Influence of Corn Steep Liquor on Feeding Value of Urea Treated Wheat Straw in Buffaloes Fed at Restricted Diets

Influence of different levels of corn steep liquor (CSL) on chemical composition of urea treated wheat straw (UTWS), ruminal characteristics, digestion kinetics, nitrogen (N) utilization, and nutrient digestibility by ruminally-cannulated buffalo bulls was studied in a 4×4 Latin Square Design. The CSL was used to ensile 5% UTWS at the rate of 0, 3, 6, and 9% on a dry matter (DM) basis. Total N and neutral detergent fibre (NDF) were increased with increasing level of CSL. Increased NDF content was attributable to increased neutral detergent insoluble nitrogen. Four diets were formulated to contain 20% concentrate and 80% UTWS ensiled with 0, (control), 3 (CSL3), 6 (CSL6), and 9% CSL (CSL9). All diets were mixed daily and fed at 1.5% of body weight twice daily. Ruminal NH3 concentration decreased with level of CSL used to ensile UTWS at 3 and 6 h post prandial, however, at 9 h post parandial it was similar across all diets and at 12 h post prandial was higher with diets containing UTWS ensiled with CSL. Concentrations of total ruminal volatile fatty acid and acetate were increased with the CSL level used to ensile UTWS. Increased rate of disappearance and reduction in lag time of DM and NDF was recorded with diets containing UTWS ensiled with CSL. Dry matter and NDF digestibilities were higher with CSL diets than on the control diet. Buffalo bulls retained more N with diets containing UTWS ensiled with CSL. The present results indicated that UTWS could be ensiled with CSL to improve its nutritive value and N utilization by ruminants. (


INTRODUCTION
Ruminants in developing countries are largely being fed on fibrous crop residues.Protein deficiency and low digestibility of crop residues often restrict animal productivity (Khan et al., 2006a).Physical, chemical, and biological treatments have been developed to weaken and break lingo-cellulosic bonds in crop residues, thereby increasing their nutritive value (Khan et al., 2006b;Nisa et al., 2006).Alkali, ammonia, and urea treatments have received much attention; however urea, which has high nitrogen (N) contents, was more cost effective (Sarwar et al., 2005a).
Usage of urea as a source of NH 3 is not perfect method because only 30 to 35% of NH 3 released from urea is retained in the straw (Sarwar et al., 2003;2004a;Khan et al., 2006c).Out of the retained N much is held as water-soluble and thus rapidly librated in the rumen and cause nutrient loss at ruminal level (Sarwar et al., 2005b).To overcome these problems, some researchers (Dass et al., 2000;Sarwar et al., 2003) have tried to fix NH 3 in straw by spraying organic acids (formic acids and acetic acid) or inorganic acids (H 2 SO 4 and HCl) with different degree of NH 3 fixation.However, fixing NH 3 with acid is costly and hazardous and thus, its use by farmers is impracticable.
Corn steep liquor (CSL) may offer a solution to the problem of escaping NH 3 and poor fermentation of urea treated wheat straw (UTWS).Because, CSL not only contains easily soluble carbohydrates, which can improve fermentation, but it's acidic, pH (3.7) can also help to fix the NH 3 (Sarwar et al., 2004b).Thus, the use of this ingredient can enhance both the fermentation process and NH 3 fixation in the ensiled UTWS.However, scientific evidences regarding CSL effect on the NH 3 fixation in UTWS and its nutritional value in buffalo are limited.Therefore, objectives of this study were to establish the amount of CSL for N fixation in UTWS, and to determine its dietary effects on ruminal characteristics, digestion kinetics, N utilization and digestibility in ruminally cannulated buffalo bulls.

Corn steep liquor
The CSL for this study was procured from Rafan Maize Products Ltd. Faisalabad, Pakistan.CSL derived from the enzymatic conversion of cornstarch.The steeping of corn is a necessary prerequisite to the fractionation of corn components in the wet-milling process.It involves the countercurrent flow of water, initially containing some SO 2 , and dried corn in a number, around 10, of steeping tanks at 50-55°C over a period of approximately 30 h.It follows that the fresh, dried corn enters the tank that contains the steep water from exposure to the partially steeped corn in the previous tanks.The volume of steep water produced in the corn wet-milling industry is large.It is primarily handled by evaporation to concentrated thick liquor that is a complex mixture of carbohydrates, amino acids, peptides, organic compounds, inorganic ions, and myo-inositol phosphates (Table 1).

Treatment of wheat straw
Wheat straw was treated with 5 kg urea (50% moisture level) and was ensiled with 0, 3, 6, and 9% CSL on DM basis in four cemented pits (each pit was 3×10×2 m) at 30-35°C for fifteen days.Each pit was covered with a 10-cmthick layer of rice straw, followed by a plastic film covering, which was plastered with a blend of wheat straw and mud to avoid any cracking on drying.When the feed was used, the plastic film was removed and the feed was withdrawn starting with the upper layer and working downwards to the lower layers.An amount of the fermented straw was taken out just sufficient for one day's feeding and the plastic film was put back to keep the pit sealed.The samples of this fermented wheat straw were analyzed for dry matter (DM), organic matter (OM), neutral detergent insoluble nitrogen (NDIN), acid detergent insoluble nitrogen (ADIN), N and ash by the methods of AOAC (1990).Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined by methods described by Van Soest et al. (1991).

Animals and diets
Four buffalo bulls of average weight 350 kg fitted with ruminal cannulae were used in a 4×4 Latin Square Design.Four diets were formulated to contain 20% concentrate and 80% UTWS ensiled with 0 (control), 3 (CSL3), 6 (CSL6), and 9% CSL (CSL9).Urea was included to make all the diets iso-nitrogenous (Table 2).All diets were mixed daily and fed twice daily at 1.5% of body weight per day in two frequencies.During the experimental period, the animals were housed on a concrete floor in separate pens.
The animals were given 20 days for adaptation to diets at the start of each experimental period, followed by a 10day collection period.Feed offered and orts were weighed and recorded twice daily.For first 3 days, of each collection period, the ruminal contents (liquid plus digesta) were sampled (500 ml) at 3, 6, 9, and 12 h after morning feed.Ruminal pH was measured immediately and samples were squeezed through four layers of cheesecloth.Three ml of 6 N HCl was added to terminate fermentation and samples were frozen.After thawing, these samples were used to determine ruminal NH 3 (Chaney and Marbach, 1962) and volatile fatty acids (VFA).On day 4, the ruminal content were sampled just before the morning feed and strained.One portion of this strained rumen fluid was used to enumerate total viable and cellulolytic bacterial counts (Olumeyan et al., 1986).Another portion was blended with a volume of saline solution containing 20% formaline for total bacterial count by microscopic examination (Suto, 1973).
Complete collections of urine and faeces were made according to the procedures described by Williams et al. (1984).Faeces were collected daily, dried at 55°C, bulked and mixed at the end of each collection period and sampled for analysis.Daily collections of urine were acidified with 50% H 2 SO 4 , stored and then mixed and sampled at the end of each period.On day 5 of each collection period, blood samples were taken at 3, 6, 9, and 12 h after feeding.Blood samples were analyzed for urea-N (Coulomobe and Favreau, 1963).Feed, orts and faecal samples were dried at 55°C and ground through a Wiley mill (2-mm screen).These samples were analyzed for DM, N, and OM using method described by AOAC (1990), and NDF, ADF, and ADL by methods of Van Soest et al. (1991).
Weight loss due to soaking was expressed as pre-ruminal DM disappearance.On day 6 of each collection period at 08.00 hours 3 bags for each fermentation time were incubated in the rumen for 0, 1, 2, 4, 6, 10, 16, 24, 36, 48, and 96 h, in reverse order and were removed all at the same time.After removal from the rumen, bags were washed in running tap water until the rinse was clear.Bags were then dried in oven at 55°C for 48 h.After equilibration with air for 8 h, the bags were weighed and the residues were transferred to 100 ml cups and stored for later analyses.The extent of digestion, rate of digestion and lag time, were determined for each incubation period individually.Degradation rates were determined by subtracting the indigestible residue, i.e. the 96 h of ruminal incubation, from the amount in the bag at each time point and then regressing the natural logarithm of that value against time (Sarwar et al., 1991) after correcting for lag time (Mertens, 1977).The lag time was calculated according to Mertens and Loften (1980).

Statistical analysis
Data were analyzed as a 4×4 Latin Square Design using the GLM procedure of SAS (1988).The ANOVA and trend comparisons were made to see the linear quadratic and cubic responses.Significance at p = 0.05 were used throughout unless otherwise noted.

RESULTS
Chemical composition of UTWS ensiled with or with out CSL is given in Table 3. Nitrogen, NDIN and ADIN contents of UTWS were increased linearly (p<0.01) with increasing level of CSL used to ensile UTWS.The percent retained of added N as NDIN was 48, 44, and 48% when UTWS was ensiled with 3, 6, and 9% CSL.The NDF and ADF contents were linearly increased with increasing CSL level.Nitrogen free NDF (NDF-NDIN×6.25)and cellulose contents were similar across treatments.
Ruminal NH 3 , total volatile fatty acid concentrations and pH were shown in Table 4. Ruminal NH 3 concentration in bulls at 3 and 6 h post parandial was linearly decreased (p>0.01) with increasing level of CSL used to ensile UTWS; at 9 h post parandial it was similar, however, at 12 h post parandial ruminal NH 3 was linearly (p<0.01)increased with increasing CSL level.Ruminal pH values were linearly reduced (p<0.01)at 3 and 6 h post parandial with higher CSL level there after ruminal pH was noted similar across bulls.Concentrations of total ruminal VFA, acetate and propionate were linearly increased (p<0.01) at all sampling times in bulls fed diets containing UTWS ensiled with higher CSL levels.Concentrations of total ruminal, viable and cellulolytic bacteria were linearly increased (p<0.01) with diets containing UTWS ensiled with CSL (Table 5).
Ruminal DM and NDF degradability and rate of disappearance were linearly increased (p<0.01) with increasing CSL level (Table 6).The extent of DM and NDF  Non-significant at p = 0.05.
disappearance at 96 h of ruminal incubation (an estimate of extent of digestion) was higher (p<0.01) with higher levels of CSL used to ensile CSL.Apparent DM digestibility was linearly increased (p<0.01) with diets containing UTWS ensiled with higher CSL level (Table 7).Crude protein digestibility was noted similar across all experimental diets.Linear increase (p<0.01) in NDF and ADF digestibilities were noted with diets containing UTWS ensiled with higher CSL levels.
Nitrogen balance was positive in all bulls; however, the bulls fed diets containing UTWS ensiled with CSL retained higher amount of N (Table 8).Urinary N excretion was reduced with higher CSL level than those; however, fecal N excretion was similar across treatments.Nitrogen balances whether expressed as grams per day, as a percentage of N intakes or, as a percentage of digestible N intakes were linearly (p<0.01)improved with CSL level used to ensile UTWS.When expressed as a percentage of N intakes, plasma urea-N was also affected by treatment.

Chemical composition of wheat straw
Higher N content of UTWS ensiled with CSL was because of high lactic acid content of CSL (Table 1).However, the provision of readily available nutrients (carbohydrates, minerals and proteins) for proper fermentation milieu by CSL might have caused a further drop in pH of UTWS.This reduced pH probably has changed free ammonia (NH 3 ) released from urea into an ionic form of ammonia (NH +4 ) that is very reactive and has the greater tendency to make bonds with fibrous materials.Consistent with the present findings higher N values in ammoniated straw were reported by different workers who trapped the excess free NH 3 by spraying organic acids (Sarwar et al., 2003), inorganic acids (Taiwo et al., 1995) or using non-structural carbohydrates (Khan et al., 2004;Sarwar et al., 2004a).Increase in NDF content of UTWS was due to increased NDIN because when NDF was calculated on a crude protein (CP) free basis (NDF-NDIN×6.25),its concentration was similar between UTWS ensiled with different levels of CSL.Similar results regarding hemicellulose were obtained when it was calculated on CP-free basis (NDF-NDIN×6.25)-ADF-(ADIN×6.25).In this study, approximately one half of the added N was bound in some form.Consistent with present findings, Lines and Weiss (1996) have reported that ammoniation increased the concentration of NDF and its entire increase was in the hemicellulose fraction and was because of increased NDIN.

Ruminal characteristics
Higher ruminal NH 3 values at 3 and 6 h post parandial with control diet indicated rapid release of N from the  Control, CSL 3, CSL 6, and CSL 9 diets contained 5%urea treated wheat straw ensiled with 0, 3, 6, and 9% CSL, respectively.
UTWS ensiled without CSL.Increased utilization of urea N by rumen micro-flora and ruminal fermentation explained the decrease in ruminal pH at 9 and 12 h post-parandial (Nisa et al., 2004).Present results indicated that urea N was fixed in the matrices of cell wall of UTWS ensiled with CSL and released slowly in the rumen.The slow release of ammonia N from UTWS ensiled with CSL was beneficial in keeping the ruminal NH 3 concentrations below wasteful levels (Nisa et al., 2004;Sarwar et al., 2004b).Increased ruminal VFA concentration with diets containing UTWS ensiled with CSL was because of increased degradability of DM and NDF in the rumen (Table 6).However, it may be suggested that changes occurred in the cell wall structure after ensiling UTWS with CSL, making more structural carbohydrates available for microbial fermentation in the rumen (Sarwar et al., 2006).Ensiling UTWS with fermentable carbohydrates brought physiochemical changes in straw and thus alleviated those factors that hindered fibre fermentation (Khan et al., 2006b).

Microbial count
Ruminal microbial response indicated more constant supply of NH 3 for ruminal microbial growth with diets containing UTWS ensiled with CSL.However, availability of carbon skeleton or energy for microbial growth might have differed in bulls fed different experimental diets.It has been proposed that the energy usually was a limiting nutrient for growth of ruminal microbes (Sarwar et al., 1991).It may be suggested that changes occurred in the cell wall structure after ensilation of UTWS with CSL, making more structural carbohydrates available for microbial fermentation in the rumen.Moreover, ensilation of UTWS with CSL retained most of the urea N as NDIN that was released slowly (Table 3) and thus synchronized with fibre fermentation to maximize the bacterial growth in bulls fed UTWS ensiled with CSL.

Digestion kinetics
Higher ruminal DM and NDF degradabilities and rate of disappearance of UTWS ensiled with CSL was due to increased surface area fermentability index (SAF i ) when calculated according to Fisher et al. (1989).Bhat and Bansil (1999) reported one of the constraints to NDF degradation either by enzymatic or chemical systems was the extensive hydrogen bonding that occurred in micro fibrils to give cellulose a crystalline nature.Increased NH 3 -N retention in UTWS ensiled with CSL might have sponify esters bonds between lignin and hemicellulose and saturates H-bonds linking the matrix polysaccharides.The NH 3 may affect the bonds linking the macromolecules, thus altering the physical rather than the chemical structure of cell wall.Similar results were reported by Sarwar et al. (2003) when they fed UTWS ensiled with CSL or acidified molasses to buffalo bulls.

Digestibility
Increased DM and NDF digestibilities with CSL diets were because of increased rate of degradation and shorter lag time of these fractions (Table 7) or increased SAF i of UTWS ensiled with CSL that had increased the fragility of UTWS.The increased surface area of lingo-cellulose resulted in increased accessibility to microbial attack (Sarwar et al., 2004) and ammonolysis of uronic ester crosslinks in the cell wall.In present study, increased NH 3 retention in UTWS ensiled with CSL might have broken either linkages between lignin and cellulose or hemicellulose, and increase extent and rate of cellulose and hemicellulose digestion (Nisa et al., 2005).

Nitrogen utilization
Higher plasma urea-N concentration with control diet was because of higher concentration of soluble N in UTWS ensiled without CSL (Table 3) that released rapidly in the rumen (Table 4).The lower blood urea N in buffalo bulls fed diets containing UTWS ensiled with CSL implies slower release of NH 3 .The slower release of fibre bound N from UTWS ensiled with CSL might have synchronized with fibre fermentation and thus utilized by the rumen micro-flora.The present results indicated that UTWS ensiled with CSL was effective in enhancing utilization of N by minimizing N loss.

CONCLUSION
Ensiling UTWS with CSL increased the N fixation in the matrix of cell wall thus slowing its release at ruminal NH 3 that maximized N synchronization with carbon skeleton, which consequently minimized N loss.Ensilation of UTWS with 9% CSL seems more effective to improve nutritive value of wheat straw.However, further research is warranted to see the effect of urea plus CSL treated wheat straw in lactating animals.

ACKNOWLEDGMENT
Authors are highly thankful to Rafan Maize Products Limited, Faisalabad, Pakistan on providing CSL for this project.

Table 1 .
Chemical composition of corn steep liquor (DM basis)

Table 2 .
Ingredients and chemical composition of the diets (DM 1Mineral mixture contained per gram 226 mg Ca, 100 mg P, 91 mg Mg, 45 mg Zn, 14 mg Cu, 0.6 mg Co, and 1 mg I, 0.4 mg Se. 2 Chemical composition is calculated.

Table 4 .
Ruminal characteristics in buffalo bulls fed diets containing wheat straw with or without corn steep liquor (CSL)

Table 5 .
Influence of varying levels of corn steep liquor (CSL) on rumen microbial counts in buffalo bulls fed diets containing urea treated wheat straw with or without corn steep liquor

Table 6 .
Influence of varying levels of corn steep liquor on dry matter (DM) and neutral detergent fiber (NDF) digestion kinetics in buffalo bulls fed diets containing urea treated wheat straw with or without corn steep liquor

Table 7 .
Nutrient intake and digestion by buffalo bulls fed diets containing urea treated wheat straw with or without corn steep liquor Dry matter. 2 Organic matter. 3ude protein.4Neutraldetergent fibre. 1

Table 8 .
Utilization of nitrogen (N) and plasma urea N by buffalo bulls fed diets containing urea treated wheat straw with or without