ENSILING CHARACTERISTICS AND NUTRITIVE VALUE OF CORN COBS AS AFFECTED BY ADDITION OF DIFFERENT LEVELS OF UREA AND SOLUBLE CARBOHYDRATES

This study was carried out in vitro to investigate the effect of addition of different levels of dates honey (DH) as soluble carbohydrates (WSC) and urea on fermentation quality and nutritive value of corn cobs silages (CCS). CC was ensiled with 4 levels of DH, 4, 6, 8 or 10% and 3 levels of urea 0, 1.5 or 3%. CCS samples were packed in double layer nylon bags and kept anaerobically for 60 days. Results revealed that color of CCS samples were ranged between yellow and brown. Smell of diluted vinegar was detected in samples made with addition of DH only, whereas, those made with addition of low and high levels of urea were characterized with smell of diluted and concentrated ammonia respectively. Increasing DH level from 4 to 10% significantly (P˂0.05) decrease in neutral detergent fiber (NDF) and hemicellulose by 2.42 and 2.69% respectively, with significant (P˂0.01) increase in crude protein (CP) and ether extract (EE). Significant (P˂0.05) decrease in crude fiber (CF) and NDF with significant (P˂0.01) increase in CP and cellulose contents were noticed due to addition of urea. Results also showed a trend for pH to be reduced (P˂0.01) with increasing level of DH, but different responses to increased urea levels were shown, where values were 5.91, 6.17 and 6.95 at levels of 0, 1.5 and 3%. Increasing DH level (6 and 8%) significantly (P˂0.01) decrease silage ammonia nitrogen concentration as a percentage of total nitrogen (18.52 and 18.35) and increase concentration of total volatile fatty acids as a percentage of silage dry matter (DM) at higher levels (6.73 and 8.24%), however both concentrations were reversely responded to increase urea level. Lower DM loss and buffering capacity (BC) were recorded at the lower level of DH (41.86 and 43.60 meq NaOH/100 g DM), whereas the higher level was associated with better Fleig point (Fp). Regarding effect of levels of urea, lower (P˂0.01) DM loss and BC with higher (P˂0.01) Fp at the lower level were observed, the later was 63.16. Results of study also revealed that increasing level of DH decreased aerobic stability of CCS samples to 41 hours (h), but higher value of 43.5 was recorded at higher level of urea. Results also showed that in vitro DM digestibility of CCS was (P˂0.05) improved due to increasing levels of DH and urea, however, lower (P˂0.05) values were obtained with lower levels of these additives.


INTRODUCTION
Animal production in middle and southern parts of Iraq is characterized with clear deficiency in diets, particularly, pastures, good quality roughages and green forages. Availability of such diets is necessary for ruminant feeding (57). This situation forced farmers and herdsman to use expensive concentrates to improve productivity of their animals. However, small herds owners are still depending on poor grazing and feeding low quality crops byproducts such as straws. These residuals are low nutritive sources due to low CP and WSC with high CF contents. Accordingly, feeding to animals will lead to poor performance unless suitable processing or supplementation was applied (22). However, high content of structural carbohydrates may render these materials as an important potential energy sources for ruminants due to their unique ability to utilize lignocellulosic complexes by anaerobic activity of rumen microbes. Tripathi,et. al. (71) reported that activity of ruminal bacterial can be reflected on improved dietary CF, consequently, increased energy available for microbial growth. According to statistics issued by general directorate of Agriculture in Babylon Province in 2014, production of yellow corn was 230706 metric tons, from which only 135418 tons were marketed to factories. Therefore, 30,000 tons of cobs were accumulated in these factories. Corn cobs comprise 20% of total corn ears (11). This ratio may reach 25% (31). Average productivity of corn crop in Iraq is about 2600 kg of corn grain/hectare or 3336 ears/hectare, and then there will be 664-832 kg cobs/hectare (21). Corn cobs as other agricultural residuals are characterized with low digestibility due to attachment of cell wall components with lignin, where ratios of cellulose, hemicellulose and lignin are about 15, 40 and 30% respectively (11). Many local attempts were conducted to improve utilization of cobs by chemical and biological treatments (7,10,32,33). Since chemical treatments were associated with many problems, such as increase basicity of rumen fluid and increased urine excretion as a result to increased quantity of water that animals have to drink (25). Dry matter loss and necessity to wash chemical treated material in addition to risk of handling with chemicals (10). Moreover, biological treatments faced challenges concerning selection of effective microbes and securing growth conditions (68). Therefore, ensiling may be suitable alternatives. Many workers concluded possibility of introducing corn cobs in ruminant feeding. Nada,et. al.,(45) successfully added 10% of corn cobs in sheep diet. Encouraging results were obtained with 5 and 10% of corn cobs (6). Raheef (51) reported that alkali treatment of corn cobs with 4% NaOH improved in vitro dry matter and organic matter digestibility (IVDMD and IVOMD). Reshan (52) noticed that addition of urea increased N and decreased CF contents of CCS which were characterized with accepted smell and low pH. The objective of this study was to investigate the possibility of improving low nutritive value of corn cobs by manipulation the deficient chemical composition by addition of DH as a source of WSC and urea as a source of N, and making use of ensiling conditions and exposure to ammonia released from hydrolysis of urea to improve digestion due to expected breakdown of cell wall.

MATERIALS AND METHODS Preparation of Silages
Corn cobs obtained from factories and stores of general company of cereals trading in Babylon Province were chopped handly to 2-3 cm. It was then treated with solutions made on DM basis by addition of 4, 6, 8 or 10% of DH, and 0, 1.5 or 3% of urea. Table 1 shows components of silages prepared in the study. Water was added to dilute treatment solutions and to bring DM content of ensiled CC to about 40%. Samples (500g each) were packed in double layers plastic bags and placed in pit silos, well compacted and kept covered for 60 days, thereafter, it were opened to determine pH and other determinations.

Sensory characteristics of silage
Sensory characteristics of silage included color, smell and presence of molds. Yellow and brown colors were based on to describe color of CCS samples. Smell was determined directly after opening bags by sense, terms of diluted or concentrated were used for precise description of smell emanated from samples. Moldiness was mentioned by (+) referring to presence of mold, number of (+) was used to describe extent, whereas, (-) was used referring to clearance of samples from molds.

Fermentation characteristics
Water extracts of each CCS sample were prepared as described by Levital,et. al.,(39) by homogenizing 50 gram samples for 10 min in 500 ml of distilled water and filtered through 4 layers of cheese cloth and filter paper thereafter. The pH of the water extract was immediately measured using a pH meter (Mi 180 Bench Meter). Extracts were then kept frozen at -20ºC until subsequent determinations. Ammonia nitrogen (NH 3 -N) and total volatile fatty acids (TVFA) were determined in water extract according to AOAC (13) (39), where, 120 g of samples were thoroughly shaken to ensure air exposure and then packed loosely in 500 ml plastic container. Samples were covered with double-layered cheesecloth to prevent drying and contaminations. Four small holes were made on top of each container to permit air exchange. Thermometer was inserted inside silage mass. An additional container filled with water to measure ambient temperature. Temperature of silage was recorded every 30 min. Aerobic stability was defined as time required to raise silage temperature by 2 • C above ambient temperature (38). BC was determined as described by Playne and McDonald (50). 20g fresh material was macerated with 250 ml of distilled water. The pH of the macerate was recorded. The macerate was titrated first to pH 3 with 0·1 N HCl in order to release bicarbonate as carbon dioxide (R 1 ), and then was titrated to pH 6 with 0·l N NaOH (R 2 ). BC was expressed as meq. of alkali required to change the pH from 4 to 6 per 100 g of DM, after correction for the titration value of a 250 ml water blank. BC (meq. NaOH/100g DM) = 390 /(R2-R1) × DM% of sample. IVDMD and IVOMD of CCS samples was determined as described by Tilley and Terry (70).

Chemical analysis
Silage samples were analyzed for proximate analysis (13). DM was determined by drying in air draft oven at 60 ºC for 48 hours. Ashing dried samples at 500 ºC for 4 hours was used to determine OM. EE was determined by hot extraction with hexane using Sohxylate appatarus. CP was determined by Kjeldahl method using S 4 Kjeltec System. NFE was calculated by difference. Fiber fractions were anlyzed according Goering and Van Soest (27).

Statistical analysis
Data obtained were analyzed as a factorial experiment in completely randomized design by analysis of variance (59). Factors were level of DH and urea with 4 replicates. Means were separated using Duncan multiple range test (24).

RESULTS AND DISCUSSION
Chemical composition: Table 2 shows the effect of level of DH and urea on chemical composition of CCS. CP content was significantly (P˂0.01) affected by both levels, where, it was increased to 6.79% with increasing level of WSC to 10%. This may due to improving ensiling condition with increasing level of WSC added at ensiling (55). As evidenced by higher (P˂0.01) concentration of TVFA (Table 4). CP content was as expected increased ascending and higher (P˂0.01) value was associated with higher level of urea. This was due to presence of ammonia released from dissociation of urea during ensiling (58). This was proved in a current study by increased pH values with increasing level of urea from 0 to 1.5 and 3%, where, it increased from 5.52 to 6.17 and to 6.95 respectively (  (11), as a result of providing silage microbes with incremental energy leading to increase rate of fermentation rate (16). Similarly, other cell wall components were significantly affected by urea treatment, NDF content was decreased (P˂0.05) by 1.64 and 1.74% at levels of 1.5 and 3% of urea, and hemicellulose by 1.52 at low level of urea only. Shoukry (65) reported 2.5 and 1.9% decrease in NDF and hemicellulose contents in corn cobs due to treatment with urea at rate of 3%. The significant decrease in NDF may due to degradation of hemicellulose and a decrease in its quantity in treated cobs (28). Release and degradation of hemicellulose was the reason for decrease its content in corn cobs (65). Whereas, cellulose content was increased (P˂0.01) by 1.49 and 0.77 due to treatment with low and high level of urea respectively. The significant increase in cellulose content can be explained by the effect of exposure to the ammonia during ensiling on links between lignin and cellulose and disconnecting of hemicellulose from these links (3,67). Alwazir (11) reported that urea treatment of corn cobs increased (P˂0.01) cellulose and total nitrogen and decreased (P˂0.01) NDF, hemicellulose and lignin. The author attributed increase cellulose content to the role of ammonia produced from degradation of urea in breakdown of covalent linkages that linked lignin with cellulose and hemicellulose leading to increase cellulose and decrease lignin as a result of releasing cellulose and hemicellulose which was bonded to lignin. Same result and conclusion were announced by Wanapat (4) reported that the positive effect of molasses in reducing proteolysis and concentration of NH 3 -N had been covered by rapid degradation characteristics of urea. Concentration of total volatile fatty acids (TVFA) adopted significant ascending (P<0.01) trend with increasing level of DH, values were 5.21, 5.52, 6.73 and 8.24% of DM in CCS samples ensiled with 4, 6, 8 and 10% of DH respectively. This agreed with results obtained by Arbabi and Ghoorchi (14) who observed that there was an increase (P<0.05) in TVFA due to increasing level of molasses. Considering these acids represented end product of degradation of sugars during anaerobic condition of ensiling (47,64), providing silage microbes with increased amounts of readily fermented carbohydrates such as DH, will increase concentration of TVFA. However, TVFA values adopted significant (P<0.01) descending trend with increasing level of urea, values were 8.15, 6.22 and 4.90% of DM in CCS samples ensiled with 0. 1.5 and 3% of urea respectively. This can be explained by the fact that addition of urea affected silage fermentation quality through elevated pH and release of ammonia (35). With regard to the effect of interaction between level of DH and urea, as shown in  (9) noted that DM loss was associated with extent of anaerobic fermentation occurred in stored materials after depletion of oxygen. Statistical analysis also showed that this DM loss was increased (P<0.05) in CCS. samples ensiled with urea as compared with those ensiled without urea, values of increase were 3.48 and 3.07% in samples ensiled with low and high levels of urea as compared with control in which CCS were ensiled without urea. This may due to effect of ammonia produced from degradation of urea during ensiling that embedding fermentation and slowing down its rates. Tapia,et. al.,(69) (12,17,42,55,62). Fleig points were markedly declined (P<0.01) to below than 25 as a result of treatment with high level of urea as compared with those ensiled without and with 1.5% of urea which gained 63.16 and 40.63 points respectively. CCS sample as affected by level of urea treatment in the current study were respectively, considered worthless, good and moderate quality according to the mentioned score. Decline of Fp may be due to increase ammonia concentration. Açıkgöz (2) attributed such decline to the buffering effect of increased ammonia content that neutralized organic acids and prevented rapid drop of pH. Regarding aerobic stability (AS) which represents stability of silage against aerobic deterioration caused by aerobic microbes that attack silage after opening the silos and oxidize end products of fermentation (23). Results (table 6) showed that AS was significantly (P<0.01) affected by level of DH, where, CCS samples ensiled with 6% of DH were superior as compared with other samples. Since there were different levels of DH used in the current study ranged from 4 to 10% on DM basis of corn cobs, then superiority of 6% level may be associated with preparation procedures rather than DH levels per se. This agreed with observation of O'Kiely and Muck (48) that AS neither associated with addition of glucose at ensiling time nor associated with DM content, pH and yeast counts. However, AS in the current study, was significantly (P<0.01) increased with increasing level of urea, increase values were 3 and 4 hours in CCS samples ensiled with 1.5 and 3% of urea respectively, as compared with those ensiled without urea. Oude Elferink, et. al., (49) reported that AS can be improved by addition of urea and ammonia. This was due to antifungal effect of ammonia released from degradation of urea during storage (37). Buffering capacity as defined by Martinez-Fernandez, et. al., (41), represents extent of resistance of materials to changes in pH. Materials with high BC are more resistant to a decrease in pH (50). Results shown in table 6 revealed that increasing level of DH to 8 and 10% decreased (P<0.01) BC by 6.43 and 7.19 meq NaOH/100 DM respectively as compared with low level of DH (4%). This may be due to role of DH in providing silage microbes with soluble sugars and stimulating organic acids producing silage fermentation. The organic acids were responsible for most of the buffering effect in herbages and silages (50). Results also showed that BC values were increased (P<0.01) with increasing level of urea. This may be attributed to increase ammonia concentration which resisted decline in pH. Moharrery (44) noticed that BC was increased due to treatment of wheat straw with ammonia; this was attributed to increase pH. The increase values in BC due to treatment with 1.5 and 3% of urea in the current study were 5.49 and 11.65 meq NaOH/100g DM respectively, as compared with CCS samples ensiled without urea. Regarding digestion of CCS, results showed there was a significant (P<0.05) increase in IVDMD with increasing level of DH, coefficients were 39.40, 40.15, 42.30 and 42.32% in CCS samples ensiled with 4, 6, 8 and 10% of DH respectively. Similar results were obtained by Hassan and Mohamad (33) in corn cobs due to addition of molasses at rate of 10%. This may be attributed to the role of WSC in stimulating silage fermentation (46). IVDMD was also increased (P<0.05) as a result of increasing level of urea, CCS samples ensiled with low and high levels of urea (42.23 and 41.63%). IVDMD of corn cobs was increased by 8.9% due to treatment with urea and NaOH (33). IVOMD in the current study was responded similarly, where; coefficients were 46.32, 49.09 and 48.70% in CCS samples ensiled with 0, 1.5 and 3% respectively. Shoukry, et. al., (66) treated corn cobs with 3% of urea and noticed a significant increase in DM and OM digestibility. Similar results were also obtained due to treatment of corn cobs with urea at rate of 5% (8). Improvement of IVDMD and IVOMD of CCS due to urea treatment can be explained by exposure of cellulose and hemicellulose to the enzymatic activity of ruminal microbes as a result of the role of ammonia to breakdown of cell wall (32). Regarding effect of interaction between level of DH and urea, results shown in table 7 that lower DM loss was recorded in CCS samples ensiled with lower level of DH, and treatment with urea increased this loss. Similar trend of DM loss was shown in other samples and for all DH levels. This may be due to the interaction between level of DH and urea on rate and extent of fermentation (69). Jarrige, et. al., (34) reported that DM loss as affected by fermentative activity was 12%, and it was attributed by these workers to catabolization of nonlipid organic compounds (mostly WSC and proteins). In the current study DM loss ranged between 6.64 and 14.65%. Silage qualities as judged by Fleig points (table 7), were satisfying, moderate and good for CCS ensiled with DH regardless to urea level, but those ensiled with urea regardless to DH levels were judged semi moderate and somewhat worthless (36). This may be due to insufficiency of DH levels, taking into account, the cellulosic structure of corn cobs. Tuah and Ørskov (72) considered most corn cobs as cell walls, 46.4% as hemicellulose and only 6.04% as cellular components. Therefore, degradable component of cell walls may not well available for ruminal microbial activity. AS is the time in which silage sustained aerobic deterioration after opening silos. Aerobic microbes will attack silage surface and degrade lactic acid produced during anaerobic fermentation to CO 2 and water, leading to increase pH, which enhance growth of many aerobic deterioration microbes (42). Lower time was recorded in CCS samples ensiled with DH levels only, where, AS were 39-40 hours. As compared with results obtained by Schmidt,et.al.,(63) who reported AS of 30.4 hours for corn silage, and 36 hours in other study (37), CCS samples prepared in the current study without addition of urea seemed that it was well preserved, but inexistence of buffering agent may weaken resistance to aerobic deterioration as compared with other samples which resisted that deterioration for relatively longer period, and attributed to existence of ammonia. BC was also affected (P<0.01) by interaction between level of DH and urea, values were ranged between 39.53 and 60.40 meq NaOH/100g DM in CCS samples ensiled with 10% of DH only and those ensiled with 4% of DH and 3% of urea respectively. In general, BC values calculated in the current study are consistent with 25-40 for tropical roughages and 40-60 40 meq NaOH/100g DM for leguminous reported by Playne and McDonald (50). Higher (P<0.05) IVDMD was recorded in CCS samples ensiled with 8 and 10% of DH with 1.5 and 3% of urea respectively (43.97, 49.67%), whereas, lower values were recorded in CCS samples ensiled with 4% of DH only. Regarding in vitro digestibility, table 7 shows that ensiling CCS samples with 4% of DH only resulted in lower IVDMD and IVOMD, higher values was associated with samples ensiled with 8% of DH and 1.5% of urea. These results may speculate importance of introducing N source together with WSC to improve digestibility of corn cobs. Moreover, using higher level of these soluble carbohydrates may not be sufficient with higher level of urea.

Table 2. Effect of level of date honey-DH (A) and urea (B) on chemical composition of corn cobs silages
Means with different letters are differed significantly * (P˂0.05) ** (P˂0.01) NS=non-significant Table 3   biological procedures to upgrade cereal straws for ruminants, Nutrition Abstracts and Reviews, 68 (5)