Application of Different Types of Lactic Acid Bacteria Inoculant on Ensiled Rice Straw; Effects on Silage Quality, Rumen Fermentation, Methane Production and Microbial Population

Bacterial inoculants are known to improve quality of silage. The objectives of the present study were to evaluate the effects of different types of lactic acid bacteria (LAB; L. plantarum, L. salivarius, L. reuteri, L. brevis and S. bovis) inoculation (106 g−1 DM) on rice straw silage quality and to examine these effects on ruminal fermentation characteristics, digestibility and microbial populations in an in vitro condition. Inoculated rice straw was ensiled for 15 and 30 days. For in vitro study, rumen liquor was obtained from two rumen fistulated mature cows fed on mixed forage and concentrate at 60:40 ratio twice daily. Inoculation of LAB improved (P˂0.05) the rice straw silage quality such as increased dry matter and crude protein contents, decreased pH and butyric acid, and increased propionic acid and LAB contents especially after 30 days of ensiling. Results from in vitro study revealed that addition of LAB to the rice straw silage improved fermentation characteristics such as increased total volatile fatty acids and dry matter digestibility (P˂0.05). LAB treatments also decreased methane production and methane/total gas ratio after 15 and 30 days of ensiling. From the rumen microbial population perspective, cellulolytic, and fungal zoospores were enhanced while protozoa and methanogens were decreased by the LAB treatments. Based on these results, it could be concluded that inoculating rice straw silage with LAB (especially for L. plantarum and S. bovis) improved silage quality, rumen fermentation parameters and microbial populations in vitro. However, in vivo studies need to confirm those effects.

* Correspondence should be addressed to Mahdi Ebrahimi: mehdiebrahimii@gmail.com Use of agricultural by-products is increasing because of limitations in food sources for livestock 61 which result in economic and environmental concerns. Rice straw, a major agricultural by-product, is 62 routinely utilized as a food source for ruminants in many regions of East and South-East Asia (Zhang 63 et al., 2017). In Malaysia, rice straw is one of the most abundant agricultural by-products (Ghazali et 64 al., 2013) . However, rice straw has very low nutritive values with low crude protein content and 65 metabolic energy for ruminants. Technologies to create high-quality animal feed from agricultural 66 residues need to be developed. Ensiling is a practical way to utilize water-soluble carbohydrates by  (Zhang et al., 2010;74 Cao et al., 2013;LIU et al., 2015;Oladosu et al., 2016). Besides, those studies mentioned that adding 75 LAB increased the lactic acid content of silage, increased dry matter digestibility, improved in vitro 76 ruminal fermentation parameters and decreased ruminal methane production. However, not all in 77 vitro studies have reported reductions in methane production (Contreras-Govea et al., 2011). 78 It has been hypothesized that LAB silage inoculants could reduce methane emissions from ruminants 79 by several modes of action; changes in the chemical composition of the silage, interaction of LAB 80 with rumen microbes and alteration of rumen fermentation (EllIS et al., 2016). Methane, as produced 81 from anaerobic fermentation in the rumen, accounts for 2-12% loss of dietary gross energy in 82 ruminants and is a potent greenhouse gas with a global warming potential 23 times higher than that 83 of carbon dioxide in trapping the heat (Jafari et al., 2018). Therefore, reducing ruminal methane production not only improves the efficiency of nutrient utilization in ruminants but also helps to 85 protect the environment from the negative consequences of global warming.  Therefore, the purpose of this experiment was to test the rumen microbial populations and 91 fermentation characteristics as well as testing methane mitigation potential of rice straw silage 92 inoculated with different types of LAB in an in vitro condition.

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The protocol for the experimental procedures were reviewed and approved by the Animal Care and 95 Use Committee of the University of Putra in Malaysia.

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Cecal contents from healthy adult, commercial broiler chickens and rumen samples from fistulated 98 male cattle (body weight: 209 kg) were used for the isolation of LAB. 1 gram of each samples were 99 dissolved in 9 ml of peptone water (0.01%) and shaken at 200 rpm for 10 min. Several dilution from 100 each sample (10 -3 to 10 -7 ) were prepared into dilution tube containing peptone water (0.01%). 100 µl 101 of each dilution were transferred into the plate containing MRS Rogosa agar (Oxoid CM 627,102 Hampshire, UK) as selective medium for LAB (Ebrahimi, 2012). Plates were anarobically incubated 103 at 37 o C for 48h. Several clones were selected from each plate and subcultured for three times. Total 104 of 80 isolates were selected and tested for Gram stain, hydrogen peroxidase and lactic acid 105 production. The LAB strains that actively produced lactic acid were chosen for the molecular 106 identification.

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Amplification was performed using a BIORAD MyCycler™ thermal cycler with the following 114 program: 1 cycle at 94°C for 4 min, 30 cycles of 94°C for 1 min, 55°C for 30s, 72°C for 2 min and a 115 final extension at 72°C for 5 min. The PCR products were mixed with loading dye and loaded on to 116 a 1.0% SeaKem® GTG® agarose (FMC BioProducts, Rockland ME, USA) containing ethidium 117 bromide, and electrophoresis was carried out at 90 V for 1 h. The PCR products were visualized 118 under UV illumination and excised from the gel and the PCR product was extracted using

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In vitro rumen fermentation and digestibility 160 Two rumen fistulated mature cows were fed (Table 1)  (e.g. pH, NH 3 -N and VFA) and the number of LAB were also determined as described earlier.

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The targeted microbes were cellulolytic bacteria such as Fibrobacter succinogenes, Ruminococcus 177 albus, Ruminococcus flavefaciens, general bacteria, general anaerobic fungi, total protozoa, total 178 methanogens and total archaea. DNA was extracted from 300 µl of fermented rumen content (fluid 179 and digesta from three syringes) by QIAGEN DNA Mini Stool Kit (QIAGEN, Valencia, CA) according to manufacturer's recommendations. Then the PCR product was purified using a QIA 181 quick PCR purification kit (QIAGEN, Inc., Valencia, CA) and cloned to the plasmid. The target 182 DNA was quantified by using serial 10-fold dilutions from 10 1 to 10 8 DNA copies of the previously 183 quantified DNA purified plasmid. Microorganisms and sequences of the primers used in this study 184 are shown in Table 2. completely randomized design and the means were compared with Duncan's Multiple Range test.

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Differences of P < 0.05 were considered to be significant.

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Chemical analyses and fermentation quality of rice straw silage 191 The contents of DM, CP, ether extract, NDF, ADF were affected (P˂0.05) by the treatments (Table   192 3). The DM contents were numerically decreased as the duration of ensiling increased. The control 193 group had higher DM content as compared with the LAB treatments at 15 and 30 d of ensilage. The

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CP content was greater in LAB treatments as compared with control (10.9-12.7 vs 9.5, respectively).

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The NDF and ADF of the LAB treatments were less than those of the control (Table 3)

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The analysis of the LAB content (log cfu/g) showed that the LAB treatments exhibited a significant 212 (P < 0.05) difference and increase as compared with control (Table 4).

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In vitro rumen fermentation characteristics, methane production and DM digestibility 214 According to the data of in vitro (Table 5) Table 3. Chen et al.

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(2019) mentioned that lower NDF content in silages could also be due to the loss of hemicellulose 247 occurred in the ensiling process. This loss could be due to a combination of enzymatic and acid 248 hydrolysis of the more digestible cell wall fractions during the fermentation. DM is the remaining 249 materials after the removal of water and contains the main nutrients for animal consumption.

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Ensilage of the forage will mostly result in the DM loss which occurs during the fermentation. In the current study, inoculations of different LAB decreased the DM loss which could be due to inhibiting 252 the clostridia and aerobic bacteria (Ni et al., 2015). The lack of DM loss in our study was also 253 consistent with the application of LAB isolated from forage paddy rice silage in China (Ni et al., .

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The previous studies showed that bacterial inoculation of silage could convert the composition of  The growing public concern over the widespread use of antibiotics in livestock production and the 312 emergence of antibiotic-resistant bacteria has stimulated interest in developing alternatives that 313 promote animal performance and health. One potential alternative is the use of direct-fed microbials In: The Association. 361 Baek Chen L, Yuan X, Li J, Dong Z, Wang S, Guo G and Shao T 2019. Effects of applying lactic acid bacteria and 370 propionic acid on fermentation quality, aerobic stability and in vitro gas production of forage-based 371 total mixed ration silage in Tibet. Animal Production Science. 59 (2)