EFFECT OF ADDITION OF DIFFERENT LEVELS OF FORMIC ACID AND UREA ON CHEMICAL COMPOSITION AND FERMENTATION CHARACTERISTICS OF WILD REED PHRAGMITIS COMMUNIS SILAGE

This study was carried out in the laboratory to investigate the effect of addition of different levels of formic acid (FA) and urea on chemical composition and fermentation characteristics of common reed silages. About 400-500g of silage samples were prepared by treating chopped reed plants (2-3 cm) with solutions containing 10% of date juice, 0.5, 1 or 1.5% of FM and 0, 1 or 2% of urea. Silage samples were packed in double nylon bags and stored anaerobically for 60 days. Results showed that green color was roughly dominant in most samples of silage with vinegar odor. Samples were well aggregated with little mold was observed in few urea untreated silages. Results revealed also that increasing level of FA from 0.5 to 1 and 1.5% increased (P˂0.01) contents of dry matter (DM) by 0.79 and 1.15%, and crude protein (CP) by 1.42 and 2.11% respectively, and decreased (P˂0.01) ether extract (EE), by 0.29 and 0.63%. About effect of urea levels, most variables pointed out that there was a decrease may be occurred in fermentations due to a significant decrease in contents of DM (P˂0.01) and EE (P˂0.05). Content of crude fiber (CF) was decreased (P˂0.01) from 44.7 to 43 and 41.1% for 0, 1and 2% levels of urea respectively. Results showed that there was a decrease (P˂0.01) in pH values from 5.90 to 4.99 and 4.88, concentrations of ammonia nitrogen (NH3-N), from 1.19 to 0.75 and 0.66% of total nitrogen and total volatile fatty acids (TVFA) from 6.56 to 4.61 and 4.14 mmol/100 g DM of silage samples as a result of addition of FA at levels of 0.5, 1 and 1.5% respectively. However, increasing urea levels from 0 to 1 and 2% associated with an increase (P˂0.01) in fermentation parameters, 5.02, 5.06 and 5.70 for pH, 0.67, 0.98 and 0.95 for NH3-N and 3.70, 5.53 and 6.07 mmol/100 g DM for TVFA respectively Key word: Reed, Silage, Formic acid, Urea, Fermentation ةيقارعلا ةيعارزلا مولعلا ةلجم 2019 : 50 ) 5 ) : 1324 1335 نورخآو ديعس ريثأت ةفاضا بصقلا جلياسل تا رمخت ريياعمو يئايميكلا بيكرتلا يف ايرويلاو كيمروفلا ضماح تايوتسم يربلا Phragmitis communis و ميرك ملاس دئا ر و نيسح دمحم مثيه و ديعس نيما يلع لضاف سابع يدهمو ةزمح ناندع دعسا يضا ر حلاص مثيهو ب دعاسم سردم ذاتسا يملع ثحاب يملع ثحا يملع ثحاب يملع ثحاب يناويحلا جاتنلاا مسق , ةعا رزلا ةيلك , ءا رضخلا مساقلا ةعماج صلختسملا تيرجا لا هذه ةسارد ةيربتخملا هبيكرتو يربلا بصقلا جلياس تا رمخت يف ايرويلاو كيمروفلا ضماح نم ةفلتخم تايوتسم ةفاضا ريثأت نع يرحتلل ( جلياسلا جذامن تعنص .يئايميكلا 400 500 ىلا عطقملا بصقلا ةلماعمب )مغ 2 3 ىلع يوتحملا ةفاضلاا لولحمب مس رمتلا ريصع ىوتسمب %10 ايرويلا نم تايوتسم ثلاث و 0 و 1 وا 2 كيمروفلا ضماح نم اضيا تايوتسم ثلاثو % 0.5 و 1 وا %1.5 نوليان سايكا يف جذامنلا تئبع . ةدمل ايئاوهلا تظفحو ةجودزم 60 جلياسلا جذامن مظعم ىلع ادئاس هبش يقب رضخلاا نوللا نا جئاتنلا ترهظا .موي ثاعبنا عم تزيمت امك .لخلا ةحئا ر جذامنلا فط دوجو نع لاضف ديج كسامتب ضماحلا ةفاضا ىوتسم ةدايز نا اضيا جئاتنلا ترهظاو .ايرويلاب لماعت مل يتلا جذامنلا ضعب يف نفعتلل في نم 0.5 ىلا 1 و %1.5 ةدايز لوصح ىلا تدا (P˂0.01) تغلب ةفاجلا ةداملا نم ىوتحملا يف 0.79 و %1.15 ماخلا نيتوربلا يفو , 1.42 و 2.11 ضافخناو ,يلاوتلا ىلع % (P˂0.01) ةبسنب رثيلاا صلختسم يف 0.29 و %0.63 دق تا ريغتملا مظعم ناف ايرويلا ةفاضا ىوتسم ريثأت اما . دامرلاو ةفاجلا ةداملا يف يونعم ضافخنا لوصحل ا رظن تا رمختلا يف عجارت لوصح ىلا تراشا (P˂0.01) رثيلاا صلختسمو (P˂0.05) امك . ضفخنا ماخلا فايللاا نم ىوتحملا (P˂0.01) ىلا 41.10 ىوتسمب ايرويلا مادختسا دنع % 2 عم ةنراقم ,% 43 و %44.7 ىوتسمب ايرويلا مادختسا دنع 1 و 0 .يلاوتلا ىلع % ضافخنا لوصح اضيا جئاتنلا ترهظاو (P<0.01) نم ينيجورديهلا سلأا ميق يف 5.90 ىلا 4.99 و 4.88 نيجورتن زيكرتو نم اينوملاا 1.19 ىلا 0.75 و 0.66 ةرايطلا ةينهدلا ضامحلاا زيكرتو ,جلياسلا جذامن يف يلكلا نيجورتنلا نم % ةيلكلا نم 6.56 ىلا 4.61 و 4.14 /لوميلم 100 ةدام مغ ىوتسمب كيمروفلا ضماح ةفاضلإ ةجيتن ةفاج 0.5 و 1 و %1.5 .يلاوتلا ىلع ةدايز تدا اميف ايرويلا ىوتسم ىلا يونعم عافترا لوصح (P<0.01) يياعم يف ,تا رمختلا ر 5.02 و 5.06 و 5.70 ,ينيجورديهلا سلال 0.67 و 0.98 و 0.95 % ا نم نيجورتنل و اينوملأا نيجورتن زيكرتل يلكلا 3.70 و 5.53 و 6.07 /لوميلم 100 ةفاج ةدام مغ ةرايطلا ةينهدلا ضامحلأا زيكرتل ةيلكلا .يلاوتلا ىلع ,كيمروفلا ضماح ,جلياس ,بصقلا :ةيحاتفم تاملك تا رمخت ,ايروي *Received:29/9/2018, Accepted:26/2/2019 Iraqi Journal of Agricultural Sciences –2019:50(5):1324-1335 Saeed & et al. 1325 INTRODUCTION Since arable land is limited, there will be high correlation between human and animal diets, hence searching for un-conventional plants or new source of green crops to feed animals should be hardly taken into account. Improve animal feeding depending on local available feed sources could enhance animal production via lowering production cost and minimizing deficiency in protein and energy. Common reed, Phragmitis communis, was the most expanded plant. Recently Al-Sultani (3) concluded that it was possible to produce good quality reed silage by addition of molasses and urea at level of 10 and 1% respectively. Silage is plant material exposed to fermentation in the silo. Ensiling is a conservation forage crops with minimum nutrient loss (1). It is based on natural fermentation under anaerobic condition in which epiphytic lactic acid bacteria (LAB) convert water soluble carbohydrates (WSC) into organic acid, as a result, pH decreases and the forage is preserved (30). To improve the ensiling process, various chemical and biological additives have been developed. Formic acid (FM) was known as effective additive. Addition of FA to silage material has been reported to have generally positive effects (42). The use of FA has been found to reduce pH, lactic acid, acetic acid and butyric acid in different kinds of silage (25, 26). Kennedy (16) reported that FA can restrict the fermentation by its ability to decrease pH and antibacterial effect that results in a limited fermentation and reduction in organic acid. The restriction of silage fermentation by FA is positively associated with higher content of residual WSC and lower proteolysis (15). Moreover, addition of FA effectively improved utilization of nutrients in silage (24). Improved silage preservation, in vitro digestibility and reduced in-silo losses (19). Molasses may be added with FA to take advantage on silage fermentation. Molasses enriches the fresh material with WSC and fills the gaseous pores, thereby reducing the influx of oxygen in the silage (7). The current study aimed to evaluate the effect of addition different levels of FA and urea as an available and cheap source of nitrogen (N) on nutritive value of wild reed silage through changes in chemical composition and fermentation nature. MATERIALS AND METHODS Making reed silage This study was carried out in nutrition laboratory based on preparing 400-500 g samples of common reed silages with 4 replicates. Reed plants (40.84% DM, 15.22% ash, 5.71% CP, 3.74% EE, 44.95% CF and 30.38% nitrogen free extract (NFE)) were obtained from the area nearby Animal Production DepartmentAlqasim Green University. Plants were chopped into 2-3 cm of length. Treatment solutions were prepared by addition of date juice as a source of WSC at level of 10%, and three levels of both FA, 0.5, 1 or 1.5% and urea as a source of N, 0. 1 or 2%. Quantities of additives were estimated on DM basis of reed plants, tap water was added to ensure DM content of about 30% in treated materials. Samples were tightly packed in double nylon bags, compacted by hand to exclude the air. Samples were then moved to pit silos, well covered, filled up with soil and stored for 60 days. By the of this period bags of samples were moved again to laboratory to perform sensory characterization, chemical and fermentation analysis. Sensory and fermentation characteristics Sensory characteristics of silage included color, odor, aggregation and presence of molds were determined as described by Saeed (32). Fermentation characteristics included pH, NH3-N and TVFA concentrations were determined in silage extract of each sample prepared as described by Levital et al. (18). pH was measured immediately using a pH meter. Concentrations of NH3-N and TVFA were determined in preserved silage extract according to AOAC (4) and Markham (21),


INTRODUCTION
Since arable land is limited, there will be high correlation between human and animal diets, hence searching for un-conventional plants or new source of green crops to feed animals should be hardly taken into account. Improve animal feeding depending on local available feed sources could enhance animal production via lowering production cost and minimizing deficiency in protein and energy. Common reed, Phragmitis communis, was the most expanded plant. Recently Al-Sultani (3) concluded that it was possible to produce good quality reed silage by addition of molasses and urea at level of 10 and 1% respectively. Silage is plant material exposed to fermentation in the silo. Ensiling is a conservation forage crops with minimum nutrient loss (1). It is based on natural fermentation under anaerobic condition in which epiphytic lactic acid bacteria (LAB) convert water soluble carbohydrates (WSC) into organic acid, as a result, pH decreases and the forage is preserved (30). To improve the ensiling process, various chemical and biological additives have been developed. Formic acid (FM) was known as effective additive. Addition of FA to silage material has been reported to have generally positive effects (42). The use of FA has been found to reduce pH, lactic acid, acetic acid and butyric acid in different kinds of silage (25,26). Kennedy (16) reported that FA can restrict the fermentation by its ability to decrease pH and antibacterial effect that results in a limited fermentation and reduction in organic acid. The restriction of silage fermentation by FA is positively associated with higher content of residual WSC and lower proteolysis (15). Moreover, addition of FA effectively improved utilization of nutrients in silage (24). Improved silage preservation, in vitro digestibility and reduced in-silo losses (19). Molasses may be added with FA to take advantage on silage fermentation. Molasses enriches the fresh material with WSC and fills the gaseous pores, thereby reducing the influx of oxygen in the silage (7). The current study aimed to evaluate the effect of addition different levels of FA and urea as an available and cheap source of nitrogen (N) on nutritive value of wild reed silage through changes in chemical composition and fermentation nature.

MATERIALS AND METHODS Making reed silage
This study was carried out in nutrition laboratory based on preparing 400-500 g samples of common reed silages with 4 replicates. Reed plants (40.84% DM, 15.22% ash, 5.71% CP, 3.74% EE, 44.95% CF and 30.38% nitrogen free extract (NFE)) were obtained from the area nearby Animal Production Department-Alqasim Green University. Plants were chopped into 2-3 cm of length. Treatment solutions were prepared by addition of date juice as a source of WSC at level of 10%, and three levels of both FA, 0.5, 1 or 1.5% and urea as a source of N, 0. 1 or 2%. Quantities of additives were estimated on DM basis of reed plants, tap water was added to ensure DM content of about 30% in treated materials. Samples were tightly packed in double nylon bags, compacted by hand to exclude the air. Samples were then moved to pit silos, well covered, filled up with soil and stored for 60 days. By the of this period bags of samples were moved again to laboratory to perform sensory characterization, chemical and fermentation analysis. Sensory and fermentation characteristics Sensory characteristics of silage included color, odor, aggregation and presence of molds were determined as described by Saeed (32). Fermentation characteristics included pH, NH 3 -N and TVFA concentrations were determined in silage extract of each sample prepared as described by Levital et al. (18). pH was measured immediately using a pH meter. Concentrations of NH 3 -N and TVFA were determined in preserved silage extract according to AOAC (4) and Markham (21), respectively.

Chemical analysis
Chemical analysis was performed in duplicate manner using methods of AOAC (4). DM content was determined by drying samples in oven at 105 ºC for 24 hours (h). Dried samples were left to cool in desiccator, grind and kept in plastic containers. Ash content was determined by ashing samples in furnace at 500 ºC for 4 h. Crude protein (CP) content was determined using S4 Kjeltec system manufactured by German Behr Company. EE content was determined by extraction with hexane in Soxhlet apparatus manufactured by Korean FINE TECH, SCMP:F50-6H Company. CF content was determined using DOSI-Fiber Extractor manufactured by Spanish Selecta Company. NFE was determined by difference.

Statistical analysis
Data obtained were analyzed as a factorial experiment in completely randomized design by analysis of variance using statistical analysis system, SAS (38). Table 1 shows sensory characteristics of reed silages as affected by levels of FA and urea. Most silage samples prepared by addition of FA at level of 0.5% without urea were greenish yellow, but addition of urea at 1 and 2% caused a light darkness, however, green color was still dominant. Similar dark green color was shown in samples prepared by addition of other levels of formic acid without urea. Other samples of reed silage showed colors ranged between greenish and yellowish. Similar observations were obtained by Al-Sultani (3) in which reed plants ensiled without or with 1% of urea were greenish yellow, whereas, those ensiled with higher level (2%) showed darker color. Degradation of urea during ensiling may affect color of silages in a current study. The slight differences in color may be due to dissociation of chlorophyll green color occurred during silage fermentation (10). Rowghani and Zamiri (30) reported that differences in concentration of organic acid as affected by addition of FA may interfere with the above observations. Regarding odor, all samples characterized with diluted to concentrated vinegar odor. This may refer to existence of different Since all samples were ensiled with similar level of WSC source, vinegar odor (though it differed in strength) was affected by level of FA rather than level of urea. Djordjević et al. (12) indicated that increased level of FA increased acidity of silage. Those workers considered acidity as a result of dissociation of lactic and acetic acids produced during fermentation in addition to added FA. Odor observation was in line with that reported by Al-Sultani (3) in reed silage prepared without urea. Similar finding was also observed in most samples of reed silage in another study (32). Results revealed that most samples were well aggregated as a result of restriction of fermentation due to addition of FA. Slight presence of mold was observed in some samples especially those prepared without addition of urea. This can be explained by the antifungal role of ammonia released from dissociation of urea during ensiling (17). Similar result was obtained by Saeed and Al-Sultani (34), moldiness was observed in ureauntreated reed silage, whereas, 2% ureatreated reed silage was clear. Clearance of mold in reed silage in a current study may also correlated with level of FA, since it limits the fermentation by acidification. Antimicrobial effect of FA may interfere with cell function and inhibit growth of both mold and bacteria (20). Table 2 shows the effect of levels of FA and urea on chemical composition of reed silages. Statistical analysis revealed that except ash, chemical composition of reed silage was significantly affected by increasing level of FA. Increasing level of acid from 0.5 to 1 and 1.5% increased (P˂0.01) DM content by 0.79 and 0.48% respectively. Increase DM content may be due to the restriction of fermentation by FA, hence samples prepared with higher levels of acid may recovered higher DM. Similar results were obtained by many other studies (30,14,16). Results showed significant (P˂0.01) increase in CP content of reed silage with increasing level of FA, increases were 1.42 and 3.11% due to increasing level of acid from 0.5 to 1 and 1.5% respectively. Rowghani and Zamiri (30) reported similar results, and attributed higher CP content of FA treated whole corn crop silage to restricted effect of this acid on fermentation. Rooke et al. (29) clarified that this finding could be due to the restriction of fermentation, deamination and decarboxylation of proteins. Moreover, Selwet (40) illustrated that silages treated with organic acids were characterized with higher protein content probably due to limited growth of microorganisms leading to reduce intensity of protein proteolysis. Results of chemical composition showed as well that there was a significant (P˂0.01) decrease in EE content of reed silages by 0.29 and 0.63% due to addition of FA at 1 and 1.5% as compared with 0.5% levels. This decrease may be due to restriction of fermentation caused by high Results of a current study showed that NFE content was significantly increased (P˂0.01) due to addition of urea. Lower (P˂0.01) NFE content was found in samples of reed silages prepared without addition of urea (31.33), whereas, higher (P˂0.01) content was found in samples prepared with addition of 1 and 2% levels of urea (35.02 and 36.77% respectively). These increases may be due to effect of ammonia produced during ensiling on degradation of cell components (32,11). Table  3 shows effect of interaction between levels of FA and urea on chemical composition of reed silages. Significant analysis showed that DM and EE contents were affected (P˂0.05) by this interaction in similar trend of main effect of both factors. It seemed that samples of reed plants ensiled with FA at level of 1% without (21.99%), or with addition of low level of urea (21.87%) recovered higher DM in comparison with other samples, especially, those prepared with addition of high level of urea. This may reflect the restricted effect of acid (8,30,27). This effect may associated with lower DM loss. Samples of reed silages prepared with addition of FA at low (0.5%) and mid (1%) levels had higher EE content (4.32-4.57%) as compared with other samples, especially those prepared with high level of acid (3.51-3.71%). This may reflects the role of FA on silage fermentation, in which higher levels were shown to be more effective. Considering that WSC was converted into fatty acids during aerobic fermentation (5). Fat content may be expected to decrease due to reduction of VFA produced as a result of restriction of fermentation as shown in table 4. Statistical analysis of interaction effect also showed higher (P<0.01) CP content in reed silages prepared with addition of urea at low and high levels and FA at high (7.21 and 8.13% respectively) and medium (7.63%) levels. This result was consistent with the synergized effects of: 1) Urea which increased N and CP contents of silages with each increase in its level (33,41,32). 2) Formic acid which may limited fermentation rate and consequently, reduced protein degradation (40). Results revealed lower (P<0.01) CF content in samples of reed silages prepared with higher level of FA and urea (37.65%). This lower content though it was not significantly differed with many other samples, it can be explained by the effect of ammonia released from dissociation of urea during 60 days-ensiling period (11, 32), together with the effect of FA (table 2). Table 4 shows effect of levels of FA and urea on fermentation characteristics of reed silages. Statistical analysis revealed that pH was significantly (P<0.01) decreased with increasing level of formic acid. Similar result was obtained by Hapsari et al. (13) in grass silage. Reduction of pH can be explained on basis of the strength of formic acid which is twice stronger than lactic acid (3.75 vs. 3.85 pKa) (43). Lorenzo and O'Kiely (19) reported that FA lowered the pH immediately after its addition and worked by reducing the activity of saccharolytic enterobacteri and clostridia bacteria. Baytok and Muruz (7) concluded that addition of FA at level of 0.5% with 2, 4 and 6% levels of molasses produced well preserved grass silages with low pH, 4.62, 4.51and 4.54 respectively.

Table 3. Effect of interaction between levels of formic acid and urea on chemical composition of reed silages (% ± SE).
Level of formic acid (%) 0 In a current study, increasing level of FA from 0.5 to 1 and 1.5% decreased (P<0.01) pH of reed silages from 5.90 to 4.99 and 4.88 respectively. The decrease in pH values associated with the increase of FA level was attributed to the dissociation of the produced lactic and acetic acid and also of the FA used (12). Results revealed that there was a significant (P<0.01) decrease in NH 3 -N concentration in reed silage with increasing level of FA. Values were 1.19, 0.75 and 0.66% of TN in samples prepared with addition of 0.5, 1 and 1.5% levels of FA. This may be attributed to restriction of fermentation as affected by the limited effect of acid (8,30,27). Since low pH limits process of protein degradation during ensiling by inhibiting plant proteolytic enzymes (22). Then, reduction in pH of reed silages due to addition of FA may be another reason for reduction of NH 3 -N concentration observed in a current study. Concentrations of TVFA were changed with similar trend as NH 3 -N concentrations. Values were significantly (P<0.01) decreased from 6.56 to 4.61 and 4.14 mmol/100 g DM with increasing levels of FA from 0.5 to 1 and 1.5%, respectively. This result confirmed that addition of acid limited silage fermentation. Similar result was obtained by Saarisalo et al. (31), in this study addition of FA resulted in immediate reduction in concentrations of NH 3 -N and TVFA in grass silage. Workers attributed reduction in these fermentation parameters to the restriction of silage fermentation caused by addition of FA.  (23) reported that about 60% of studies he surveyed mentioned to the increase in fermentation acids due to addition of nonprotein nitrogen (NPN) sources at ensiling. Moreover, Bolsen et al. (9) found that an increase in concentration of lactic acid was detected as a result of addition 0.5% of urea or 0.35-0.4% of ammonia. Effect of interaction between levels of FA and urea on fermentation characteristics of reed silages was shown in table 5. Lower pH values were recorded in samples of reed silage prepared with higher level of FA regardless to levels of urea, 0, 1 or 2% (4.80, 4.87 and 5.98, respectively), samples prepared with lower level of acid without urea (4.88), and those prepared with medium level of acid and lower level of urea (4.88). Samples prepared with lower level of acid and urea at levels of 1 and 2% were characterized with higher concentrations of NH 3 -N, 1.52 and 1.51 % of TN, respectively. This may be attributed to dissociation of urea in medium of lower level of FA, in which degradation of protein may not be effectively inhibited. Higher concentration of TVFA was recorded in samples prepared with addition of 0.5% level of FA and 2% level of urea (9.82 mmol/100 g DM), whereas, lower concentrations were recorded in samples of reed silages prepared without addition of urea and all levels of FA. This can be explained on basis of integration of lesser effect of lower level of acid as compared with other levels in restricting fermentation and consequently slight reduction in acid produced during fermentation (31), and effect of addition of high level of urea which enhanced acid production.

Table 5. Effect of interaction between levels of formic acid and urea on fermentation characteristics of reed silages (as appeared ± SE).
Level of formic acid (%) 0