Introduction

India has a very large population of livestock and the major constraint in the livestock production is the scarcity of fodder and inconsistent quantity and quality of livestock feed for year-round feed supply. Ruminants are mostly fed on high fibrous feeds, which are poor in protein, energy, minerals, and vitamin contents. Consequently, it results in low productivity, poor growth and reproduction of animals (Krishnamoorthy and Moran, 2011). Several reasons, including human population pressure on the land, scarcity of high cost concentrate feeds and the economic need to match the livestock production system with available resources, justify the increased use of non-conventional feed resources for animal feeding (Devendra, and Leng, 2011). The anticipated high demand for animal products could only be met in a sustained manner through the efficient use of crop residues and unconventional feed resources that do not compete with human food (Makkar, 2003; Qrskov et al., 1998). The ruminants can make efficient use of industrial by-products, crop residues, and other unconventional feed sources. Rain tree pods are one such unconventional feed and rain tree (Samanea saman) is a fast-growing tropical tree. The pod, also known as cow tamarind or monkey pod and often surfed by ruminants (Devendra, 1989). An adult tree can produce 500 – 600 kg pods in a year (Cruz, 2003; Idowu et al., 2006). It is palatable and nutritious for livestock and thus can make an excellent feed supplement (George and Craig, 2006). Several aquatic plants such as water hyacinth are commonly grown in flooded rice fields, ponds, canals, road side ditches and other water reservoirs. Due to deprived understanding of their nutritional and economic value, the usage of water hyacinth is limited. Some of the studies have disclosed that these plants are rich in protein and mineral contents (Chumpawadee et al., 2007). Thus, the knowledge concerning the nutrient composition of different feedstuffs is the need of the hour in preparing sensible rations for livestock. Among all the biological methods, In vitro Gas Production Technique (IVGP) is the best alternative to In vivo method in determining the nutritive value of feedstuffs, since rate and extent of degradation, and rumen fermentation can be determined by measuring the volume of gas production (Dhanoa et al., 2000). Therefore, the objective of this study was to evaluate the unconventional feeds using in vitro gas production technique which would be helpful in predicting the rumen fermentation characteristics of these feeds and future incorporation of these ingredients more effectively and judiciously in the livestock feeds for optimal or enhancing ruminant’s productivity.

Materials and Methods

Samples Collection and Preparation

The experimental plants, water hyacinth and Mimosa pudica, were harvested from the College of Forestry, Kerala Agricultural University. Water hyacinth leaves have higher concentration of macro and micro minerals in relative to water hyacinth (Khan et al., 2002) and hence water hyacinth was categorised into two samples, viz. water hyacinth leaves (WHL) and water hyacinth petioles (WHP). Rain tree pods (Samanea saman) were collected by hand picking from the tree, in and around the College of Forestry, Kerala Agricultural University, India. Rain tree pod is found to have different secondary metabolites at different concentration in whole pod (RTP), only seeds (RTS) and pod without seed (PWS) and hence need to be assessed separately before incorporation as feed ingredient (Rath et al., 2014). Therefore, Rain tree seeds (RTS) were collected by cracking out the rain tree pods and left out pods without (PWS) seeds were collected separately. Five kg of all the samples were dried in a hot air oven at 55 to 60 ̊C for 48 h, and ground to pass a 1mm screen and stored in air-tight containers.

Chemical Analysis

Proximate composition of six unconventional feed samples were analysed in duplicate as per the standard procedures (AOAC, 2012). The cell wall constituents namely, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, cellulose, and hemicellulose of the feed samples were determined (Goering and Van Soest, 1991) and expressed inclusive of residual ash.

In vitro Gas Production and Substrate Digestibility

A total of two incubations in sextuplicate for each sample were kept for assessing 24h GP, digestibility and other fermentation attributes. For in vitro gas production test, the rumen fluid was collected from three 6-year-old male Holstein Friesians crossbred cattle (Average BW 380±15 kg) fitted with permanent rumen fistula in the morning before feeding, the donor animals were fed on a maintenance diet comprising of green roughage and concentrate mixture @ 40 and 2 kg/d, respectively. The rumen fluid sample was brought to the laboratory under anaerobic condition in a thermos flask and strained through a four layer of muslin cloth, pooled and used as inoculum for IVGP test. About 200 mg of samples were weighed into 100 ml calibrated glass syringes in quintuplicate. Petroleum jelly was applied to the piston of the syringes to ease movement and to prevent escape of gas. The syringes were pre-warmed (39°C) for 1 h, before addition of 30±1.0 ml of buffered rumen fluid into each syringe under CO₂ flushing as per Menke and Steingass (1988). The syringes were then placed in a BOD incubator at 39^oC. Four blank syringes containing only 30 ml of buffered rumen fluid were incubated to estimate gas production due to endogenous substrates for the blank corrections. The syringes were gently shaken every hour during the first 8 h of incubation and readings were recorded at the end of 24 hr incubation. In vitro true DM and OM digestibility were estimated using methods suggested by Van Soest et al. (1991). Metabolizable energy (ME) content was calculated using the equation of Menke et al. (1989) as follows-

For Concentrate Feeds

ME (MJ/kg DM) = 1.06 + 0.1570 × Gas produced (ml/200 mg DM) + 0.0084 × CP (g/kg DM) + 0.022 × EE (g/kg DM) – 0.0081 × Ash (g/kg DM)

Miscellaneous Feeds

ME (MJ/kg DM) = 2.20 + 0.1357 × Gas Produced (ml/200 mg DM) + 0.0057 × CP (g/kg DM) + 0.0002859 × EE² (g/kg DM)

Where, CP = crude protein, EE = ether extract, TA = total ash, GP = corrected gas production for 24 hrs.

Microbial Biomass Production (MBP) was calculated from TDOM using equation-

MBP (mg) = TDOM (mg) – (Corrected gas production for 24 hrs×2.20)

Where, 2.20 is the stoichiometric factor for roughages (Blümmel et al., 1997) and for mixed diets (Blümmel and Lebzien, 2001).

Partitioning Factor (PF) was calculated by the method of Blümmel et al. 1997-

PF = (mg TDMD) / corrected gas production for 24 hrs

Where, TDOM = truly digestible organic matter

Fermentation Metabolites

In a separate run, the incubations were terminated after completion of 24hr incubation to measure individual VFA and total VFA concentration. On completion of the incubation, the buffered rumen liquor was filtered through four layers of muslin cloth and approximately 0.8 ml of the sample was preserved with 200 μl of 25%metaphosphoric acid. The samples preserved in this way were immediately analysed or stored at -20 °C temperature for the future analysis. Samples were centrifuged at 20000 rpm at 4°C, for 20 mins and the supernatant was analysed for volatile fatty acids. The analyses were conducted on a 7890A GC System gas chromatograph, Agilent Technologies as per standard procedure described by Filípek and Dvořák 2009.The amount of methane production was calculated from VFA composition according to Moss et al., 2000 as CH₄production= 0.45 (acetate) − 0.275 (propionate) + 0.4 (butyrate).

Statistical Analysis

The data obtained were subjected to one way analysis of variance using SPSS version 18.0 software. Treatment means were separated using Duncan’s multiple range test (Duncan, 1955).

Results and Discussion

Chemical Composition

The chemical composition of the evaluated feedstuff is shown in Table 1. All feedstuff had high CP contents (>17%), ranging from 17.01% (WHP) to 29.00% (RTS). Protein content was greater in RTS and WHL (25.40%) than in PWS (17.80%), WHP and Mimosa pudica (19.30%). CF ranged from 7.13% (RTS) to 19.90% (WHP) and TA content was highest in WHP (18.80%) and lowest in RTS (3.21%).

Table 1: Chemical composition (%) of evaluated unconventional feedstuffs

PWS: pods without seeds; RTS: raintree seeds; WHL: water hyacinth leaves; WHP: water hyacinth petioles

Cell wall composition of the feedstuffs is described in the Table 2. NDF was found having the lowest values for PWS (52.49%) followed by Mimosa pudica (53.60%) and WHP (55.90%), and RTS (63.19%) and WHL (64.70) had similar NDF content. ADF ranged from 29.70% (Mimosa pudica) to 36.50% (WHL). Based on their chemical composition, RTS, WHL and Mimosa pudicacould be classified as highly pretentious feeds. Rath et al., 2014 reported similar values of chemical composition for rain tree pod meal, who found 25.25, 1.7, 4.1, 12.2 and 56.6% for CP, EE, TA, CF and NFE respectively.

Table 2: Cell wall constituents (%) of evaluated unconventional feedstuffs

PWS: pods without seeds; RTS: raintree seeds; WHL: water hyacinth leaves; WHP: water hyacinth petioles; NDF: neutral detergent fibre; ADF: acid detergent fibre; ADL: acid detergent lignin

The nutritional value of raintree pods and its potentiality to serve as animal feed have been assessed by Oduguwa et al., 2000; Hosmani et al., 2010; and Idowu et al., 2006. It is well reported fact that rain tree pods can be combined very well in the ration of ruminants (Hosmani et al., 2010). The CP content of RTS and PWS are in agreement with Babayemi et al., 2010. The difference in the composition of the raintree pods may be due to change in the soil, pod age and the age of the tree producing the pod (Durr, 2001). The CP content of different parts of water hyacinth plant namely its leaves (18.03 per cent), shoots (18.04 %) and the entire plant (8.53 %) was reported by Aboud et al., 2005 in their study and its content in the present study was almost similar to the value reported by the workers. The higher CP content of WHP, PWS and Mimosa pudica reported in the present study is comparable to some of the common leguminous fodders like cowpea (16.58%), berseem (17.05%) which are fed to livestock (Kumar et al., 2015). WHP and WHL are also rich source of minerals with TA content of 18.80 and 13.71% respectively, and similar value was also reported by Aboud et al., 2005 in their study. These feedstuffs can be fed to different class of livestock as supplemental feeding to meet their partial protein requirement.

In vitro Gas Production

In vitro gas production profile is depicted in Table 1. The statistical analysis of the data on average gas production (ml/200mg) revealed a significant difference between RTS (40.14±1.04), PWS (31.52±0.49) and WHL (15.97±0.28) (p<0.05). No significant difference was found between WHP (13.26±0.28) and Mimosa pudica (13.48±0.26). The GP of other feed samples arranged from lowest to highest were- WHP, Mimosa pudica, WHL, PWS and RTS. Semae et al.,2013 found a GP of 45.03 ml/200mg DM for rain tree pod which is higher than the value obtained in the present study for PWS and RTS. The reduced gas production may be due to the less availability of residual fractions to the microbes and also may be due difference in the ripeness of raintree pod collected. Higher concentration of tannins could have resulted in lesser gas production. The gas production from the incubation of WHL and WHP with buffered rumen fluid in the present study is in agreement with that of Khan et al., 2002, who reported average gas production of 18.5 and 14.5 ml/200mg DM for water hyacinth and water hyacinth leaf, respectively. Sallam et al., 2008 opined that gas production from the incubation of feed in the buffered rumen fluid is directly associated with feed fermentation and carbohydrate fraction which is evident in the incubations of PWS and RTS.

Energy Content, True DM and OM Digestibility

The estimated metabolizable energy (ME), TDMD and TOMD are presented in Table 3. The ME contents were particularly higher in RTS (10.36±0.16), PWS (7.79±0.08) and Mimosa pudica, WHL and WHP had significantly lower values of ME (MJ/kg DM; respectively), however the ME values WHL and Mimosa pudica were similar. Kumar et al., 2015 reported similar metabolisable energy values for some of the oilseed cakes/meals such as groundnut cake, mustard oil cake, cotton seed cake, and soybean meal and it ranged from 7.71 to 9.44 (MJ/kg DM). Menke and Steingass 1988 have demonstrated that there exists a positive correlation between metabolisable energy calculated from in vitro gas production along with CP and EE content with metabolisable energy value of conventional feeds measured through in vivo experiments. The differences in the ME of feedstuffs reflect the difference in the content of fermentable carbohydrates and nitrogen available.

Statistically a significant difference (p<0.05) was found in both the dry matter and organic matter digestibility and followed the same trend in all the feed samples. TDMD ranged from 52.34±0.82 for Mimosa pudica to 86.88±0.70 for RTS and the TOMD ranged from 52.16±0.80 to 87.46±0.67 per cent respectively. The TDMD of PWS and RTS were more than the value reported by Semae et al., 2013 who found TDMD of 65.09%. Higher DM digestibility of PWS and RTS in the present study can be attributed to relatively lower ADL (Table 2) and high NFE levels (Table 1). Khan et al., 2002 reported 463 and 454 g/kg DM of organic matter digestibility for water-hyacinth and WHL respectively, and the values obtained in the present study is higher than the values reported by them. The higher value obtained in this study may be because of the lower fibre and ADL levels in the substrate.

Microbial Biomass Production and Partitioning Factor

The MBP (mg/200mg DM) and PF (OM fermented/ml gas), calculated from the gas production data are listed in Table 3. The statistical analysis of the data on MBP (mg/200mg DM) revealed a significant difference (p<0.05) between WHL (87.44±1.30), WHP (76.54±2.87) and Mimosa pudica (67.77±0.97).

Table 3: In-vitro gas production, digestibility, metabolisable energy, microbial biomass production and partitioning factor of unconventional feedstuffs

a, b, c, d, e: means with different letters in a row different significantly (P<0.05); PWS: pods without seeds; RTS: raintree seeds; WHL: water hyacinth leaves; WHP: water hyacinth petioles

There was no significant difference between PWS (81.61±1.16) and RTS (79.23±2.49). PF of PWS, RTS, WHL, WHP and Mimosa pudica were 4.67±0.05, 4.11±0.12, 7.51±0.16, 7.82±0.30 and 7.19±0.05, respectively. Microbial nitrogen is the major source of protein for ruminants and is utilized to meet the maintenance requirement of the animal. Microbial biomass is produced in the rumen from the degradation of feed material (substrate). In the in vitro gas production system, the substrate (200 mg) kept for incubation is either degraded by the rumen microbes into short chain fatty acids or incorporated into microbial biomass with liberation of fermentation gases. Microbial biomass production is more when substrate is used for the growth of microorganisms and less for the fermentative gas. The MBP in all the samples was better and is indicative of improved fermentable substrate which is favourable for the growth and development of rumen microorganisms. The PF for PWS and RTS were within the theoretically possible value of 4.41 (Blummel et al., 1997). Higher values in rest of the feedstuffs may be due tannins and other incrementing factors (Getachew et al., 2004).

Fermentation Metabolites

Individual volatile fatty acids (mmol/L) such as acetate, propionate, butyrate, valerate, Total Volatile Fatty Acids (TVFA), acetate to propionate and estimated methane production of different feedstuffs are presented in Table 4.

Table 4: Short chain fatty acids (mM/L) production and estimated methane production from different unconventional feedstuffs

a, b, c, d Means with different letters in a row different significantly (P<0.05); PWS: pods without seeds; RTS: raintree seeds; WHL: water hyacinth leaves; WHP: water hyacinth petioles; ND: not detected

The concentration of acetate was higher (p<0.05) in RTS (40.68±1.19) and PWS (35.87±0.73), and lower in Mimosa pudica (26.26±0.76). While intermediate values were observed in WHP (31.96±0.37) and WHL (32.33±1.41). However, the trend among the feed samples for propionate was somewhat different to that of acetate production. The concentration of propionate was significantly (P<0.05) higher for PWS (22.57±0.86) and lower in Mimosa pudica (9.44±0.42) and intermediate values was observed in WHP (15.28±0.22) and WHL (14.94±0.89). The concentration of butyrate was higher (P<0.05) for RTS (3.69±0.15) and lower for Mimosa pudica(1.69±0.08). The concentration of valerate was also significantly (P<0.05) higher in RTS (0.36±0.05) and lower in WHP (0.11±0.04). The TVFA (mmol/L) concentration was higher (P<0.05) for PWS (61.65±1.78) and RTS (62.76±1.99) and lower for Mimosa pudica(31.90±6.16). Incubations of WHL (50.36±2.39) and WHP (49.93±0.64) showed intermediate values. The acetate to propionate ratio was wide (P<0.05) in RTS, WHL, WHP and Mimosa pudica. PWS showed lower value of 1.59±0.03. The narrower ratio indicating more propionate production thus less hydrogen is available for methane production.

Higher acetate concentration in PWS and RTS may be due to higher content of soluble and easily fermentable carbohydrates. The non-structural carbohydrates promote the production of propionic acid whereas the fibrous carbohydrates stimulate the production of acetic acid in the rumen. In addition, the non-structural carbohydrates yield more VFA since they are fermented faster and further completely. Hungate et al., 1952 reported that the rations containing large amounts of cellulose are characterized by high numbers of gram-negative cocci, which are responsible for more acetate production and the same phenomenon is evident in this study for RTS (24.43% cellulose) sample and the lower production of acetate in Mimosa pudica may also be due to less cellulose (13.79%) available for fermentation. The higher concentration of propionate production in PWS and RTS may be due to higher gas production with high proportion of carbon dioxide gas in the incubation media (Johns 1951a; 1951b). Estimated methane (Table 4) (mol %) production was higher (P<0.05) in RTS (14.82±0.49) and lower in Mimosa pudica (9.90±0.28).

Conclusion

The results in the present study allows us to conclude Mimosa pudica, WHL and RTS as medium to high protein containing feeds and can be considered as protein supplement to low quality diets. PWS, RTS and WHL are highly digestible. However, roughage source is required when these are included in the diet. Based on this study, high rumen fermentation potential ranked from the highest to the lowest were; RTS, PWS, WHL, Mimosa pudica and WHP. Nevertheless, more research on animal responses, through in vivo trials are needed to support the nutritional characteristics reported in this study.

Acknowledgements

The corresponding author is thankful to the Director, ICAR-NIANP, Bangalore, for providing necessary facilities to work in Food Safety and Quality Laboratory. The financial support in the form of institutional scholarship from KVASU is duly acknowledged by the corresponding author.

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