In vitro Evaluation of Phalaris minor Seeds as Livestock Feed

The nutritional worth of Phalaris minor seeds was assessed in comparison to conventional cereal grains like maize and wheat. P. minor seeds had higher total ash and cell wall constituents as compared to wheat and maize grains. The CP content of P. minor was comparable to wheat grains but higher than maize grains. The in vitro studies revealed that the net gas production and availability of ME from P. minor was comparable to that of maize but the digestibility of nutrients was significantly (p<0.05) lower than that of conventional cereal grains. The digestion kinetic parameters for DM and CP revealed that P. minor had the highest (p<0.05) soluble fraction (a) followed by wheat and maize. Reverse trend was observed for insoluble but potentially degradable fraction (b). The effective and true DM and CP degradability was significantly (p<0.05) higher in wheat grains followed by that in P. minor and maize grains. The digestibility of OM and NDF was not affected by replacing cereal grains in concentrate mixture with P. minor seeds up to 75 per cent level. But the availability of ME from concentrate mixtures was comparable to control only up to 50% level of replacement. Replacement of cereal grains with P. minor did not affect the rapidly soluble fraction and insoluble but potentially degradable fraction of concentrate mixture containing P. minor up to 75 per cent, but it was depressed significantly at 100% replacement level. The effective and true degradability of DM of concentrate mixtures containing P. minor from 50 to 100 per cent was comparable to that of conventional concentrate mixture (CCM). The wheat based concentrate mixtures showed higher net gas production (208 vs. 201 ml/g DM/24 h), digestibility of nutrients and ME availability (9.64 vs. 9.54 MJ/kg DM) as compared to maize based concentrate mixture. The wheat based concentrate mixture had significantly (p<0.05) higher rumen undegradable fraction and effective degradability. The data conclusively revealed that conventional cereal grains could be replaced with P.minor seeds up to 75 per cent without affecting the availability of nutrients. (Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 3 : 363-367)


INTRODUCTION
Phalaris minor (family, graminae) is a fast spreading weed in wheat fields, especially in the Northern States of India, where rice-wheat rotation is predominant.Each plant produces about 300-400, shiny black, very small, flat seeds, which usually contaminate wheat grains.Wheat being the staple food throughout the world, no compromise with it's yield or quality can be made, therefore, it becomes imperative for the scientists to check the propagation of Phalaris minor or to find an alternate use of this weed.Different methods have been employed to control weed, but none could eliminate the weed completely.Since it emerges with the germination of wheat seedlings and resembles very closely, making it difficult to recognize in the initial stage of growth.The P. minor has many medicinal properties viz.acts as carminative, flushes out the worms and above all it improves the immunity of the animals (Oudhia, 2003).However, information about its nutritive value is lacking.The present study was, therefore, undertaken to assess the nutritive value of Phalaris minor, for ruminants.

MATERIALS AND METHODS
In the 1 st trial nutritional value of Phalaris minor seeds was assessed in comparison to conventional energy supplements like wheat and maize.In the 2 nd trial cereal based conventional concentrate mixture (CCM) containing either wheat or maize 30, mustard cake 15, de-oiled mustard cake 15, rice bran 15, de-oiled rice bran 5, wheat bran 10, molasses 7, mineral mixture 2 and common salt 1 par each was prepared.The cereal grains in the concentrate mixture were replaced with P. minor seeds on weight basis, at 25, 50, 75 and 100%.The nutritive value of P. minor seeds and the concentrate mixtures was assessed by in-vitro gas production technique (Menke et al., 1979) and in sacco method (Mehrez and Orskov, 1977).

In vitro gas production studies
About 375 mg of the substrate was incubated at 39°C for 24 h in triplicate in 100 ml calibrated glass syringes (procured from M/s Haberle Labortechnik, Germany) containing feedstuff and a buffered rumen fluid.Blank, sample of standard hay and standard concentrate (procured from International Atomic Energy Agency, Vienna) were run in triplicate with each set.Rumen contents were collected from 3 buffalo calves maintained on 2.0 kg conventional concentrate mixture (maize 25, maize oil cake 10, de-oiled mustard cake 20, rice bran 15, de-oiled rice bran 15, wheat bran 12, mineral mixture 2 and common salt 1 part each), 4.0 kg green fodder and 6.0 kg wheat straw.The rumen contents were collected at 0 hr in a thermos flask flushed with CO 2 and maintained at 39°C.The rumen contents were blended for 2-3 minutes in blender maintained at 39°C and then strained through 4-layered muslin cloth.The solution, containing 960 ml distilled water, 0.16 ml micro-mineral solution (13.2 g CaCl 2 2H 2 O, 10.2 g MnCl 2 4H 2 O, 1.0 g CoCl 2 6H 2 O and 8.0 g FeCl 3 6H 2 O per 100 ml distilled water), 660 ml bicarbonate buffer (35 g NaHCO 3 and 4.0 g NH 4 HCO 3 per 1,000 ml distilled water), 330 ml macro-mineral solution (5.7 g Na 2 HPO 4, 6.2 g KH 2 PO 4 , 0.6 g MgSO 4 7H 2 O per 1,000 ml distilled water) and 1.6 ml Resazurine (0.1%), was mixed in a Woulff flask (3 l cap) mixed with magnetic stirrer in a water bath at 39°C, followed by addition of freshly prepared reducing (373.0 mg Na 2 S H 2 O, 2.6 ml of 1 N NaOH in 62.0 ml distilled H 2 O) solution and flushing of CO 2 through a submerged tube.The light bluish solution first turned pinkish then became colourless.Then strained rumen liquor (SRL) was added to the buffer media in 1:2 ratio.The flushing of CO 2 was continued till the last syringe was filled.The tube on the capillary attachment to the syringe was firmly fixed on to the top dispenser bottle.30 ml (SRL: buffer) solution was pumped in each syringe.The contents in the syringe were mixed by gentle shaking.Air bubbles were brought to the surface and removed through the capillary by careful upward movement of the piston.The clip was closed immediately and exact volume of contents in the syringe was noted and kept in a water bath maintained at 39°C.The contents in all the syringes were swirled at 60 minutes interval for first few hours of incubation so that material does not stick to the walls of the syringe or bottom of the plunger.If at 8 h after incubation the volume of the gas exceeded 70 ml, the actual volume of the gas produced was recorded and then the gas was taken out.After 24 h, volume of gas produced in each syringe was recorded and the contents of syringes were transferred to spout-less beaker, boiled with neutral detergent solution (which dissolves microbial biomass) for assessing the true organic matter (OM) and neutral detergent fiber (NDF) digestibility.The metabolisable energy (ME) was worked out by using the equation developed by Menke and Steingass (1988).
Where GP is net gas production in ml/200 mg substrate DM/24 h.
XP is crude protein in g/kg DM XL is ether extract in g/kg DM

In sacco degradability
For determination of in sacco degradability and kinetic parameters, three buffalo calves fitted with permanent rumen fistulae were selected and maintained on 2 kg concentrate mixture (maize 25, maize oil cake 10, de-oiled mustard cake 20, rice bran 15, de-oiled rice bran 15, wheat bran 12, mineral mixture 2 and common salt 1 part each) supplemented with 4 kg green fodder and 6 kg wheat straw.The nylon bags of 8×17 cm stitched with monofilament polyester thread with pore size of 50±10 µ were used.Five grams sample was placed in the nylon bags, which were incubated in the rumen for 2, 4, 6, 8, 10, 12, 24, 36 and 48 h in triplicate.The 'zero h' bags were not incubated in the rumen but were washed in the same manner as incubated bags.After the stipulated period, bags were taken out, washed under tap water, until rinsing water from the bag became colorless.The bags were dried in a forced air oven maintained at 60°C for 48 h.The disappearance of dry matter (DM) was measured as the loss in weight of the bag contents.The residue was analyzed for DM, crude protein (CP) and NDF content.The different physical constants characterizing extent and rate of ruminal degradation, i.e. rapidly soluble fraction (a), insoluble but potentially degradable fraction (b), degradation rate (c) and effective degradability (ED) were calculated according to McDonald (1981).The rumen fill was calculated according to Van Eys (1982).The DM intake was predicted by using the equation of Orskov et al. (1988).

Chemical analysis
Samples of Phalaris minor seeds, cereal grains (wheat and maize) and concentrate mixtures were ground to pass through 1 mm sieve and analyzed in duplicate for nitrogen, ether extract and total ash (AOAC, 1995), cellulose (Crampton and Maynard, 1938) and other cell wall constituents (Robertson and Van Soest, 1981).

Statistical analysis
The data of the 1 st trial were analyzed by completely randomized design and that of the 2 nd trial by 2×5 factorial design (Snedecor and Cochran, 1994) using STATGRAPHICS version 5.0.The means were compared for statistical significance by using Duncan's multiple range test (Duncan, 1955).

Chemical composition of Phalaris minor seeds in comparison to conventional cereal grains
In order to meet the nutritional requirements of livestock, as well as for manufacturing compounded feed, precise knowledge of feedstuff composition is a prerequisite.The P. minor seeds being unconventional feed resource have never been evaluated for their nutritional worth.The P. minor seeds had higher total ash, NDF, acid detergent fiber (ADF), cellulose and lignin content and considerably low OM content than that of wheat and maize grains.The CP content of P. minor seeds and that of wheat grains was higher than that in maize grains.The hemi-cellulose content in P. minor and that of wheat grains was considerably lower than that in maize grains (Table 1).

In vitro/in sacco evaluation
The net gas production from P. minor was lower (p<0.05)than that from wheat but equivalent to that produced from maize grains (Table 2).Amongst the seeds/grains tested, the P. minor seeds had the lowest (p<0.05)digestibility of NDF and OM.The higher digestibility of nutrients in wheat resulted in higher (p<0.05)availability of metabolizable energy (ME) as compared to other grains.The availability of ME from P. minor seeds was similar to that of maize.
The digestion kinetic parameters for DM, assessed by nylon bag technique, revealed that P. minor had the highest (p<0.05)soluble fraction (a) followed by wheat and maize (Table 3).Reverse trend was observed for insoluble but potentially degradable fraction (b).The amount of potentially degradable fraction in wheat and maize was comparable, but the degradation rate (c) was higher (p<0.05) for wheat followed by that for P. minor and maize grains.The P. minor seeds had higher (p<0.05)rumen undegradable fraction (UDF) as compared to conventional cereal grains (wheat and maize).The effective and true degradability of P. minor was higher (p<0.05)than that of maize grains, but lower (p<0.05)than that of wheat grains.
The digestion kinetic parameters for CP revealed that P. minor had the highest (p<0.05)rapidly soluble fraction followed by that in wheat and maize (Table 3).But, the potentially degradable fraction followed the reverse trend.The potentially degradable fraction in P. minor was 25 and 50% less than that in wheat and maize grains, respectively and was degraded at the lowest rate.The minimum potentially degradable fraction with slowest degradation rate resulted in highest rumen undegradable protein fraction in P. minor.It indicated that P. minor seeds could be used as potential source of by pass protein in comparison to conventional cereal grains.The effective degradability of crude protein of P. minor was in between the two cereal grains.The true degradability of crude protein of P. minor was the lowest.The in vitro and in sacco studies exhibited promising results.

Replacement of cereal grains with Phalaris minor seeds in concentrate mixture
The net gas production was depressed significantly (p<0.05) when the cereal grains were replaced with P. minor (more than 25 per cent).The net gas production from concentrate mixtures containing 50 and 75 percent P. minor was comparable, but higher (p<0.05)than that produced from concentrate mixture where cereal grains were completely replaced by P. minor (Table 4).The digestibility of OM and NDF was not affected by inclusion of P. minor, up to 75 per cent level in concentrate mixture.The availability of ME (MJ/kg DM) from concentrate mixtures containing P. minor seeds, varied from 9.3 to 9.6 as compared to control concentrate mixture (9.8).The data conclusively revealed that conventional cereal grains could be replaced without affecting the availability of nutrients, by P. minor seeds up to 75 per cent.
On an average, the wheat based concentrate mixture, irrespective of the level of P. minor, showed higher net gas production, digestibility of nutrients (NDF and OM) and ME availability as compared to maize based concentrate mixture (Table 5), which supports the in vitro and in sacco results of the individual cereal grains.
The in sacco study revealed that level of P. minor showed no significant impact on the rapidly soluble fraction (Table 6).The insoluble but potentially degradable fraction of concentrate mixture containing P. minor up to 75 per cent was comparable with that of CCM.The concentrate mixture containing only P. minor (100%) had the lowest potentially degradable fraction, but was statistically comparable to that of concentrate mixtures containing 50 and 75 per cent of P. minor seeds.The potentially degradable fraction of the concentrate mixtures was degraded at a comparable rate, except that it was significantly depressed at 25 per cent level of replacement.The rumen UDF increased linearly with the increase in level of P. minor in the concentrate mixture.However, it was exceptionally low in concentrate mixture where 25 per cent of cereal grains were replaced by P. minor seeds.The effective and true degradability of DM of concentrate mixtures containing P. minor from 50 to 100 per cent was similar to that of CCM.But, at 25 per cent level of replacement, effective degradability was highest.
The digestion kinetic parameters for CP, irrespective of the type of cereal grains used, in the concentrate mixture showed no consistent trend.The rapidly soluble fraction and insoluble but potentially degradable fraction were comparable in all the groups except at 25 and 50 per cent level of replacement, which differed significantly (Table 6).The degradation rate of CP from the concentrate mixture containing 50 percent P. minor was highest but comparable to that of control.The undegradable fraction was observed to be highest in concentrate mixture containing only P. minor.The effective degradability of CP of concentrate mixtures containing P. minor up to 75 per cent level was statistically comparable to that of CCM but it was depressed (p<0.05) at 100 per cent level of replacement.
Irrespective of level of P. minor, the digestion kinetic parameters for DM of concentrate mixtures revealed that  wheat based concentrate mixture had higher (p<0.05)rapidly soluble fraction and lower (p<0.05)insoluble but potentially degradable fraction as compared to maize based concentrate mixture (Table 7) with no significant effect on the protein fractions.The DM and CP of wheat based concentrate mixture was degraded at a much faster rate (p<0.05) as compared to that of maize based concentrate mixture.Wheat based concentrate mixture showed significantly (p<0.05)higher true degradability of nutrients (DM and CP).The in vitro gas production and in sacco studies conclusively revealed that conventional cereal grains (maize and wheat) in the concentrate mixture could be replaced with P. minor seeds, up to 75% without any adverse effect on the availability of nutrients.

Table 1 .
Chemical composition of Phalaris minor seeds (% DM basis)

Table 3 .
Digestion kinetic parameters for DM and CP of Phalaris minor seeds a: Rapidly soluble fraction; b: Insoluble but potentially degradable fraction; c: Degradation rate; ED: Effective degradability; UDF: Undegradable fraction; TD: True degradability.Figures with different superscripts in a row differ significantly (p<0.05).

Table 2 .
In vitro evaluation of Phalaris minor seeds NGP: Net gas production; NDFD: Neutral detergent fiber digestibility; TOMD: True organic matter digestibility; ME: Metabolisable energy.Figures with different superscripts in a row differ significantly (p<0.05).

Table 4 .
In vitro evaluation of concentrate mixtures containing different level of Phalaris minor seeds (irrespective of type of cereal NGP: Net gas production; NDFD: Neutral detergent fiber digestibility; TOMD: True organic matter digestibility; ME: Metabolisable energy.Figures with different superscripts in a row differ significantly (p<0.05).

Table 6 .
Digestion kinetic parameters of concentrate mixtures (irrespective of cereal grain used) containing P. minor seeds a: Rapidly soluble fraction; b: Insoluble but potentially degradable fraction; c: Degradation rate.ED: Effective degradability; UDF: Undegradable fraction; TD: True degradability.Figures with different superscripts in a row differ significantly (p<0.05).

Table 5 .
In vitro evaluation of conventional cereal grains based concentrate mixtures (irrespective of the level of P. minor seeds)

Table 7 .
Digestion kinetic parameters of conventional cereal grains based concentrate mixtures (irrespective of level of P. Rapidly soluble fraction; b: Insoluble but potentially degradable fraction; c: Degradation rate; ED: Effective degradability; UDF: Undegradable fraction; TD: True degradability.Figures with different superscripts in a row differ significantly (p<0.05). a: