Effect of combined lactic acid bacteria at the ensiling of rice straw with whey or molasses plus urea on degradability, palatability, digestibility, and nutritive values.

Objective
This study was designed to ensiling rice straw by using some additives and assessing its effect on the fermentation, degradability, feed consumption, and digestibility.


Methods
Ensiling rice straw using either water (50ml/kg v/w), or molasses plus urea (45 ml + 5g /kg), or whey (55ml/kg v/w) was conducted in this study. Each of these treatments inoculated with three levels of lactic acid bacteria. The best treatments from in-vitro results were used to compare with urea treated rice straw in the digestibility trials. Twelve adult Ossimi rams (average weight =61.4±0.16 kg) randomly distributed on four experimental diets in a 4 x 4 Latin square (3 in each block), each period lasted 28 d.


Results
Added whey or molasses plus urea (MU) showed well properties for ensiled rice straw (ERS), especially with the high level of lactic acid bacteria addition. Accumulative gas production has corresponded with the degradability of OM, NDF, and CP (%). The feed consumption of ERS and palatability increased with the whey or MU as an additive, especially with a high level of lactic acid bacteria. Despite the similarity of the apparent digestibility coefficients of NDF, and ADF for a diet including ERS with lactic bacteria compared that containing 3% urea-treated rice straw (UTRS), the digestibilities of protein, organic matter, and ether extract were significantly higher when emulated to the group fed UTRS. The total digestible nutrients and digestible protein of the groups fed on diets that included any of ERS with whey or MU were superior to a diet contain UTRS.


Conclusion
The results concluded that added either whey or molasses plus urea can be successful in improving the quality of the ensiling rice straw.


INTRODUCTION 42
At a time when the population increase and depletion of some important agricultural resources, 43 especially those used in animal feed the demand for animal protein grows, principally in poor 44 countries within the arid area. On the opposite hand, a lot of ingredients accumulate and are not 45 being optimally exploited, especially rice straw (RS), which is produced in large quantities. 46 These lots of RS unused left improperly disposed of by burnt directly, wasting resources and 47 A c c e p t e d A r t i c l e by incinerating in a muffle furnace at 550°C for 4h. All of the chemical examines were executed in triplicate and expressed on a dry matter basis. Neutral detergent fiber (NDF) was 179 assayed with the addition of a heat-stable amylase but without sodium sulfate and acid 180 detergent fiber, procedures were performed as a description of [20]. Water-soluble 181 carbohydrate (WSC) consistency was determined by the colorimetric method [21]. 182 Nonfibrous carbohydrates (NFC) was calculated by difference, where: NFC = 100 -(%NDF 183 + %CP + %Fat + %Ash). The chemical composition of ERS and the experimental rations are 184 presented in Table (2&5). The buffering capacity (BC) was determined according to the 185 method described by [22]. The ensilability of RS was assessed by calculating the 186 fermentation coefficient (FC) according to the formula described in [23]. 187

Statistical analysis: 188
The digestibility trials subjected to ANOVA for a 4x4 Latin square design using the 189 General Linear Models (GLM) procedures of the Statistical Analysis System Institute (SAS 190 version 9.4, SAS Institute, Inc. 2002) according to the following model: Y = Y = μ + α + β + 191 γ(β) + p + ε, where μ is the general mean, α is the fixed effect of treatment, β is the random 192 effect of the square, γ(β) is the random effect of the animal within a square, p is the random 193 effect of the period and ε is the random error. The data of silage fermentation quality, chemical 194 composition, and gas production kinetics were analyzed separately for variance using a general 195 linear model (GLM). Differences between treatment means were determined by Duncan's 196 multiple range test. Differences among means with P<0.05 were accepted as representing 197 statistically significant differences. 198

Physical characteristics evaluation 200
The results of the physical quality evaluation of ERS in terms of colour, odor, texture, and 201 molds are shown in Table 1. The ERS was loose and had no clumps and indicated that the 202 A c c e p t e d A r t i c l e fermentation occurred as in the good quality silage, which gives a good impression. Regarding 203 the temperature, there were no differences among all types of tested ERS. While the pH values 204 were decreased (P<0.05) by adding the LAB, the decline of pH significantly (P<0.05) better 205 with a high level of LAB addition. Acetic acid was significantly (P<0.05) increased in the ERS 206 with the increased level of added LAB. Ammonia concentration in ERS and buffering capacity 207 were significantly (P<0.05) increased with the presence of MU or whey and was significantly 208 (P<0.05) more at the higher level of addition of LAB. The values of buffering capacity and 209 Flieg Score significantly (P<0.05) improved with the presence of MU or whey. The higher 210 level of addition of the LAB was significantly (P<0.05) higher than the low levels. 211 Regarding the number of microorganisms in ERS, no differences were found with mold 212 counts among different ensiled types of RS and the numbers were in the normal range. Lactic 213 acid bacteria, aerobic bacteria, and yeasts significantly (P<0.05) augmented with MU or whey, 214 while were significantly (P<0.05) raised when added a higher level of LAB. The probability for 215 the effect of silage type had a significant (P<0.05) effect on the concentration of LA, ammonia, 216 buffering capacity, and fermentation coefficient, while it did not affect the pH, acetic acid, 217 butyric acid, and Flieg Score. The probability for the effect of LAB had a significant (P<0.05) 218 effect on pH, acetic acid, butyric acid, and Flieg Score, while it did not show an effect on LA, 219 buffering capacity, and fermentation coefficient. Concerning the microbial count, the 220 probability for the effect of silage type had a significant (P<0.05) effect on the total number, 221 LAB, anaerobic bacteria, and yeast, whereas the probability for LAB showed an effect on a 222 total count, except on the number of LAB and yeast. 223

Chemical composition of ERS 224
The chemical composition of the tested ERS in Table 2  As anticipated, the NDF and ADF content of the different ERS types, there was a 237 significant (P<0.05) declined in its content in particular with those containing MU or whey. 238 The content of NDF and ADF of ERS significantly (P<0.05) lowered with a higher level of 239 addition of LAB bacteria. As for the content of different ERS types of NFC or WSC, it was 240 significantly (P<0.05) higher when added either of MU or whey, while significant (P<0.05) 241 furthers more with the addition of LAB. As for the effect of the ensiling method for RS on the 242 chemical composition of the tested ERS, the ensiling process had a significant (P<0.05) moral 243 effect on all components of the ERS, except for DM and EE content. While, LAB bacteria had 244 a significant (P<0.05) effect on all ERS components except for OM, CP, and Ash content. 245 No significant difference was found in potential gas production (b) among all tested silage 246 except RWL1 was decreasing (p<0.05). The gas production rate constant for the insoluble 247 fraction b (h -1 ) was not different among all experimental ERS incubated. 248

In-vitro incubation of ERS 249
The Basic pattern fermentation, GPt, and degradability of different types of ERS are 250 shown in Table 3. The values of pH were significantly (P<0.05) observed lower when the ERS 251 contained MU or whey compared to their absence when in-vitro incubation. A significant 252 A c c e p t e d A r t i c l e (P<0.05) decline was also noted with the increase in the level of addition of the LAB. An 253 opposite trend was observed with NH 3 -N (g kg -1 TN). Although the total of SCFA's increased 254 with ERS which included MU or whey at in-vitro incubation, the content of acetate, propionate, 255 and butyrate (mmol/l) was not different. These results indicate that the fermentation efficiency 256 in the rumen will increase with feeding on ERS which includes either MU or whey, especially 257 when the addition of a high level of LAB. 258 The number of bacteria and protozoa significantly (P<0.05) increased with in-vitro 259 incubation of ERS that included either MU or whey. On the other hand, found that adding LAB 260 to the ERS significantly (P<0.05) increased the numbers of bacteria and protozoa. While 261 significantly (P<0.05) increasing in volume of GPt with added MU or whey in ERS, the gas 262 rate released was comparable among the different forms of ERS. 263 The gas released corresponds to the degradability of OM, NDF, and CP (%). The gas 264 released corresponds to the degradability of OM, NDF, and CP (%). The degradability rates of 265 IVOMD, IVNDFD, and IVCPD (%) increased (P<0.05) significantly of RMU and RWh 266 compared to RW, especially with a high LAB level. The same trend was observed with ME 267 (MJ/kg DM), MCP (g/kg DM), and RFV. The higher DM consumption with the ERS reflects 268 the higher IVOMD, IVNDFD, and IVCPD. 269

Chemical composition, intake, and palatability 270
The content of DM and OM in the control diet were significantly (P<0.05) higher 271 compared to those diets containing the experimental ERS. At the same time, the content of DM 272 and OM decreased (P<0.05) in diets containing ERS especially with that employing MU or 273 whey. The same trend was seen with the content of NDF and ADF in the ERS. In opposite, the 274 control diet contents of CP, EE, and ash decreased (P<0.05) compared to feeds containing 275 tested ERS. In the same context, the content of these components was significantly (P<0.05) 276 A c c e p t e d A r t i c l e high in diets containing ERS, which included both MU or included whey. The same trend was 277 seen with the contents of NFC, and WSC. 278 Although there were no differences in offered of all EAR types, the consuming and 279 palatability for RWL3, RMUL3, and RWhL3 were significantly (P<0.05) higher compared to 280 the control group fed UTRS. The consumption of RWhL3 was significantly (P<0.05) higher 281 compared to RWL3 and RMUL3. The residues of feed (kg) were significantly (P<0.05) 282 reduced when sheep fed ERS compared to fed on UTRS. While the feed residue of RWhL3 was 283 lower (P<0.05) compared to RMUL3, which was significant (P<0.05) higher than the residual 284 when fed RWL3. This result was reflecting on the eating time (min/d) of feed which decreased 285 (P<0.05) at fed UTRS compared to the time spent on eating for fed ERS, except for RWL3. 286 Reversing the trend was observed for actual feed consumption (AFC) and palatability 287 compared to the time animals spent chewing feed (min/d). While the same trend of chewing 288 time was observed with feed residue. 289

Apparent Nutrient Digestibility 290
The results obtained in Table 5 indicate that there were no differences (P>0.05) in the 291 digestibility coefficient of NDF and ADF among all different tested groups. Feeds RMUL3 and 292 RWhL3, which contained ERS, whether treated by MU or whey, showed a significant (P<0.05) 293 increase in the digestibility coefficients of OM, CP, and EE compared to control. No 294 differences are shown for CP and EE content of RWL3 compared to RMUL3, RWhL3, and 295 control. The results appear that diet RWhL3, which contained ERS with whey, was 296 significantly (P<0.05) superior for total digestible nutrients (TDN) and digestible crude protein 297 (DCP) over the other tested groups, while there was no difference between RMUL3 and the 298 control group. 299 DISCUSSION 300

Physical quality evaluation 301
A c c e p t e d

A r t i c l e
Several studies have demonstrated that increasing dietary concentrate contents would increase 302 the feed intake of ruminant animals, although they are typically fed high-fiber diets due to 303 physiological and economic considerations [24]. The extent of fermentation and quality of 304 ensiled crops hinges on pH value and well-preserved silage usually has a low pH but the LA 305 concentration may be high [25,26]. In our current study, the ERS preservation was good, as 306 evidenced by the relative confidente pH, butyric acid, and NH 3 -N values, and the relatively 307 suitable ratio of LA to acetic acid, these data are in agreement with the results found by [27]. 308 Whey is considered to be of high quality if the quality of the protein is taken into account, 309 as it contains all the essential amino acids, vitamin D, and lactose [28] as the effects of lactose 310 increase on the final products of fermentation with L. plantarum strain. From this, it can be 311 speculated that the improvement of the fermentation which occurred with added whey at ERS, 312 due to the type of sugars present in the whey compared to those in molasses. Generally, the 313 acceleration of homofermentative attributable to adequate WSC during the initial stage of 314 ensiling with L. plantarum addition, thus producing more LA, as was reported by [1]. The 315 factors affecting the quality of fermentation include not only the physiological properties of 316 epiphytic bacteria but also the chemical composition of ensiled material [26]. Microorganisms 317 can be assorted according to their efficiency to grow at low, reasonable, or altitude 318 temperatures (psychrophilic, mesophilic, and thermophilic microorganisms, respectively). In 319 general, the proper temperature for good silage fermentation is <25°C [25]. Silage pH is a 320 substantial factor in the long-range stability of the ensiled plant substance. A report by [28] 321 stated that improvements in the aerobic stability of wheat silage were as a resulted of added 322 LAB where leads to a decline in pH slowly. 323 The concentration of LA increased by the addition of whey or MU, compared to the 324 non-addition and further was shown with an increased level of LAB. Rapid production of LA is 325 important to obtain high-quality silage, as it is responsible for inactivating plant enzymes and 326 A c c e p t e d A r t i c l e unwanted microorganisms that may inhibit fermentation or lead to deterioration of silage even 327 after the end of fusion, i.e. silage with low stability [25]. The pH values of ERS reported in our 328 study at 45d are within the range reported for well-ensiled materials [5] who reported that the 329 potential of sugarcane molasse and whey as additives to ensile lemongrass leaves was 330 investigated. The presence of acetic acid in silage indicates that fermentative bacteria were 331 active during ensiling, where acetic acid resulting from WSC fermentation [26]. The acetic 332 acid concentration in any subsequent period was lower than the LA concentration. The 333 concentration of acetic acid at any given ensiling period was lower than that of LA. WSC, therefore molasses, a source of WSC, is often used along with urea to help to prevent 338 silage instability [29]. A study by [26] states that the buffering capacity of silages increases 339 with the addition of urea. In the same context [30] found that the addition of 0.5% urea or 0.5% 340 urea plus 5% molasses to the silage significant effect on buffering capacity. 341 In the current study, the ERS was well preserved as indicated by good fermentation 342 characteristics especially with MU or whey, such as pH value and NH 3 -N/TN ratio, as well as 343 Flieg points after 45 d of ensiling. In work by [1] reported lower yeasts and molds, and greater 344 aerobic stability in corn silage treated with MU and added LAB. Lower WSC content of RS in 345 the harvest stage restricts LAB growth during silage fermentation [29]. Therefore, adding MU 346 or adding whey resulted in the necessary nutrients for the LAB, which improved the 347 fermentation efficiency in silage. High numbers of yeasts in ensiled materials tend to spoilage 348 more quickly than that with lower numbers of yeasts when exposed to air [6], because many of 349 these yeasts are lactate assimilators. A report by [28] observed an increase in the LA 350 concentration in silage 15% with added molasse while increased 25% with whey and was 351 A c c e p t e d A r t i c l e higher than in silage supplemented with commercial additive. Added whey or MU [5] 352 improved the action of LAB, which is capable of amino acid. In the present study inoculation 353 with LAB reduced P<0.05) pH, as well as the acetic acid, butyric acid, and NH 3 -N contents, but 354 increased the LA content. Current results imply that LAB inoculation promoted rapid 355 acidification, which inhibited the proteolytic activities of plant enzymes [25]. The difference in 356 the fermentation quality of silage among rice varieties was mainly because of the WSC 357 concentration in the straw; LAB application and selection of rice varieties whose straw 358 contains high levels of WSC are a good strategy for ERS. Therefore, the strain of bacteria used 359 in the preparation of ERS can promote the propagation of LAB, decrease pH, inhibit the growth 360 of clostridia and aerobic bacteria, and improve the quality [7]. The improvement in the quality 361 of the wheat silages attributed to decrease major spoilage microorganisms such as aerobic, 362 yeasts, which can utilize both WSC and LA, followed by molds [6]. 363

Chemical composition of ERS 364
The ERS at appropriate forage ratios is a good option for making well-preserved RS as 365 In the current study perhaps the higher content of degradable nutrients in ERS provides an 400 appropriate substrate for microorganisms which resulted in relatively high gas production. This 401 A c c e p t e d A r t i c l e indicates that a combination of MU or whey may enhance the quality of the silage. In work of 402 [3] indicated that the gas potential extent of gas production (b) and cumulative gas production 403 at 120 h was increased by MU supplementation regardless of used dose (P<0.001). The community with an increase in NFC and WSC. This may be since LAB maintains a good 419 range of acidity [25], which helps to increase the activity of bacteria and protozoa, thus 420 increasing their numbers [6]. 421 The effect of silage source on various microbial species and the characteristics of 422 fermentation are linked to differences in the chemical composition between ERS. Generally, 423 the ERS has a higher concentration of non-structural carbohydrates, also RMUL3 and 424 RWhL3 contain higher concentrations of CP (Table 2) and degradable fiber fractions (Table  425 3). The diversity in physiology might be the reason why the significant effect of the silage 426 molasses to silages are consistent with [26] reporting that the addition of molasses to the 435 silage increased digestibility due to increasing cell wall hydrolysis. The addition of whey or 436 MU to the silages increased the IVDCP compared with the control. They attributed this rise to 437 increasing the availability of soluble carbohydrates and nitrogen [5,28] in additives (MU,or 438 with whey) resulting in increased activity of proteolytic microorganisms in silage [4]. 439

Chemical composition, intake, and palatability 440
Changes in the chemical composition of the tested diets are due to nutrient contents of ERS and 441 consumption. Although silage produced in warm climates tends to present greater 442 concentrations of NDF and less starch in comparison with that produced in temperate areas, the 443 digestibility of silage is expected to be associated with feed intake [26]. Higher consumption 444 and raise protein in ERS led to an increase in the protein content in the experimental diets. The 445 finding obtained in the current study on chemical composition is supported by [25] who found 446 that protein content and EE, were increased with feeding silage, and attributed that to silage 447 consumption and raise protein and EE content in silage. In contact, [5] reported that increasing 448 protein content and EE in the experimental diets are due to total intake and the levels of both in 449 the silage. According to [26] the restricted factor to forage intake will be the ruminal fill 450 provided by the fiber. The volume of the plant cell has been linked to the filling effect [25,26]. 451

A c c e p t e d A r t i c l e
Hence, this effect attributed to the lower intake of treatments containing ERS (i.e. RWL3, 452 RMUL3, and RWhL3) compared with control one and may be ascribed to the interaction 453 among filling, the rumen-distension capacity, and the energy density because no differences 454 (P>0.05) were found between the treatments regarding the time used for feeding. Raising feed 455 consumption and palatability of ERS compared to the control diet, attributed to the higher 456 in-vitro degradability of OM, NDF, and CP, these results are typical of the findings of [25]. A 457 similar intake of ERS was observed to that reported by [4], and may partially reflect their 458 similar NDF concentrations. The NFC of the diets was higher for the treatments containing 459 ERS (Table 2). Thus, the availability of more fermentable nutrients might have influenced the 460 intake of ERS-based diets [1,3,25]. The finer chop length and the fiber content of the ERS may 461 also have contributed to its improved intake, which also can be explained that by the 462 compensating effect of the higher DM content [26]. The decrease eaten of UTRS is due to the 463 bitter taste caused by the treatment, which corresponds to the low palatability. The increased 464 chewing rate of UTRS compared to RMUL3 and RWhL3 is due to it being one of the means to 465 stimulate saliva production when feeding on urea-treated feeds. Saliva contributes to regulating 466 pH levels and is involved in the recycling of nitrogen to the rumen. Higher content of NDF as 467 shown in Table 4 corresponds to [24] who found that increasing proportion of NDF and length 468 of particle size of forage leading to the increased time spent chewing. 469    RW=ensiled rice straw by water; RMU=Ensiled rice straw by molasses & urea and RWh=Ensiled rice straw by whey; RWL1= ensiled rice straw with water and low level of LAB; RWL2= ensiled rice straw with water and a medium level of LAB; RWL3= ensiled rice straw with water and high level of LAB; RMUL1= ensiled rice straw with molasses and urea+ low level of LAB; RMUL2= ensiled rice straw with molasses and urea + the medium level of LAB; RMUL3= ensiled rice straw with molasses and urea + high level of LAB; RWhL1= ensiled rice straw with whey and low level of LAB; RWhL2= ensiled rice straw with whey and a medium level of LAB, and RWhL3= ensiled rice straw with whey and high level of LAB.
A c c e p t e d A r t i c l e 19 20 A c c e p t e d A r t i c l e