Appraisal of Spatial Distribution and Fibre Degradability of Cereal–Legume Fodders to Enhance the Sustainability of Livestock Feed Supply in Sub-Tropics

Abstract

Fodder scarcity, inadequate nutritional quality, and lack of degradation kinetics research are among the serious concerns hindering sustainable development of livestock globally. Rumen degradation kinetics data on neutral detergent fibre (NDF) in buffaloes are lacking for most tropical forage species. This study evaluated the effect of forage species, family, and growing location on NDF concentration and in situ degradability of six tropical cereal and four legume fodder crops. The fodder crops were grown following uniform recommended agronomic practices at three different agroecological locations and harvested at the appropriate growth stage: cereals at booting and legumes at 50% flowering. Later, the dried ground forage samples were incubated in the four rumen-cannulated Nili-Ravi buffalo cows in a four × two × three split-plot design for 0, 4, 8, 16, 24, 48, 96, and 168 h. The degradation fractions and degradability, expressed either on an NDF or dry matter basis, were considerably affected by forage family (cereal vs. legume) and growing locations and their interaction. Legume fodders degraded more rapidly but to a lesser extent than cereal fodders. The chemical components, notably the NDF, showed a significant but moderate negative relationship with the effective NDF degradability. Among studied fodders, the legumes had a significantly lower NDF concentration and subsequent degradability than the cereals. Although the cereals showed a slower rate of NDF degradation, their overall degradability was higher. The agro-climatic variability among three locations strongly impacted the NDF concentrations and fractions in the tropical forages. Effective NDF degradability was also correlated with the fodders’ NDF concentration, especially in cereals where the nature of the correlation was negative. In conclusion, the nutritional composition and NDF degradation parameters of the fodders were significantly affected by the species, family, and location of growth and their interactions. These results will help to improve agronomy and usage of these important fodder crops.

1. Introduction

World fodder supplies are insufficient to fulfill the livestock demand. The global forage feed market is projected to register a 4.5% increase in fodder demand by 2030. A huge portion of the rural communities raising livestock in sub-tropical areas of Asia and Africa will be challenged with fodder insecurity, restricted supply, and poor quality, especially in the dry seasons, owing to climate change [1]. Forages have been and should be the base of modern animal production systems due to competition between ruminants and humans for an increasing demand for food and feed [2]. Forage species constitute the main source of feed for ruminants because they prefer fibrous plant material consumption owing to the presence of microbial fermentation. Although forages are cheap to use as a feed source for animals, the quality and composition of forages is a matter of great concern for meeting the requirement of the animals for optimal performance. Significant fluctuations in the quality of the forages might be responsible for the variable performance of the ruminants [2,3].

The dietary concentration and the digestibility of neutral detergent fibre (NDF) is the most important measure in defining the quality of forage resources with respect to animal performance [4]; the digestibility is determined by the rate and extent of degradation of the NDF fraction [5,6]. These digestibility parameters vary among the forage species and families due to bindings among and within various nutrients [7,8]. In general, the dry matter digestibility values of legume forages are better than those of cereal ones when studied in vivo or in situ, considering rumen as a single compartment [9]. The legumes’ cell wall bonding and chemical structure are different from the cereal forages, containing more lignified material and fewer free bond structures, thereby offering more resistance to degradation [7].

Other than the species and family of the forage, several studies have demonstrated the effects of growing locations in terms of temperature and precipitation on soil conditions and forage quality for ruminants [10,11,12]. It has been suggested that growing location and conditions, by affecting the duration of the growth phase and growth dynamics, impact most chemical parameters of forages [12,13]. Several classical studies also revealed that increased environmental temperatures and reduced precipitation caused extended fibre growth and reduced its availability to the ruminants, along with a reduction in the protein contents [14].

Pakistan is a country with varied agro-ecological zones endowed with a range of climatic conditions with a significant livestock production footprint. However, we do not have much information on fodder quality and nutrition attributes as affected by geo-climatic and production conditions. We therefore designed this study to demonstrate the impact of three geographically different locations based on annual rainfall, temperature, and altitude from the sea level (about 400 km apart) on the forage fibre concentrations and fractions in tropical forages. The impact of agro-climatic variability was further combined with forage family (cereal vs. legume) in order to see which forage family was more affected than the other. We hypothesized that forages grown in different agro-ecological zones with inconsistent rainfall and temperature patterns would contain fluctuating fibre concentrations and degradability that would affect the nutritional quality and acceptability by the ruminants. The outcomes of this research will help to identify and develop a strategy for sustainable high-quality fodder supply for livestock in the sub-tropics.

2. Materials and Methods

2.1. Forage Sampling

Forage species studied were barley (Hordeum vulgare L.), maize (Zea mays L.), millet (Pennisetum glaucum L.), oats (Avena sativa L.), sorghum (Sorghum bicolor L. Moench), wheat (Triticum aestivum L.), berseem (Trifolium alexandrinum L.), jantar (Sesbania bispinosa Jacq.), lucern (Medicago sativa L.), and mustard (Brassica napus L.). Further details on these forage species have been previously described [9]. Their plant height and dry biomass yields are presented in Figure 1a,b. Summer crops were sown in mid-March and winter crops in late November by following uniform locally recommended agronomic practices for land preparation, seeding, irrigation, and plant nutrition. All forage species were sown in three agro-ecological zones, i.e., Bahawalpur (29.39° N, 71.68° E), Lahore (31.55° N, 74.35° E), and Rawalpindi (33.58° N, 73.04° E), representing southern, central, and northern regions of the Punjab Province of Pakistan, respectively (Figure 2). At each zone, each species was sown in 3 plots ~100 m apart to produce local replicates. The soil profiling attributes of the three locations are presented in

Table 1. Soil profiling attributes of three growing locations.

Location	OM (%)	Soil Texture	EC (dS/m)	Soil pH	Available P (mg/kg)	Available K (mg/kg)	Available N (mg/kg)	PP (mm)	Temp (°C)	Topography	Ht (m)
Rawalpindi	1.42	Clay loam	0.36	7.9	11.12	112	0.062	1200–1400	−3.9 to 42.0	Hilly subterrain	530
Lahore	0.87	Clay loam	0.67	7.8	14.3	158	0.054	900–1100	−2.0 to 45.0	Flat and slopes towards south	210
Bahawalpur	0.57	Sandy loam	1.03	8.2	10.5	134	0.038	150–250	4.0 to 48.0	Flat and slopes towards south-east	117

OM = organic matter; EC = electrical conductivity; PP = annual precipitation; Temp = temperature ranges; and Ht = Average height from the sea level.

. The agro-climatic conditions of the three growing locations are presented in Figure 3.

Around 10 kg of herbage samples were harvested from each plot and all materials were pooled. The cereals were harvested at the booting stage whereas the legumes at 50% flowering. The fresh herbage was chopped with a chaff cutter (Toka 510, Patiala Agri-Industries, Faisalabad, Pakistan) to a nominal length of 20 mm. The chaffed material was dried for 3 to 7 days under shade to reduce moisture content. The dried samples were passed through a hammer mill (POLYMIX PX-MFC, Kinematica AG, Germany) to obtain a particle size of 2 mm to be used for in situ evaluation and 1 mm to be used for chemical analyses. The sieved materials were stored in small, plastic jars at room temperature.

2.2. Chemical Analyses

The ingredients of the diets offered to the animals were sampled fortnightly during the entire study period of 70 days including the adaptation period of 14 days. Fresh, chopped forage DM was determined at 60 °C for 48 h and that of dry ingredients at 105 °C for 16 h according to ref. [15]; method 7.003. Ash, CP (6.25 × N), ether extract, and NDF were determined according to ref. [15]; method 923.03, method 7.015, and method 7.062, and [16,17], respectively.

2.3. Maintenance of Cannulated Animals

This study was conducted at the Department of Livestock Management and the Livestock Farm of the Directorate of Livestock Farms, The Islamia University of Bahawalpur (29.39° N, 71.68° E), Bahawalpur, Pakistan. All experiments were conducted according to the criteria of the University’s Animal Care and Management Committee (The IUB, 2015).

The in situ incubations were conducted in four rumen-cannulated (Bar Diamond, Parma, ID, USA) dry, non-pregnant Nili-Ravi buffalo cows (mean live weight = 529 ± 33.4 kg, age = 2635 ± 49.5 days, parity no. = 3.5) according to a 4 × 2 × 3 split-plot design with factorial arrangements of treatments. The animals were offered a standard diet based on fresh maize/sorghum fodder, commercial concentrate and cotton seed cake of the mean chemical composition, and feed intakes of various nutrients shown in

Table 2. Mean chemical composition and intakes of the diets offered to rumen-cannulated animals (g/kg DM unless otherwise stated).

Item
Chemical composition (g/kg diet DM)
DM (as fed)	508.5
CP	119.25
EE	27
NDF	494.5
NFC	215
Ash	144
Intake (g/d/animal)
DM	9430
CP	1256
EE	284
NDF	5207
NFC	2264
Ash	1516

DM = dry matter; CP = crude protein; EE = ether extract; NDF = amylase-treated neutral detergent fibre; and NFC = non-fibre carbohydrates.

. The animals were individually fed and confined to individual stalls (2 × 2.5 sq. m.) with all-time access to fresh drinking water.

2.4. In Situ Incubations and Degradation Profiles

The NorFor standards [18] were used to determine the NDF degradation profiles and the procedures have been described in detail previously [5]. The in situ incubations were conducted from June to November 2022 in batches. The time duration for each batch consisting of five feeds was approximately one week, with a one-week interval between the batches. The sieved fodder samples (one g feed giving 15 mg per cm² of the bag surface) contained in sewn and glued polyester bags (11 × 8.5 cm (10 × 7.5 effective size), pore size 33 µm (PES material 140/37), and 25% free bag space (Sefar AG, Heiden, Switzerland)) were incubated inside the rumen of each cannulated animal for 0, 4, 8, 16, 24, 48, 96, and 168 h using an all-in system. The placement of the samples was such that one sample per incubation period was placed into each animal making four experimental replicates per feed and incubation interval. The bags were retrieved, washed, and then stored at –18 °C at the end of each incubation interval. All frozen bags were thawed and washed with tap water at room temperature (20 °C) at the completion of each batch and the residues were analysed for amylase-treated NDF using the Van Soest et al. [17] technique later modified by Mertens et al. [16].

2.5. Data Analyses and Curve Fitting

The in situ degradation data were categorised as washing loss or washable fraction (a, 0 h values for washed samples) and non-washable fraction. The non-washable fraction was further divided into potentially degradable (b) and indigestible fractions, which corresponded to the degradation and residue at the final incubation interval, respectively. A first-order kinetic model with intercept and lag [19,20] was fitted to the data obtained from in situ degradation:

$$Y_t=a+b\times (1-\mathrm{e}\mathrm{x}\mathrm{p}(-K_d(t-L)\left)\right)$$

(1)

where Y_t denotes the degraded fraction at a given time t, K_d denotes the fractional degradation rate of fraction b, L denotes the lag time (h) for t > L, and t denotes the time of incubation (h). Table Curve 2D, version 5.0 (Systat Software, Inc., San Jose, CA, USA) was used for model fitting. Effective ruminal NDFD was calculated as:

$$NDFD=a+\left[b\times \frac{K_d}{K_d+K_p}\right]$$

(2)

assuming the fractional rate of passage (K_p) to be 0.05/h (a 20 h rumen retention time) according to a single-pool model for forages [21].

The statistical analyses were performed using the GLM procedure of Minitab 16.1.1.0. The data on in situ parameters were pooled and averaged against buffalos making four experimental replicates against a single feed using the model

Y_ijkl = µ + F_i + Sj(F_i) + L_k + (F × L)_ik + [S(F) × L]_ijk + E_ijk

(3)

in which Y_ijkl is the dependent variable, µ is the overall mean, F_i (n = 2) shows the effect of ith forage family (cereal vs. legume), Sj (n = 10) shows the effect of jth forage species, L_k (n = 3) shows the effect of kth location of sown forage, (F × L)_ik and [S(F) × L]_ijk show the effect of the interaction of main factors, and E_ijkl is the residual error. Results were considered significant when p ≤ 0.05 and trends when 0.05 ≤ p ≤ 0.10 and are presented as least square means with standard error of the means.

3. Results and Discussion

3.1. Forage Species and Family

The variability in terms of significance among feeds and locations was presented in

Table 3. Effect of forage family, forage species, and location of growth on in situ neutral detergent fibre degradation kinetics and effective degradability of cereal and legume fodders sown at three locations viz. Bahawalpur, Lahore and Rawalpindi. The p values are presented in this table where p ≤ 0.05 shows a significant effect.

In Situ Parameters 1	NDF	aNDF	bNDF	KdNDF	LNDF	NDFD24 3	NDFD48 3	NDFD168 3	aDM	bDM	KdDM	LDM	NDFD%DM
F 2	<0.001	0.084	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.016
S(F) 2	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
L2	0.006	0.012	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
F × L2	<0.001	0.002	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.015	<0.001
S(F) × L2	<0.001	0.043	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

NDF = neutral detergent fibre (NDF: g/kg dry matter); a = washable fraction representing the portion of NDF that had disappeared at time 0; b = potentially degradable NDF fraction. The estimate of K_d from the in situ method represents the fractional rate of degradation of fraction b; NDFD24, NDFD48, and NDFD168 = NDF degradability at 24, 48, and 168 h of incubation, respectively; DM = dry matter. ¹ Degradation parameters described according to the model by Ørskov and McDonald [20]. ² F = forage family; S(F) = forage species nested under forage family; L = location of growth; F × L = interaction effect of F and L; S(F) × L = interaction effect of S(F) and L. ³ Effective NDFD calculated from data assuming the fractional rate of passage (K_p) to be 0.05/h for forage as used by the protein evaluation system of Hvelplund and Weisbjerg [21].

. The experimental feedstuffs showed a wide variation in the chemical composition and degradation characteristics of NDF. The NDF composition of these forages ranged from 442 to 686 g/kg of DM for cereals and 350 to 553 g/kg DM for legumes. All degradation data were analysed collectively; however, tables were arranged based on a wide classification of cereals and legume fodders (

Table 4. Effect of forage family, forage species, and location of growth on in situ neutral detergent fibre degradation kinetics and effective degradability expressed on an NDF basis of cereal and legume fodders sown at three locations viz. Bahawalpur, Lahore and Rawalpindi. The values are presented as least square means (Location × Species (Family)) and (Location × Family) with standard error of mean (SEM) unless otherwise stated.

				In Situ Parameters 1
Location	Family	Species	NDF Content	aNDF	bNDF	KdNDF	LNDF	NDFD24 2	NDFD48 2
Bahawalpur	Cereal	Barley	563.4 d	0.004 ± 0.009 c	0.91 ± 0.009 ab	0.060 ± 0.002 b–g	0.80 ± 0.010 no	0.39 a–f	0.45 a–f
		Maize	596.1 b	0.004 ± 0.0160 c	0.83 ± 0.010 c–g	0.048 ± 0.002 d–k	0.81 ± 0.031 m–o	0.29 f–m	0.35 g–l
		Millet	626.8 a	0.004 ± 0.004 c	0.78 ± 0.004 f–i	0.043 ± 0.001 e–k	0.89 ± 0.011 i–n	0.24 i–n	0.31 h–m
		Oat	442.4 f	0.013 ± 0.009 bc	0.94 ± 0.011 a	0.078 ± 0.002 ab	0.76 ± 0.010 o	0.50 a	0.55 a
		Sorghum	585.1 c	0.024 ± 0.008 bc	0.86 ± 0.003 a–e	0.058 ± 0.002 c–i	0.85 ± 0.015 k–o	0.39 b–g	0.44 b–g
		Wheat	541.8 e	0.062 ± 0.009 bc	0.84 ± 0.009 b–g	0.062 ± 0.001 b–f	0.86 ± 0.023 j–o	0.42 a–e	0.49 a–c
	Legume	Berseem	375.4 i	0.022 ± 0.007 bc	0.77 ± 0.005 g–i	0.090 ± 0.004 a	0.91 ± 0.029 h–n	0.45 ab	0.47 a–e
		Jantar	402.2 h	0.002 ± 0.002 c	0.61 ± 0.006 mn	0.060 ± 0.004 b–h	1.09 ± 0.023 c–e	0.23 k–n	0.27 k–o
		Lucern	351.6 j	0.028 ± 0.006 bc	0.63 ± 0.005 l–n	0.089 ± 0.007 a	1.01 ± 0.018 d–h	0.37 b–h	0.38 d–j
		Mustard	433.80 g	0.015 ± 0.004 bc	0.74 ± 0.005 h–j	0.067 ± 0.001 b–d	0.93 ± 0.010 f–l	0.34 c–k	0.38 d–j
Lahore	Cereal	Barley	685.8 a	0.055 ± 0.006 bc	0.79 ± 0.004 e–i	0.037 ± 0.001 jk	0.95 ± 0.010 f–l	0.27 h–m	0.33 h–m
		Maize	685.0 a	0.006 ± 0.003 c	0.79 ± 0.006 e–i	0.052 ± 0.002 c–k	0.86 ± 0.013 j–o	0.29 f–l	0.35 g–l
		Millet	640.1 c	0.010 ± 0.005 c	0.79 ± 0.004 e–i	0.052 ± 0.003 c–k	0.91 ± 0.021 g–n	0.29 f–l	0.34 h–m
		Oat	631.7 d	0.027 ± 0.005 bc	0.81 ± 0.005 d–h	0.048 ± 0.002 d–k	0.89 ± 0.004 i–n	0.31 e–l	0.36 f–k
		Sorghum	671.7 b	0.017 ± 0.007 bc	0.79 ± 0.009 e–i	0.043 ± 0.004 e–k	0.89 ± 0.014 i–n	0.26 h–n	0.29 i–n
		Wheat	573.9 e	0.080 ± 0.002 a–c	0.81 ± 0.002 d–h	0.061 ± 0.004 b–g	0.94 ± 0.014 f–l	0.44 a–d	0.48 a–d
	Legume	Berseem	458.1 h	0.024 ± 0.004 bc	0.73 ± 0.005 i–k	0.063 ± 0.002 b–e	0.92 ± 0.026 g–m	0.35 b–i	0.38 e–j
		Jantar	540.1 g	0.007 ± 0.006 c	0.69 ± 0.007 j–l	0.058 ± 0.003 c–i	0.94 ± 0.025 f–l	0.28 g–m	0.33 h–m
		Lucern	429.9 i	0.048 ± 0.006 bc	0.66 ± 0.007 k–m	0.066 ± 0.003 b–d	1.02 ± 0.025 d–g	0.35 b–j	0.38 d–j
		Mustard	552.6 f	0.091 ± 0.005 ab	0.44 ± 0.009 p	0.041 ± 0.004 h–k	1.44 ± 0.017 a	0.22 l–n	0.26 l–o
Rawalpindi	Cereal	Barley	592.1 b	0.070 ± 0.002 a–c	0.81 ± 0.005 d–h	0.039 ± 0.002 i–k	0.96 ± 0.020 f–j	0.30 f–l	0.36 f–k
		Maize	610.0 a	0.022 ± 0.001 bc	0.88 ± 0.006 a–d	0.054 ± 0.003 c–j	0.85 ± 0.013 l–o	0.35 b–j	0.41 b–h
		Millet	610.2 a	0.016 ± 0.006 bc	0.75 ± 0.003 h–j	0.042 ± 0.002 g–k	0.94 ± 0.012 f–l	0.24 k–n	0.31 h–m
		Oat	593.8 b	0.028 ± 0.003 bc	0.89 ± 0.004 a–c	0.044 ± 0.002 e–k	0.82 ± 0.005 m–o	0.33 d–l	0.39 c–i
		Sorghum	560.1 c	0.017 ± 0.004 bc	0.73 ± 0.005 i–k	0.043 ± 0.002 f–k	0.96 ± 0.025 f–k	0.24 i–n	0.29 j–o
		Wheat	481.3 e	0.148 ± 0.005 a	0.85 ± 0.012 b–f	0.045 ± 0.004 e–k	0.99 ± 0.018 e–i	0.45 a–c	0.49 ab
	Legume	Berseem	405.0 g	0.001 ± 0.002 c	0.56 ± 0.008 no	0.068 ± 0.006 bc	1.11 ± 0.010 cd	0.24 j–n	0.27 k–o
		Jantar	464.4 f	0.008 ± 0.006 c	0.59 ± 0.016 m–o	0.043 ± 0.002 f–k	1.03 ± 0.036 d–f	0.18 mn	0.24 m–o
		Lucern	350.1 h	0.005 ± 0.002 c	0.45 ± 0.002 p	0.048 ± 0.004 d–k	1.18 ± 0.017 bc	0.16 n	0.19 o
		Mustard	506.1 d	0.039 ± 0.004 bc	0.53 ± 0.004 o	0.033 ± 0.002 k	1.23 ± 0.013 b	0.16 n	0.20 no
		SEM	1.55	0.015	0.014	0.004	0.021	0.020	0.018
Bahawalpur	Cereals		559.3 a	0.11 ± 0.006 a	0.75 ± 0.007 a	0.061 ± 0.002 b	0.82 ± 0.017 b	0.37 a	0.43 a
	Legumes		390.8 b	0.01 ± 0.007 b	0.66 ± 0.007 b	0.079 ± 0.002 a	0.97 ± 0.019 a	0.35 b	0.38 b
Lahore	Cereals		598.0 a	0.06 ± 0.007 a	0.71 ± 0.008 a	0.049 ± 0.003 b	0.91 ± 0.020 b	0.31 a	0.36 a
	Legumes		495.2 b	0.01 ± 0.009 b	0.63 ± 0.010 b	0.057 ± 0.003 a	1.08 ± 0.025 a	0.30 ab	0.34 ab
Rawalpindi	Cereals		574.6 a	0.07 ± 0.007 a	0.73 ± 0.008 a	0.045 ± 0.003 ab	0.92 ± 0.020 b	0.32 a	0.38 a
	Legumes		442.7 b	0.03 ± 0.009 b	0.59 ± 0.010 b	0.048 ± 0.003 a	1.14 ± 0.025 a	0.18 b	0.23 b
		SEM	1.54	0.006	0.006	0.002	0.010	0.009	0.008

NDF = neutral detergent fibre (NDF: g/kg dry matter); a = washable fraction representing the portion of NDF that had disappeared at time 0; b = potentially degradable NDF fraction. The estimate of K_d from the in situ method represents the fractional rate of degradation of fraction b; NDFD24, NDFD48, and NDFD168 = NDF degradability at 24, 48, and 168 h of incubation, respectively; SEM = standard error of the mean. ¹ Degradation parameters described according to the model by Ørskov and McDonald [20]. ² Effective NDFD calculated from data assuming the fractional rate of passage (K_p) to be 0.05/h for forage as used by the protein evaluation system of Hvelplund and Weisbjerg [21]. The different letter (a–p) within the same column show significant differences among the treatments.

and

Table 5. Effect of forage species and location of growth on in situ neutral detergent fibre degradation kinetics and effective degradability expressed on a dry matter basis of cereal and legume fodders sown at three locations viz. Bahawalpur, Lahore and Rawalpindi. The values are presented as least square means (Location × Species (Family)) and (Location × Family) with standard error of mean (SEM) unless otherwise stated.

					In Situ Parameters 1
Location	Family	Species	aDM	bDM	KdDM	LDM	NDFD168DM 2
Bahawalpur	Cereal	Barley	0.44 ± 0.008 k–n	0.95 ± 0.004 a	0.060 ± 0.002 c–h	2.18 ± 0.185 b–f	0.73 c
		Maize	0.41 ± 0.005 m–p	0.90 ± 0.007 b–d	0.048 ± 0.007 d–j	3.25 ± 0.546 a–c	0.67 e–h
		Millet	0.38 ± 0.009 p–r	0.86 ± 0.017 b–e	0.043 ± 0.002 f–j	3.92 ± 0.341 ab	0.63 j–l
		Oat	0.56 ± 0.010 c–e	0.97 ± 0.004 a	0.077 ± 0.004 a–c	−1.23 ± 0.605 gh	0.78 b
		Sorghum	0.43 ± 0.007 k–n	0.92 ± 0.00 a–c	0.058 ± 0.001 c–i	1.96 ± 0.250 b–f	0.70 cd
		Wheat	0.49 ± 0.009 h–j	0.92 ± 0.008 b–d	0.062 ± 0.004 c–g	0.10 ± 0.625 fg	0.71 cd
	Legume	Berseem	0.64 ± 0.005 ab	0.91 ± 0.002 no	0.091 ± 0.003 a	−4.56 ± 0.170 ijk	0.82 a
		Jantar	0.59 ± 0.006 bc	0.84 ± 0.003 jk	0.060 ± 0.001 c–h	−7.49 ± 0.203 l	0.67 e–g
		Lucern	0.66 ± 0.004 a	0.87 ± 0.006 o	0.089 ± 0.001 ab	−6.61 ± 0.140 kl	0.78 b
		Mustard	0.57 ± 0.010 c–e	0.89 ± 0.007 g–i	0.067 ± 0.002 c–e	−3.51 ± 0.233 h–j	0.70 cd
Lahore	Cereal	Barley	0.35 ± 0.008 q–s	0.86 ± 0.005 c–e	0.037 ± 0.001 j	5.64 ± 0.403 a	0.60 l
		Maize	0.32 ± 0.009 s	0.86 ± 0.005 d–g	0.052 ± 0.003 d–j	5.38 ± 0.306 a	0.64 i–k
		Millet	0.37 ± 0.009 p–r	0.88 ± 0.005 b–e	0.047 ± 0.002 e–j	3.59 ± 0.357 a–c	0.64 h–k
		Oat	0.39 ± 0.006 o–q	0.88 ± 0.002 b–e	0.048 ± 0.004 e–j	3.65 ± 0.308 a–c	0.65 g–j
		Sorghum	0.34 ± 0.011 rs	0.86 ± 0.008 d–g	0.043 ± 0.007 f–j	5.44 ± 0.220 a	0.61 kl
		Wheat	0.47 ± 0.011 h–k	0.89 ± 0.007 d–f	0.061 ± 0.002 c–h	0.50 ± 0.426 e–g	0.69 d–f
	Legume	Berseem	0.55 ± 0.004 d–f	0.88 ± 0.008 f–i	0.063 ± 0.002 c–f	−2.62 ± 0.180 hi	0.69 d–f
		Jantar	0.46 ± 0.002 i–l	0.83 ± 0.001 i	0.058 ± 0.004 c–i	−0.01 ± 0.189 fg	0.65 g–j
		Lucern	0.59 ± 0.003 b–d	0.86 ± 0.008 ij	0.066 ± 0.004 c–e	−4.99 ± 0.543 i-k	0.67 d–f
		Mustard	0.49 ± 0.006 g–i	0.69 ± 0.016 mn	0.041 ± 0.004 h–j	−7.97 ± 0.174 l	0.54 m
Rawalpindi	Cereal	Barley	0.45 ± 0.009 j–m	0.89 ± 0.002 d–g	0.039 ± 0.002 ij	1.49 ± 0.473 c–f	0.64 i–k
		Maize	0.40 ± 0.006 n–p	0.92 ± 0.005 ab	0.054 ± 0.004 d–j	2.85 ± 0.463 b–e	0.69 d–f
		Millet	0.40 ± 0.005 n–p	0.85 ± 0.004 e–h	0.042 ± 0.003 g–j	2.93 ± 0.200 b–d	0.62 j–l
		Oat	0.42 ± 0.005 l–o	0.94 ± 0.002 a	0.044 ± 0.006 f–j	3.38 ± 0.434 a–c	0.69 d–f
		Sorghum	0.45 ± 0.002 j–m	0.85 ± 0.006 hi	0.043 ± 0.002 f–j	0.65 ± 0.049 d–g	0.62 j–l
		Wheat	0.59 ± 0.010 cd	0.93 ± 0.007 b–e	0.045 ± 0.002 f–j	−4.16 ± 0.597 ij	0.69 de
	Legume	Berseem	0.59 ± 0.004 b–d	0.82 ± 0.006 k	0.068 ± 0.002 b–d	−6.85 ± 0.192 kl	0.66 f–i
		Jantar	0.54 ± 0.006 e–g	0.81 ± 0.004 jk	0.043 ± 0.004 f–j	−5.10 ± 0.609 jk	0.62 j–l
		Lucern	0.46 ± 0.003 i–l	0.69 ± 0.006 lm	0.048 ± 0.002 d–j	−2.62 ± 0.638 hi	0.55 m
		Mustard	0.51 ± 0.009 f–h	0.76 ± 0.009 l	0.033 ± 0.002 j	−7.76 ± 0.389 l	0.57 m
		SEM	0.008	0.005	0.004	0.431	0.006
Bahawalpur	Cereals		0.45 b	0.45 a	0.058 b	1.697 b	0.70 a
	Legumes		0.62 a	0.30 b	0.077 a	−5.544 a	0.74 b
Lahore	Cereals		0.37 b	0.42 a	0.048 b	4.033 b	0.64
	Legumes		0.53 a	0.35 b	0.057 a	−3.9 a	0.64
Rawalpindi	Cereals		0.45 b	0.43 a	0.045 b	1.191 b	0.66 a
	Legumes		0.53 a	0.32 b	0.048 a	−5.582 a	0.60 ab
		SEM	0.534	0.00	0.0016	0.19	0.70

aDM = washable fraction representing the portion of neutral detergent fibre (NDF) that had disappeared at time 0 expressed on dry matter (DM); bDM = potentially degradable NDF fraction expressed on DM. The estimate of K_dDM from the in situ method represents the fractional rate of degradation of fraction b; NDFD168DM = NDF degradability at 168 h of incubation expressed on DM; SEM = standard error of the mean. ¹ Degradation parameters described according to the model by Ørskov and McDonald [20]. ² Effective NDFD calculated from data assuming the fractional rate of passage (K_p) to be 0.05/h for forage as used by the protein evaluation system of Hvelplund and Weisbjerg [21]. The different letter (a–s) within the same column show significant differences among the treatments.

) to better discuss the results. The regression model (Equation (1)) fitted well to the data with R² and fit standard error of (0.980 and 0.0565) and (0.975 and 0.0452) for cereal and legume fodders, respectively. The forage species ranked in order of decreasing effective NDFD were wheat > oats > barley > maize > sorghum > millet (cereals) and berseem > lucern > jantar > mustard (legumes), averaged against all growing locations. Forage species significantly influenced all degradation fractions and degradability expressed either on an NDF or DM basis. The degradation behaviour of winter cereal (barley, oat, and wheat) fodders was different from that of summer cereal (maize, millet and sorghum) fodders in the sense that winter cereals showed a greater washable (p < 0.05) and potentially degradable fraction (p = 0.057) resulting in an increased extent of degradability (p < 0.05) at 24, 48, or 168 h of incubation than the summer cereals; however, no differences (p > 0.05) were noticed for K_d values. The same degradation pattern was observed for winter legume (berseem, lucern, and mustard) fodders when compared with the summer legume (janter) fodders except that the K_d was greater in the summer legume fodders.

When cereal fodders were compared with legume fodders, we noticed that the legumes were degraded more rapidly (p < 0.001) but to a lesser extent (p < 0.001) than were cereals when expressed on an NDF basis. All other degradation fractions such as the washable fraction and the degradability were superior in cereals (p < 0.001). Our results for superior cereal fodders’ NDF degradability agree with the results of Sarwar et al. [22] for sub-tropical cereal and legume forages reported in cannulated Nili-Ravi buffalo calves. These results also agree with those obtained in vitro by Calabrò et al. [23] which compared the NDF degradation of some cereal and legume forages using the gas production technique. The values of K_d and NDFD reported in our study and those by Sarwar et al. [22] and Khan et al. [5] are also comparable. Our reported degradability values for legumes are also similar to those of Aufrère et al. [24] for temperate lucern. However, the values of a, K_d, and NDFD were superior for legumes than cereals when expressed on the basis of DM. The reason why the results of the degradation fractions and the degradability were reversed when presented on an alternative basis (NDF vs. DM) is unknown. We might expect that greater values of a might have caused this variation.

When degradabilities at various incubation intervals were compared, it was noticed that a 15 and 12 percentage unit increase in NDFD was recorded for winter cereals when incubation time intervals were raised from 24 to 48 h (p < 0.001) and then from 48 to 168 h (p < 0.001), respectively. Similarly, the corresponding values for summer cereals were recorded as 19 and 18 percentage units for NDFD for the same incubation intervals (p < 0.001). While legume fodders showed linear increases of 12 and 13, and 20 and 18 percentage units in the NDFD of winter and summer legume fodders, respectively, when raised from 24 to 48 h and then from 48 to 168 h of incubation, overall, cereal fodders were recorded to show a greater percentage unit increase in NDFD in the first interval (24 to 48 h; 16%; p > 0.05) than the legume fodders (14%); however, this value was same for the second interval (48 to 168 h; 15%; p > 0.05).

3.2. Growing Location

The growing location significantly influenced the NDF concentration, degradation fractions, and effective degradability of the cereal and legume forages, expressed either on an NDF or DM basis. The interaction effects between main factors were also significant (Table 3). Location-wise mean values of various degradation fractions (Location × Species (Family)) and (Location × Family) are presented in Table 4 and Table 5. Comparative degradability and degradation curves of the potentially degradable NDF fraction of cereals and legumes fodders at different locations at various incubation periods are presented in Figure 4 and Figure 5, respectively. Different forage species presented an apparently similar dry biomass yield at varying locations except for maize, sorghum, berseem, and lucern, whose yields corresponded well to the plant height (Figure 3). Among the cereal fodders, oat showed the highest NDFD, and it was highest at Bahawalpur. However, potential NDFD at the other two locations was lower than for the other cereal fodders. Wheat showed a similar NDFD at all three locations within the cereal fodders. Berseem showed the highest NDFD at Bahawalpur within the legume fodders. The agro-climatic area Bahawalpur remained favourable for plant growth and subsequent composition and degradability for all the forage species studied. All degradation fractions and NDFD were substantially reduced in the forage species grown at Rawalpindi, with more prominent reduction in the values of legume fodders.

Different environmental conditions lead to the location effect on the fodders’ digestibility because the climatic conditions including rainfall and temperature modulate the soil fertility modifying the growth dynamics, leaf to stem ratio, and chemical and nutrient composition of forages [10,11,13]. Perotti et al. [12] suggested that temperature can affect the growing season length. Warmer temperatures can extend the growing season, allowing for more biomass accumulation and potentially higher forage yields. However, if temperatures are too high, certain plants may become stressed, leading to decreased digestibility and nutrient content. Temperature can influence plant metabolism and nutrient cycling. Warmer temperatures can increase metabolic rates, potentially leading to faster growth but also affecting nutrient allocation within the plant. Extreme temperatures can disrupt metabolic processes, affecting the synthesis of structural components and secondary metabolites. Temperature can influence the composition of plant cell walls, which affects forage digestibility. Warmer temperatures may result in increased lignin content in some plants, making them less digestible. Conversely, cooler temperatures may favour the accumulation of more-digestible carbohydrates.

Melo et al. [25] have described the factors of variability in forage production and digestibility. They mentioned that adequate rainfall was essential for plant growth and nutrient uptake. The drought stress caused by insufficient rainfall may lead to reduced forage yield and quality. During drought conditions, plants may allocate more resources to survival mechanisms, such as producing defensive compounds or reducing growth, which can affect digestibility. Rainfall can affect soil moisture levels, which in turn influence nutrient availability to plants. Adequate moisture promotes nutrient uptake, leading to higher protein and mineral content in forages. However, heavy rainfall can cause leaching of soluble nutrients from the soil, potentially reducing their concentration in forage plants. This “dilution effect” can impact the overall nutrient content and digestibility of forages.

Altitude generally correlates with cooler temperatures. Cooler temperatures can slow down plant growth rates and affect the types of plants that thrive at higher elevations. This can impact the maturity and composition of forage plants, potentially influencing their digestibility. Cooler temperatures may also affect microbial activity in the rumen of grazing animals, which can further influence digestibility [26]. Altitude can shorten the growing season due to colder temperatures and a shorter period of favourable weather conditions. This shorter growing season can result in forage plants reaching maturity at an earlier stage, potentially leading to lower digestibility compared to forages grown at lower altitudes with longer growing seasons. Forage species composition can vary with altitude due to differences in climate, soil, and other environmental factors. Plants that are adapted to high-altitude environments may have different chemical compositions compared to those found at lower elevations. Some high-altitude forage species may produce compounds such as tannins or lignins as defence mechanisms against environmental stressors, which can affect digestibility. Kumar et al. [27] noted that to assure long-term sustainability, economic viability, and efficient forage production utilization, a dynamic agronomic crop–animal model should be adopted. Thus, the variation due to agro-climatic zones would allow for the ability to select the most desirable forage species at a specific location.

3.3. Relationship of NDFD to NDF Content

Figure 6 (a and b) shows relationship of NDF content with NDFD on an NDF basis for cereal and legume fodders, respectively. We found a moderate (R² = 0.57) but significant (p < 0.001) negative relationship in cereals and a borderline (R² = 0.07) but non-significant (p = 0.42) negative relationship in legumes between NDFD and NDF content. The relationship of b with NDF was also investigated with poor (R² = 0.28) but significant (p < 0.05) negative relationship in cereals and a borderline (R² = 0.02) but non-significant (p = 0.66) negative relationship in legumes.

The results of present study and those of other studies [3,18] highlight that ruminal degradability of forages is negatively correlated with the NDF concentration of the plants. This can be an impeding factor in efficient utilization of forage resources by the ruminants. Figure 6c,d clearly indicates that principally, there was poor relationship between the NDF concentration and the b in cereal forages and no relationship in legume forages, which means that it is both a (the washing loss) and K_d which have caused the NDFD to change in proportion to the NDF concentration.

The results of present study and those of other in situ studies [5,9,28] highlight that ruminal degradability of tropical forages can be accurately predicted using the relevant K_d, which has proven to be the single most important parameter among all fractions of degradation. However, this prediction can have a much more trusted value when predictions are made for cereal forages rather than legume forages [9]. Similarly, many feed evaluation systems [29,30] inaccurately predict the feed intake and nutrient utilization in dairy cows when legumes form a substantial portion of the forage fraction. This reflects the atypical degradation of the legumes in the rumen. Although they are characterized by larger quantities of soluble and rapidly degradable protein [29] and greater K_d [18], legumes present a lower extent of NDF degradation compared to grasses due to the lower b and the greater K_p [31].

4. Conclusions

Nutritional composition and NDF degradation parameters of different cereal and legume fodders were significantly different and were affected by growing locations as well. Cereals compared to legumes showed a slow but higher extent of NDF degradation. Soil characteristics modified in response to annual temperature and rainfall strongly changed the fibre concentrations and fractions in tropical forages. The NDFD was negatively correlated with the fodders’ NDF content, especially in cereals where the nature of correlation was negative. It is further indicted that agronomic conditions and forage quality parameters should be considered for ration formulation for optimal availability of nutrients to the animals.