	5.84^bcd	580 ^cd	5.24^bc	2.54 ^cd	342 ^cd	0.57 ^bcd
MA	5.59^bc	594^cde	7.08^e	2.00^abc	407 ^defg	0.66 ^d
AS	4.49^a	394^a	5.53^bc	1.64^a	238^b	0.58 ^bcd
DV	6.53^cd	409^a	3.45^a	2.77^de	142^a	0.32^a
RM	6.25^cd	547^bc	4.82^abc	2.60 ^cde	302 ^bc	0.53^bc
CT-b	6.43^cd	602^de	4.37^ab	3.13^e	301 ^bc	0.48^b
LLP	6.73^d	660^g	5.56^bcd	2.76^de	402 ^defg	0.58 ^bcd
Mean	5.88	578	5.61	2.42	352	0.58
SEM	0.080	10.4	0.151	0.059	11.00	0.013
Significance	0.001	<.0001	<.0001	<.0001	<.0001	<.0001

DMD, dry matter degradability; ME, metabolisable energy; MBM, microbial protein production; EMBP, efficient of microbial protein production; PF, partition factor (gas mL/mg DM); SFCA, short chain fatty acids; SEM, standard error of means.

Table 6. Gas production, methane production and loss of energy as methane from in vitro fermentation of range legumes in sheep inoculum.

Legumes	Gas mL/g	CH₄ mL/g	Gas mL/g DDM	CH₄ mL/g DDM	CH₄% Gas	CH₄%DE	CH₄% ME
AG	110^cde	15.2^d	170^ab	23.5^b	13.9 ^bc	5.77^ef	7.03 ^ef
AH	109^cde	15.1^d	164 ^ab	22.7 ^b	13.9 ^bc	5.23 ^cdef	6.37 ^cdef
CP	99.1^bcd	11.0^bc	192 ^abc	21.4 ^b	11.1 ^abc	4.14 ^abc	5.04 ^abc
DB	114^de	13.0^cd	199 ^abc	22.6 ^b	11.5 ^abc	5.31 ^def	6.47^def
CT-w	81.3^b	8.24^a	145 ^a	14.7 ^a	10.1 ^ab	3.23 ^a	3.94^a
SSe	108^cde	11.5^bc	188 ^abc	20.1 ^ab	10.8 ^abc	6.10^f	7.43 ^f
SH	107^cde	11.5^bc	166 ^ab	18.1^ab	11.2 ^abc	4.61 ^bcd	5.61 ^bcd
SSc	117^de	12.8^cd	186 ^abc	20.4 ^b	11.3 ^abc	5.37 ^def	6.54 ^def
SSco	109^cde	11.9^bc	169 ^ab	18.6^ab	11.4 ^abc	4.75 ^bcde	5.79 ^bcde
SV	115^de	11.4^bc	197 ^abc	19.7 ^ab	10.0 ^a	4.60 ^bcd	5.60 ^bcd
MA	90.4^bc	12.4^cd	153 ^a	21.0^b	14.1 ^c	4.72 ^bcde	5.75 ^bcde
AS	55.4^a	9.1^ab	188 ^abc	23.2^b	12.5 ^abc	3.17 ^a	3.86^a
DV	122^def	10.8 ^bc	309^d	26.4 ^c	8.99^a	3.24 ^a	3.94^a
RM	117de	11.4 ^bc	215^bc	21.0^b	9.72 ^a	3.92 ^ab	4.78 ^ab
CT-b	141^f	13.4^cd	235 ^c	22.3 ^b	9.51^a	5.27 ^cdef	6.42 ^cdef
LLP	124^ef	12.8 ^cd	190 ^abc	19.4 ^ab	10.4^abc	4.49 ^bcd	5.46 ^bcd
Mean	107	12.0	192	21.0	11.3	4.62	5.63
SEM	1.80	0.219	5.86	0.495	0.315	0.134	0.163
Significance	<.0001	<.0001	<.0001	0.010	0.041	<.0001	<.0001

DDM, dry matter; ME, metabolisable energy; SEM, standard error of means.

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