The effect of chemical treatment with NaOH and urea on chemical composition, in vitro gas production and in situ dry matter degradability of sesame residues

M Malekkhahi, M Danesh Mesgaran and A M Tahmasbi*

* Department of Animal Science, Faculty of Agriculture, Ferdowsi University of
Mashhad, P.O. Box: 91775-1163, Mashhad, Iran
Mmalekkhahi@gmail.com

Abstract

The aim of the current study was to determine the effect of chemically treatment of sesame (Sesamum indicum L.) residues including stem, capsules and leaves on chemical composition, in vitro gas production parameters and in situ disappearance. Samples were taken from different forms and then composited to provide two different type of the residues as high leaves and low leaves. Chemically treated samples were prepared by adding NaOH (NaOH as 4g in 100 ml water/ 100g DM), keeping for 48 h, followed by adding urea (urea as 4g in 100 ml water/ 100g DM). Experimental treatments were high leaves and low leaves in forms of untreated and chemically treated in a 2×2 factorial arrangement of treatments. Gas production and in situ DM disappearance were measured at 2, 4, 8, 12, 16, 24, 36, 48, 72 and 96 h of incubation. In vitro gas production and in situ DM degradation kinetics were described using the exponential equation. 

Chemical treatment increased (P < 0.05) crude protein (CP) content of both high leaves and low leaves. In addition, neutral detergent fiber (NDF) and acid detergent fiber (ADF) of the residues significantly (P < 0.05) decreased following the chemically treatment. Whereas, organic matter content of the samples treated was not affected (P > 0.05) by the chemical treatment. Slowly degradable fraction (b) and constant rate (c) of the gas production were significantly higher in high leaves untreated samples than those of the low leaves (P < 0.05). High leaves sample had higher the in situ quickly DM degradable fraction (a) than those of the low leaves sample. Chemically treatment positively affected on the gas production and in situ DM degradation parameters. In the way that NaOH + urea treatment increased (P < 0.05) the in situ soluble DM fraction and slowly degradable fraction of gas production. Our result indicates that high leaves residual had higher degradability potential than those of the low leaves residual. Also, chemically treatment is an effective method of altering the rumen degradation characteristics of DM in by products.

Keywords: leaves, oilseed, ruminant, stems, straw

Introduction

Sesamum indicum L. is an annual plant of numerous types and varieties belonging to the family pedaliacea, cultivated since antiquity for its seeds, which are used as food and flavoring and from which a prized oil is extracted (Rahnama and Bakhshandeh 2006). Depending on conditions, varieties grow from about 0.5 to 2. 5 m tall; some have branches, others do not (Rahnama and Bakhshandeh 2006). One to three flowers appear on the leaf axils and the seed capsules opened when dry, allowing the seed to scatter (Rahnama and Bakhshandeh 2006). Sesame residual has been traditionally used as a basal feed for small ruminants in Iran (Danesh Mesgaran et al 2010).                                          

 The quantity and quality of available feedstuffs are major factors influencing productivity of ruminants in many parts of the world, especially regions with high animal numbers (Prasad et al 1993). Ruminants in such areas depend largely on crop residues at least during the long dry period of year for maintenance as well as production of meat, milk and skin. However, animal performance with such feedstuffs can be poor due to low voluntary intake and digestibility, which results from low protein concentration and high level of indigestible or slowly degradable fiber (Prasad et al 1993).                                              

Various physical, chemical and biological treatments have been used to improve utilization of low quality forage such as crop residues (Abebe et al 2004). Chemicals to improve the utilization of cereal straw may be alkaline, acidic or oxidative agents. Among these, alkali agents have been most widely investigated and practically accepted for application on farms. Basically, these alkali agents can be absorbed into the cell wall and chemically break down the ester bonds between lignin and hemicellulose and cellulose, and physically make the structural fibers swollen (Chenost and Kayouli 1997). These processes enable the rumen microorganisms to attack more easily the structural carbohydrates, enhancing degradability and palatability of the rice straw (Prasad et al 1998; Shen et al 1999). Urea treatment is popular because it is non- hazardous, it can serve as a delignification agent through ammoniation and it is a source of nitrogen. In addition, urea removes the silica polymerised with cuticle waxes from the leaf blade and leaf sheath (Shen et al 1999) and exposes the underlying tissues to bacterial colonization (Bae et al 1997). For many by-product feeds, there is insufficient information available regarding the effect of sample of feed used on rumen degradability values and little research has characterized individual by-product feeds (De Peters et al 1997). Studies on degradation kinetics have been conventionally carried out using the nylon bag (Mehrez and Ørskov 1977) or the in vitro gas production techniques (Menke and Steingass 1988). Both techniques require fistulated animals and a high degree of standardization.

The objective of this experiment was to evaluate the influence of the chemically treatments of two type of sesame residues (high leaves and low leaves) on chemical composition and digestive kinetic parameters by using in vitro gas production and in situ techniques.  

Materials and Methods

Experimental samples and chemically treatment 

Sesame plant was harvested by hand cutting from Jovein farms (Khorasan Razavi Province, Iran) which is located at 56^o30' North latitude and 36^o15' west longitude. The climate of the area is described as semi-arid with average annual rainfall of 218 mm and mean temperature of 16.7^oC. Sesame residual was obtained after separating of the seeds and sun-drying. The residual component (including the leaves, capsule and stem) was separated by hand into two portions in a basis of leaves percentage; high leaves (more than 40% leaves) and low leaves (lower than 40% leaves). Also this residual were treated by  Urea + NaOH [NaOH as 4g in 100 ml water/ 100g DM was sprayed on the samples and kept for 48h, then urea (3g in 100 ml water/100g of initial DM) was added]. The chemically treated samples were filled into plastic bags with 0.5 mm thickness, then tied up and kept at room temperature on cemented floor. After 30 days storage, silages were opened, the dry matter (DM) was determined by drying the samples at 60 ^oC in an air forced dry oven (Memmert 854) for 48 h.After drying, the samples were ground through a 1 mm screen (Cyclotec 1883; Sample Mill). 

Chemical analyses 

Samples were analyzed for Kjeldahl N and crude protein was calculated as Kjeldahl N × 6.25 (AOAC 1990). Dry matter (DM), organic matter (OM) (AOAC 1990) and fibrous component contents were determined according to methods described by Van Soest et al (1991). No sulfite and heat stable amylase were included in procedure for NDF. Both NDF and ADF were excluded of residual ash.

In vitro gas production

In vitro gas production was carried out as described by Menke and Steingass (1988) procedure.  Rumen content was obtained from three adult sheep via their rumen fistula prior to the morning feeding. The donor animals were fed with 0.8 kg DM alfalfa hay and 0.4 kg DM concentrate (165 g CP/kg DM) per head per day, at 07.00 and 17.00 h. Rumen fluid was pooled into a pre-warmed thermos flask, capped and immediately transported to laboratory. Rumen content was then strained through 4 layers of cheesecloth. The laboratory handling of rumen fluid was carried out under a continuous flow of CO₂. Approximately, 200 mg DM of each sample was measured into each syringe. Then, it was filled with 30 ml of medium consisting of 10 ml of rumen fluid and 20 ml of buffer solution as described by Menke and Steingass (1988). Four blank syringes were also provided. The syringes were then placed in a water bath at 39^◦C. Gas production was recorded directly after 2, 4, 8, 12, 16, 24, 36, 48, 72 and 96 h. 

In situ disappearance in the rumen

In situ degradation of DM was studied following the nylon bag technique described by Mehrez and Ørskov (1977). Four sheep (49.6±2 kg) fitted with ruminal fistulae were used. The animals were fed with 1.5 kg DM alfalfa hay and 0.4 kg DM concentrates (165 g CP/kg DM) per head per day, at 08.00 and 17:00 h. Dried samples (5 g) were weighed into 9 cm×17 cm polyester bags (52 μm pore size),and 8 bags were prepared for each sample and each incubation time. Ruminal incubation times were 2, 4, 8, 16, 24, 48, 72 and 96 h. All bags were inserted at the same time, just before the morning feeding (i.e., 08:00 h). Bags representing 2 and 4 h were soaked in water (39 ◦C for 15min), before incubation. At the end of each incubation period, bags were rinsed with cold tap water until the rinse water was clear. Zero time disappearance was obtained by washing unincubated bags in a similar way. All washed bags were dried by using oven dryer (60 °C, 48 h). Disappearance of DM at each incubation time was calculated from the proportion remaining after incubation in the rumen. 

Statistical analysis

Cumulative gas production data were fitted to an exponential equation:

P = b (1 – e^-ct)

Where P is the gas produced at time, b is gas production from soluble and insoluble but fermentable fraction (ml/ 200mg), c is the gas production constant rate for b and t is the incubation time. The values of organic matter digestibility (OMD) and Metabolizable Energy (ME) of the samples were calculated by the equation of Menke and Steingass (1988):

OMD (g/100gDM) = 14.88+0.889×GP+0.45×CP+0.0651×XA

ME (MJ/ kg) = 2.20 +0.136 GP + 0.057 CP +0.0029 CP²

Where CP is crude protein (g/100 g DM), XA is ash as g/100 g DM and Gp is the net gas production (ml) from 200 mg after 24 h of incubation.

Degradation of DM was calculated using the equation of Ørskov and McDonald (1979) as: P = a + b (1-e^-ct), where P is the disappearance rate at time t, a rapidly degradable DM fraction, b the slowly degradable DM fraction in the rumen, c the rate constant of degradation of b, and t is the time of incubation. Effective degradability of DM (EDDM) was calculated using the equation of Ørskov and McDonald (1979) as: EDDM P = a + [b × c/ (c + k)], where k is the fractional outflow rate from the rumen (per hour) and a, b, and c are as described above. The k values used to calculate EDDM were 0.02, 0.04, and 0.06h^-1, which is a normal range of rates observed in ruminants fed forages (Ørskov and McDonald 1979).  

Data on chemical composition, in vitro gas production and in situ disappearance were analyzed completely randomized design with a 2×2 factorial arrangement and means were compared using the Least squares means procedure (LSMEANS). Analysis of variance was carried out using the general linear model procedure (PROC GLM) of SAS (1999). The statistical models are shown below:

Yij =µ+ Pi + Tj + (Pi ×Tj) + eij

 Where: Yij = dependent variable; µ= general mean; Pi = residual type (i = 1–2); Tj = chemically treatment (j = 1–2); (Pi ×Tm) = interaction residual type × chemically treatment and e_ij = residue. 

Results and Discussion

Chemical composition

The chemical composition of treatments is shown in Table 1. The low leaves  residual contained more fiber and less crude protein than the high leaves  residual.  Crude protein was increased for all samples following chemically treatment and ranged from 5.9 to 13.9 (% DM). Canblat et al (2007) showed that NaOH treatment of tree leaves had significant effect on the CP content. Oji et al (2007) determined that aqueous ammonia and urea were more effective in increasing protein content in maize stalks and husks than cobs. Also, Vadiveloo (2000) demonstrated that treatment of leaf rice straw with urea increase CP content than those of the untreated samples.

 In the present study, NDF and ADF concentrations of the untreated samples decreased remarkably when they were treated with NaOH + urea. However, OM was not affected by chemical treatment (P > 0.05). Similar results were found by Madrid et al (1998). Their investigation showed that NDF content decreased in treatment where barely straw was treated by NaOH + urea. The reduction in NDF of wheat straw by NaOH treatment was mainly due to a reduction in hemicullose content (Chaudhry 1998).  

Gas production and estimated parameters

Cumulative gas production profiles from the in vitro fermentation of sesame residues (high leaves and low leaves) and samples treated are shown in Figure 1 and the estimated parameters are given in Table 2. There were significant differences among residual type in terms of gas production at all incubation times. In vitro ruminal cumulative gas production (ml) of high leaves was higher than those of the low leaves (Figure 1). This may be attributed to more nutrients having been made available for the microbes to utilize in vitro (Osuga et al 2008). This is consistent with the findings of Kamalak et al (2004) and Karabulut et al (2007) who reported negative correlations between gas production (GP) and cell wall contents (NDF, ADF) and positive relationships between CP, ash and GP of legume hays. Therefore the estimated parameters (b and c) of high leaves were significantly (P < 0.05) higher than those of the low leaves. There are many factors that may affect the amount of gas to be produced during fermentation, such as nature and level of fiber, the presence of secondary metabolites (Babayemi et al 2004) and potency of the rumen liquor for incubation. Also, Blümmel and Ørskov (1993) described that gas production is associated with volatile fatty acid production following fermentation of substrate so the more fermentation of a substrate, will be the greater gas production.

Metabolizable energy (ME) value and organic matter digestibility (OMD) of the high leaves residual were significantly (P < 0.05) higher than those of the low leaves residual. It  may be due to increase in concentration of cell wall contents (Wilson et al 1991), lignin content in mature plant (Morrison et al. 1983).Salem et al (2007) showed that leaves of Eucalyptus Camaldulesis have lower in vitro organic matter digestibility (IVOMD) than the other species. They also described it may be due to adverse effects of secondary compounds on ruminal and intestinal bacterial activity.

Chemically treatment of sesame residues with NaOH + urea affected cumulative gas production (fig. 1), gas production parameters (b and c), ME and OMD (Table 2). Urea + NaOH treatment significantly (P < 0.05) increased the slowly degradable fraction (b) in high leaves and low leaves treated samples than those of the untreated samples. However, the constant rate of fermentation (c) was significantly increased for all samples after the chemically treatment. Current findings supported the Vadiveloo (2000) results, who observed urea treatment of leaf rice straw increased the slowly degradable fraction (b). Canbolat et al (2007) described that NaOH treatment linearly deceased gas production rate in Arbutus andrachne leaves. These findings are agree with Liu et al (2002) who found NaOH treatment of rice straw increased gas production at all incubation time. Alkali treatment causes swelling and changes in the crystalline structure of cellulose (Guggolz et al 1971; Klopfenstein 1978) and makes it susceptible to ruminal microbial degradation. Chemically treatment with NaOH + urea affected (P < 0.05) the ME and OMD of both high leaves and low leaves samples (Table 2). Vaiveloo (2000) showed that NaOH treatment increased in vitro dry matter digestibility (IVDMD) of both leaf and stem rice straw. 

In situ DM disappearance and estimated parameters

In the present study in situ results showed that residual type had a significant effect on DM degradability at all the incubation times (Table 3). Rapidly degradable fraction (a) of DM was highest (P < 0.05) for high leaves residual. The fractional rate of DM degradation rate (c) was lowest (P < 0.05) for low leaves at 0.028 h^-1. The effective degradable dry matter (EDDM) calculated at 0.02, 0.04 and 0.06 h^-1 outflow rates showed that high leaves residual had higher EDDM than those of the low leaves. Mupangwa et al (1997) observed ED of DM to decrease as the outflow rate increased. The observed difference in the DM degradation characteristics of the residues (high leaves and low leaves) may be due to the difference in chemical composition. Salawu et al. (1999) observed the Calliandera leaves had slowly degradable fraction and higher ED (701 and 399 (g/kg), respectively) than the pods (246 and 262 g/kg, respectively).

Our study results indicated that chemical treatment significantly (P < 0.05) effected on DM degradability of sesame residues (Table 3). Quickly degradable fraction (a) , constant rate (c) and the effective degradable dry matter (EDDM) of dry matter were greatest for chemically treatment in the both high leaves and low leaves compared with untreated samples. In contrast, the slowly degradable fraction b of dry matter was high in untreated samples. Present findings were contrary to findings of Vatandoost et al (2012), who showed that urea treatment increased slowly degradable fraction (b) of DM of barley silage. Chaudhry (2000) described that the treatment with NaOH+Ca (OH)₂ caused the greatest disappearance of DM, OM and NDF. In addition, he showed that the quickly degradable fractions of DM, OM and NDF were greatest for NaOH + Ca (OH)₂ treated straw compared with the untreated. This was similar to the present findings.

Conclusion

• High leaves residual appear to have more beneficial chemical nutrient components and nutritional value than low leaves residues

  
  

• Chemically treatment of residues caused increase of crude protein concentration and decrease of fiber fractions.

  
  

• High leaves chemically treated residues with NaOH + urea improved of  in vitro gas production parameters and in situ degradable DM at all time of incubation. Slowly degradable fraction (b), constant rate (c) and EDDM were increased by chemical treatment.

  
  

• Therefore, it is concluded that chemical treatment with NaOH + urea can enhance nutritive value of crop residues for ruminants.             

Acknowledgements

The authors would like to acknowledge the Department of Animal Science of Ferdowsi University of Mashhad for their cooperation.

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Received 30 September 2012; Accepted 18 November 2012; Published 2 December 2012

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