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Interaction effect of silo density and additives on the fermentation quality, microbial counts, chemical composition and in vitro degradability of rice straw silage
Journal Pre-proofs Interaction effect of silo density and additives on the fermentation quality, microbial counts, chemical composition and in vitro degradability of rice straw silage Jipeng Tian, Nengxiang Xu, Beiyi Liu, Hailin Huan, Hongru Gu, Chenfei Dong, Chenglong Ding PII: DOI: Reference:
S0960-8524(19)31642-6 https://doi.org/10.1016/j.biortech.2019.122412 BITE 122412
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2019 6 November 2019 8 November 2019
Please cite this article as: Tian, J., Xu, N., Liu, B., Huan, H., Gu, H., Dong, C., Ding, C., Interaction effect of silo density and additives on the fermentation quality, microbial counts, chemical composition and in vitro degradability of rice straw silage, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122412
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Interaction effect of silo density and additives on the fermentation quality, microbial counts, chemical composition and in vitro degradability of rice straw silage Jipeng Tiana,b, Nengxiang Xua,b, Beiyi Liua,b, Hailin Huana,b, Hongru Gua,b, Chenfei Donga,b, Chenglong Dinga,b aInstitute
of Animal Science, Jiangsu Academy of Agricultural Science, Nangjing
210014, China bKey
Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture,
Jiangsu Academy of Agricultural Science, Nangjing 210014, China
Corresponding author at: Institute of Animal Science, Jiangsu Academy of Agricultural Science, No. 50, ZhongLingJie Road, Nangjing 210014, JiangSu, China. E-mail address: [email protected] (C. Ding).
Abstract: This research evaluated the effect of molasses (M), cellulosic enzymes (E) and lactic acid bacteria (LAB) alone or in combination (M+LAB and E+LAB) on the fermentation quality, microbial counts, chemical composition and in vitro digestibility of rice straw silages in different silo densities (200, 300, 400 and 500 kg/m3). The M or E groups alone increased the dry matter (DM) losses at low silo densities. Acetic acid produced by LAB-related groups significantly inhibited yeast and mould at the silo density of 300 kg/m3. Under high silo densities (> 400 kg/m3), LAB-related additives significantly improved the fermentation quality and reduced the DM losses. The use of E+LAB further improved the in vitro degradability of rice straw silages at high silo densities. In conclusion, higher silo density and appropriate complex additives were of great significance to improve the quality of rice straw silage. Keywords: silo density, lactic acid bacteria, cellulosic enzymes, rice straw silage, in vitro digestibility
1. Introduction Rice cultivation is the main form of agricultural production in the plain area of South China (Chen et al., 2011). Much of the rice straw is used for biofuel or roughage. In view of the humid and rainy climate in Southern China, ensiling is a feasible processing method for the storage of rice straw. The low water-soluble carbohydrate (WSC) content and lactic acid bacteria (LAB) counts in rice straw prevent the pH from decreasing rapidly, and the pH will not be low enough to inhibit harmful microorganisms (Oladosu et al., 2016). As a result, it is difficult to obtain high-quality silage from rice straw alone (Li et al., 2010). In a previous study, the use of exogenous LAB was indicated to have good effects on rice straw silage (Gao et al., 2008). At present, most LAB additives used have been mixtures of homofermentative LAB and heterofermentative LAB (Oliveira et al., 2017). Complex LAB additives used in this study were shown to be effective for gramineous crops or grass silages (Liu et al., 2019). There are two ways to improve the WSC content in rice straw. One method is to add cheap sources of exogenous WSC, such as molasses and distiller grains (Yuan et al., 2016). The other method is to degrade cellulose into monosaccharides or oligosaccharides, which can be used by LAB with the addition of cellulose degradation enzymes (Tian et al., 2014). Sufficient WSC not only provides sufficient substrates for lactic acid bacteria fermentation but also increases the nutritional quality of rice straw silage. The hollow stalk of rice straw is difficult to compact, resulting in a large amount of
oxygen remaining in silage during early ensiling which made Rice straw silages more susceptible to the negative effects of low silo densities in the actual production process than corn silages (Oladosu et al., 2016). Insufficient compacting density during silage production may lead to air permeability and aerobic deterioration of silage and then lead to the proliferation of yeasts, moulds, and other undesirable microorganisms, thereby increasing the loss of dry matter (DM) and nutrition (Anesio et al., 2017; Sucu et al., 2016). Yeast, mould and mycotoxins produced by moulds in rice also have potentially negative effects on animal and human health (Sun et al., 2017). The studies mentioned above about the effect of additives on rice straw silages were mostly conducted under the condition of high silo density, while few studies have been conducted on whether different additives have effects under the condition of low silo density. On the basis of high silo density, higher density may not have any significant effect on the silage (Yildiz, 2017). It is of great practical significance to select the right additive combination for rice straw silage under several different silo density conditions, and more research is needed on the interaction between silo density and different additives. In this study, the effects of single and combinations of LAB, molasses and enzymes on the fermentation quality, nutritional value, microbial counts and in vitro digestibility of rice straw silage with different silo densities were analysed. 2. Materials and methods 2.1 Preparation of rice straw silages
The rice (Nanjing 46, hybrid rice supplied by the Institute of Food Crops, Jiangsu Academy of Agricultural Science, Jiangsu, China) was harvested at the full ripening stage at the experimental station of the Jiangsu Academy of Agricultural Science in November. The rice straw was obtained after threshing and cut to 1 cm using a guillotine cutter. The additive was evenly sprayed onto the rice straw, and the DM content was adjusted to 33.75% fresh matter (FM) (original DM content of rice straw was 36.58% FM). The rice straw materials contained WSC of 6.86% DM, starch content of 17.71% DM, crude protein (CP) of 5.09% DM, neutral detergent fibre (NDF) of 57.46% DM, acid detergent fibre (ADF) of 32.68% DM, acid detergent lignin (ADL) of 3.58% DM, hemicellulose of 24.78% DM and cellulose of 28.90% DM. The counts of LAB, yeasts and moulds was 4.95, 4.78 and 6.39 log10 colony-forming units per gram (log10 CFU/g) in rice straw materials, respectively. The additives were as follows: (1) LAB inoculants of our own consisted of Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus buchneri, the details of which can be found in our previous study (Liu et al., 2019); (2) molasses (purchased in the market of Nanjing, China); and (3) complex enzyme preparations (Guangdong VTR Bio-Tech Co., Ltd., guangdong, China) containing cellulase, xylanase, glucanase, and pectinase (overall activity > 800,000 U with a proportion of 9:9:2.4:0.7 was used in this study). The five treatments were as follows: LAB group (added at 5 × 105 CFU/g of fresh material), M group (molasses, added at 1% of fresh material), E group (added at 21.1 U/g of fresh material with Tween 80 at 0.1% of fresh material), M+LAB group (mixture of
molasses and LAB), E+LAB group (mixture of complex enzyme and LAB) and control group (supplemented with the same volume of distilled water). The rice straw was ensiled in triplicate for each treatment in 1 L silos. All forages were ensiled for 90 days before opening. 2.2 Fermentation quality Twenty grams of the silage was homogenized at room temperature (25°C) with 180 mL of sterilized distilled water for 30 pulses of 2 s and then filtered through four layers of cheesecloth. The pH of the filtrate was measured using a glass electrode pH metre (Mettler Toledo, Zurich, Switzerland). The lactic acid (LA), acetic acid (AA), propionic acid (PA), iso-butyric acid (ISOBA) and butyric acid (BA) were determined by an Agilent 1260 HPLC system equipped with a UV detector (Agilent Technologies, California, America). The analytical conditions were as follows according to the our previous study Tian et al. (2014): column, Shodex RSpak KC-811S-DVB gel C (8.0 mm × 30 cm, Shimadzu, Tokyo, Japan); oven temperature, 50°C; mobile phase, 3 mM HClO4; flow rate, 1.0 mL/min; injection volume, 5 μL. 2.3 Microbial Counts Twenty grams of each sample was homogenized in 180 mL of sterilized normal saline for 30 min in an orbital shaker. The LAB, yeast and mould counts of samples were performed according to the study of Liu et al. (2019) while the media selected were respectively de Man, Rogosa, Sharpe (MRS) agar (Beijing Aoboxing Bio-tech Co., Ltd., Beijing, China)
for LAB count and Dichloran Rose Bengal Chloramphenicol Agar (DRBC, Beijing Aoboxing Bio-tech Co., Ltd., Beijing, China) for the counts of Yeast and Mould. The results were expressed as log10 CFU/g. 2.4 Chemical analysis Approximately 100 g of samples in the silo were dried at 65°C for approximately 48 h and weighed to determine the DM content. The weight and DM content before and after ensiling were weighted to calculate the rate of DM loss. The rice straw forages and silages were ground to pass a 1 mm screen with a Wiley mill for compositional analysis. Kjeldahl Nitrogen (i.e., total nitrogen) was analysed with method 954.01 of the Association of Official Analytical Chemists (AOAC, 1990). CP was calculated as Kjeldahl N × 6.25. The ammonia nitrogen (AN) content was determined according to the method of of Broderick and Kang (1980). The NDF (Van Soest et al., 1991), ADF and ADL (973.18 of AOAC, 1990) contents were determined by using a semi-automatic fibre analyser (Ankom 200i, Ankom Tech Co. USA). The hemicellulose and cellulose contents were calculated by the difference between NDF, ADF and ADL. The WSC content was estimated by the method described by Mcdonald and Henderson (1964). The starch content was determined using hydrolysis with 30% perchloric acid (Rose et al., 1991). 2.5 In vitro degradability The in vitro digestibility of DM (IVDMD) was estimated by the method of Goto and Minson (1977) by using the pepsin-cellulase assay. Ground and dried samples (1g, passing
a 1-mm-screen) of rice straw silages were incubated in pepsin–HCl solution for 16 h, followed by hydrolysis with a cellulase-acetate buffer (pH 4.6) solution for another 48 h. After 30 minutes of inactivation at 90℃, dried and weighed the residue. The residue of enzymatic hydrolysis was used to determine the residual NDF and residual ADF, and then the in vitro digestibility of NDF (IVNDFD) and in vitro digestibility of ADF (IVADFD) were calculated. 2.6 Statistical analysis Analysis of variance was used to test the statistical significance of the silo density effect (D), additive effect (A), and the silo density × additive effect interaction (D × A) using the general linear model of SPSS 20.0 for Windows. A simple effects test was conducted when the interaction was significant (P<0.05). Polynomial contrasts were used to test the effects of silo density on rice straw silages (P<0.05). Correlation analyses were used to test the correlations among the fermentation quality, nutritional value, microbial counts and in vitro digestibility of rice straw silage. 3. Results and discussion 3.1 Raw materials of rice straw The WSC (6.86% DM) content of rice straw materials in our study was suitable for silage fermentation (Woolford and Pahlow, 1998) and was much higher than that reported (1.59% DM) in the study of Li et al. (2017), but similar to that reported (6.38% DM) in the study of Zhao et al. (2019). Study of Dong et al. (2012) have shown that there were
significant differences of non-structural carbohydrates including soluble sugars and starches in rice straw among different varieties. The combination of WSC and starch content of rice straw used in this experiment exceeded 20%, which may be related to the variety character and the delay of harvest. The counts of LAB attached to the rice straw raw material were insufficient (4.95 log10 CFU/g). In contrast, fungi, especially moulds (6.39 log10 CFU/g), tend to consume more WSC in the aerobic state at the beginning of ensiling. 3.2 Fermentation quality The silo density, additives and their interactions significantly affected the pH value and organic acid contents of the rice straw silage (P<0.01, Table 1). With increasing silo density, the contents of LA, PA, ISOBA and BA showed a linear increasing trend (P<0.05), while the pH decreased linearly (P<0.05). The content of AA changed quadratically (P<0.05) with increasing silo density (P<0.05), and the highest AA reached in 300 kg/m3. A study of Sucu et al. (2016) showed a decrease in AA but no change in LA with an increase in the silo density of corn and sorghum silages. This may be because the heterofermentative LAB, such as L. buchneri, used in the compound LAB additives in this study were inhibited to some extent with increasing silo density. There was no significant difference between the M group and the control group in pH value, or LA and AA contents (P>0.05). The classic explanation is that molasses does not decrease the final pH value unless the WSC content is too low (Leibeinsperger and Pitt, 1988). Except for the silo density of 200 kg/m3 and a few exceptions (the difference between M+LAB and the control for AA content at 500 kg/m3 was not significant), the use
of LAB, M+LAB, E+LAB significantly increased the LA, AA and ISOBA contents and decreased the pH value compared to the control group at the same density (P<0.05), which is similar to the study of Fang et al. (2012). The final pH values of rice straw silages at high silo density (>400 kg/m3) and treated with LAB, M+LAB, E+LAB were lower than 4.2, which is required for high quality silages. L. plantarum and L. paracasei were the main strains of the LAB group used in our study. The study of Gao et al. (2008) showed that L. plantarum, L. paracasei and L. fermentum were the dominant species during the fermentation of rice straw silages and could tolerate the high acidity prevalent after 30 days of ensiling. At high silo densities (400 and 500 kg/m3), the PA and BA contents of the LAB-, M+LAB- and E+LAB-treated groups were lower (P<0.05) than the control group under the same density conditions. At a silo density of 500 kg/m3, the PA and BA contents of the M and E groups were significantly higher than those of the control group (P<0.05). BA can be produced by Clostridium butyrate (Szymanowska-Powalowska et al., 2014). Under anaerobic conditions, the inhibition of C. butyrate requires a rapid decrease in pH and a very low final pH value (Emerstorfer et al., 2011). In this experiment, groups M and E failed to reduce the pH value of rice straw silage to an appropriate level, and the content of BA increased. The pH value of the LAB-related groups (LAB, M+LAB, E+LAB) was low enough to inhibit C. butyrate in rice straw silage. 3.3 Microbial counts The silo density, additives and their interactions significantly affected the counts of LAB,
yeast and mould (P<0.01, shown in Table 2). The count of LAB changed quadratically (P<0.05) and was similar to the variation in AA. The use of additives significantly increased the LAB counts (P<0.05) compared with the control group at the same density, with a few exceptions in the M group at silo densities of 400 and 500 kg/m3. At higher densities, the rice straw itself retained sufficient soluble sugars for anaerobic fermentation, and it did not improve the LAB counts as well as the M did in soybean silage (Ni et al., 2017). Yeast and mould counts showed a linear decrease with increasing silo density (P<0.05). The use of the LAB (P<0.05, at a silo density of 400 kg/m3), M+LAB (P<0.05 for mould, at a silo density of 400 kg/m3) and E+LAB (P<0.05, at a silo density of 400 and 500 kg/m3) groups in high silo densities can be expected to inhibit the counts of yeast and mould to obtain the low final pH value (Alonso et al., 2013). At a silo density of 300 kg/m3, the use of three LAB-related additives can significantly reduce the counts of yeast and mould, which is attributed to the activity of L. buchneri contained in LAB, and the AA produced has a significant inhibitory effect on yeast and mould (Weinberg et al., 2011). The E group also inhibited the counts of yeast (at silo densities of 300 and 500 kg/m3) and moulds (P<0.05, at all silo densities). Studies have shown that the use of cellulose degradation enzymes can promote the increase in yeast counts (He et al., 2018). A possible explanation is that AA at low density (300 kg/m3) and BA at high density (500 kg/m3) have the ability to inhibit yeast counts. 3.4 DM content, DM losses, nitrogen and non-structural carbohydrate components of rice straw silages
Silo density and additives have significant effects on DM, DM losses, nitrogen and non-structural carbohydrate components of rice straw silages (P<0.01, shown in Table 3). As the silo density increased, the DM, AN and starch contents showed a linear increasing trend (P<0.05), while the DM losses, CP and WSC contents showed a linear decreasing trend (P<0.05). DM losses of more than 10% in the control groups predicted that aerobic bacteria and fungi consumed large amounts of WSC (6.86% to lower than 2%) and starch (17.7% to lower than 9%) in the early stages of silage. The increase in silo density led to a decrease in oxygen content in rice straw silage. In the control, M and E groups, there were not enough LAB to make the pH low enough to inhibit the Clostridium spp. which could increase the AN content (Mcdonald, 1981). The interactions between the silo density and additives had effects on the DM losses (P<0.01), CP (P<0.05), AN (P<0.01) and starch (P<0.05) contents. The DM losses and AN contents of the LAB and E+LAB treatment groups at silo densities of 400 and 500 kg/m3 were significantly lower (P<0.05) than those of the control group. At a silo density of 500 kg/m3, the use of M+LAB and E+LAB significantly (P<0.05) increased the starch content of rice straw silage. This result was similar to the study of Zhang et al. (2010), which showed that the LAB (containing L. buchneri and Pediococcus pentosaceus) additives could decrease the DM losses and AN contents. The M and E groups resulted in a significant increase in the DM losses (P<0.05, except at the silo density of 500 kg/m3) and AN contents (P<0.05, except for the E groups at a silo density of 200 kg/m3) but a significant decrease in the CP (P<0.05 at a silo density
of 300 kg/m3) contents compared to the control group. At low silo densities (200 and 300 kg/m3), DM losses in the M+LAB group were also significantly higher (P<0.05) than those in the control group. This is similar to the study of Lynch et al. (2014), which showed that the use of exogenous fibrolytic enzymes could increase the DM losses of silages, but contrary to the result of Chen et al. (2014) and Guney et al. (2018), which showed that the use of molasses reduced the DM losses of silage. In this experiment, large nutrient consumption by yeast and mould occurred in the M group at low silo densities. At high silo densities, groups M and E consumed more nutrients due to the higher final pH and the active C. butyrate, which could use cellulose and exogenous molasses for butyric fermentation (Sushkova et al., 2013). 3.5 Structural carbohydrate components of rice straw silages For NDF, ADF and hemicellulose contents, silo density (P<0.05, P<0.01 for NDF), additives (P<0.01) and their interactions (P<0.01, P<0.05 for hemicellulose) have significant effects (as shown in Table 4). Only the E+LAB group significantly decreased (P<0.01) and reached the lowest (P<0.05) cellulose content. With increasing silo density, the NDF, ADF and hemicellulose contents of rice straw silage showed a quadratic trend (P<0.05), and rice straw silage at a silo density of 300 kg/m3 reached the peak (P<0.05). At high silo densities (400 and 500 kg/m3), the E+LAB group significantly reduced the levels of NDF, ADF and hemicellulose compared to the control group. The NDF and ADF contents of the M and M+LAB treatment groups were also lower (P<0.05) than the control group at a silo density of 500 kg/m3. These results were similar to the study of
Chen et al. (2017). However, a previous study (Li et al., 2014) has also shown that the addition of cellulase can reduce the content of NDF and ADF in king grass silages. The positive effect of E+LAB on NDF and ADF was similar to that in L. chinensis silage (Tian et al., 2014; Zhang et al., 2016). Further analysis on the effect of composite additives of LAB and cellulolytic enzymes is necessary in the future. 3.6 In vitro digestibility of rice straw silages The silo density (P<0.01), additives (P<0.05, P<0.01 for IVDMD) and their interactions (P<0.01, P<0.05 for IVADFD) significantly affected the IVDMD, IVNDFD and IVADFD of rice straw silage (shown in Table 5). As the silo density increased, the IVDMD, IVNDFD and IVADFD showed a linear increase (P<0.05). At a silo density of 500 kg/m3, the M group showed a significant increase (p<0.05) in IVDMD compared with the control groups, but the LAB and E treatment groups had no significant effect on IVDMD. At a silo density of 500 kg/m3, the M+LAB and E+LAB groups showed significant increases (p<0.05) in IVDMD, IVNDFD and IVADFD, while E+LAB exhibited higher (P<0.05) IVDMD, IVNDFD and IVADFD than the M+LAB group at a silo density of 400 kg/m3. The effect of M was similar to the study of Chen et al. (2016). However, M was considered to have no effect on the IVDMD of rice straw silage in the study of Zhao et al. (2019), while the cellulase had a positive effect on the IVDMD, IVNDFD and IVADFD of silages (Li et al., 2018; Nawaz et al., 2016). LAB can also be considered to have a positive effect on the IVDMD of fresh rice straw silages in the study of Kim et al. (2017). The use of a single type of additive is highly uncertain and needs to
be carefully selected according to the characteristics of the raw material. The use of complex additives with different types would be better. 3.7 Correlations among the fermentation quality, DM losses, chemical composition, microbial counts and in vitro digestibility of rice straw silages The correlations among fermentation quality, DM losses, chemical composition, microbial counts and in vitro digestibility of rice straw silages are shown in Figure 1. In the present study, the increase in DM losses of rice straw silages was mainly due to the increase in yeast (r=0.569, P<0.01) and mould (r=0.573, P<0.01) counts, large consumption of starch (r=-0.603, P<0.01) and the deficiency of LA (r=-0.697, P<0.01), AA (r=-0.294, P<0.05) and BA (r=-0.294, P<0.05). In this process, starch was consumed by yeast (r=0.543, P<0.01) and mould (r=-0.632, P<0.01). As a result, the in vitro digestibility of DM (r=-0.496, P<0.01), NDF (r=-0.377, P<0.01) and ADF (r=-0.544, P<0.01) in the rice straw silage decreased with the change of DM losses. Although starch is beneficial to improving the nutritional quality of rice straw silage, given that the current collection and processing level of rice straw is more difficult than that of corn silage, the retention of more starch in rice straw without targeted measures may be more conducive to aerobic deterioration caused by yeast and mould in the silage (Wilkinson and Davies, 2013). AA was mainly produced by LAB (r=0.739, P<0.01) that consume WSCs (r=-0.482, P<0.01) and have a significant inhibitory effect on yeast (r=-0.622, P<0.01) and mould (r=-0.620, P<0.01). LA is also produced by LAB (r=0.449, P<0.01), but in addition to
WSC (r=-0.469, P<0.01), the degradation of hemicellulose (r=-0.260, P<0.05) and cellulose (r=-0.327, P<0.01) could also be related to the production of LA. Corresponding, IVDMD (r=0.563, P<0.01), IVNDFD (r=0.500, P<0.01) and IVADFD (r=0.540, P<0.01) could also be enhanced by LA. The count of LAB itself is only significantly negatively correlated with WSC (r=-0.455, P<0.01). This suggests that the differences in LAB counts after ensiling may be mainly due to the differences in L. buchneri (Weinberg et al., 1999). On the other hand, the degradation of hemicellulose and cellulose is beneficial to improve fermentation by L. plantarum in rice straw silage (Zhao et al., 2018). The emergence of BA can significantly inhibit the counts of LAB (r=-0.391, P<0.01), yeast (r=-0.287, P<0.05) and mould (r=-0.354, P<0.01), while the increase in AN had a significant inhibitory effect on LAB (r=-0.309, P<0.01) and mould (r=-0.260, P<0.05). The increases in BA (r=-0.396, P<0.01) and AN (r=-0.573, P<0.01) are mainly due to the consumption of CP in the rice straw silage. More attention should be paid to the BA content in legume forages (Zhang et al., 2018) and gramineous forages with an earlier growth period (Zhang et al., 2016). 4. Conclusions The quality of rice straw silage was determined by the characteristics of raw materials, silo density and additives. The use of the M or E group alone is risky for rice straw silages. L. buchneri is active at 300 kg/m3, while L. plantarum and L. paracasei gradually take the lead with increasing silo density. The use of complex additives, E+LAB, further improved the IVDMD, IVNDFD and IVADFD of rice straw silages. It is important to select
compound additives according to the raw material and silo density to improve the quality of rice straw silage. Acknowledgments This work was financially supported by the Agricultural Innovation Fund of Jiangsu Province (grant no. CX[17]3037). Conflict of interest Authors declare that they have no competing interests. Ethical approval This article does not contain any study with human participants or animals reported by other authors. References 1.
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Bioresour. Technol. 266, 158–165. Figure Captions Figure 1 Correlations among the fermentation quality, DM losses, chemical composition, microbial counts and in vitro digestibility of rice straw silages. DM, dry matter; CP, crude protein; AN, ammoniacal nitrogen; WSC, water-soluble carbohydrates; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; IVDMD, in vitro dry matter digestibility; IVNDFD, in vitro neutral detergent fibre digestibility; IVADFD, in vitro acid detergent fibre digestibility; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; ISO-BA, isobutyric acid; LAB, lactic acid bacteria; *, Significant at P<0.05; **, Significant at P<0.01.
Table 1 The pH values and contents of organic acids in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
7.47abA
6.86bA
7.01abA
6.91bA
7.86aA
6.71bA
0.17
**,L
**
**
300
7.86aA
4.94bcB
7.41aA
5.47bB
4.83bcB
4.51cB
400
5.54aB
4.14bC
5.34aB
5.17aB
4.09bC
4.07bB
500
5.27aB
4.06cC
5.08aB
4.77bC
3.87cC
4.03cB
200
0.09
0.48C
0.17B
0.18C
0.12D
0.66C
0.20
**,L
**
**
300
0.10d
1.15bC
0.18cdB
0.7bcdBC
0.86bcC
1.90aB
400
0.56b
3.58aB
0.76bAB
0.99bB
3.62aB
4.12aA
500
0.63d
4.48bA
1.21dA
2.10cA
5.79aA
4.40bA
200
0.23B
0.40B
0.59
0.47B
0.32C
0.65B
0.06
**, Q
**
**
Items
Density
treatment
kg/m3
Control
pH value
200
LA,%DM
AA,%DM
PA,%DM
ISO-BA,%DM
BA,%DM
300
0.22dB
1.35bcA
0.56d
1.01cA
2.15aA
1.68bA
400
0.73bA
1.28aA
1.00ab
1.16abA
1.32aB
1.32aA
500
0.62cAB
1.35aA
0.70c
0.96abcA
0.89bcB
1.26abA
200
NDB
ND
0.04C
NDB
0.01
ND
300
NDbB
NDb
0.14aAB
0.03bB
NDb
NDb
400
0.27aA
0.04bc
0.12bBC
0.10bcB
NDc
NDc
500
0.09bB
NDb
0.24aA
0.24aA
NDb
NDb
200
1.35aA
0.69bB
0.80bB
0.49bc
0.27cC
0.67bB
300
0.61bB
1.31aA
0.74bB
0.74b
1.33aB
1.41aA
400
0.74bcB
1.42aA
0.98bAB
0.51c
1.52aAB
1.63aA
500
0.78cB
1.54abA
1.20bA
0.70c
1.72aA
1.64aA
200
NDC
NDB
0.03D
NDC
0.01
ND
300
NDdC
0.07bcA
0.11bC
0.17aB
NDd
0.03cd
0.01
**,L
**
**
0.06
**,L
**
**
0.02
**,L
**
**
400
0.29aA
0.05cAB
0.34aB
0.21bB
0.05c
NDc
500
0.18cB
0.08dA
0.65aA
0.32bA
0.02de
0.02e
LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; ISO-BA, isobutyric acid; LAB, lactic acid bacteria additive; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-CValues
show significant (P<0.05) differences in the same column of each item; ND, not detected; *, Significant at P<0.05;**, Significant at
P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 2 Microbial counts in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
7.61c
8.16abB
8.07abA
7.87bcB
7.68cC
8.37aB
0.05
**,Q
**
**
300
7.43c
8.78aA
7.94bA
7.99bAB
8.74aA
8.74aA
400
7.55b
8.43aB
7.45bB
8.26aA
8.41aB
8.46aAB
500
7.44b
8.40aB
7.54bB
8.25aA
8.20aB
8.37aB
200
6.87AB
6.50A
7.38A
6.98A
7.23A
6.56A
0.14
**,L
**
**
300
7.19aA
5.96bAB
7.42aA
4.41dC
5.32bcC
4.81cdB
400
6.17aBC
4.14cC
5.34abB
5.43aB
5.85aBC
4.50bcB
500
5.42bC
5.48bB
4.96bcB
4.43cC
6.39aAB
4.39cB
200
6.21aA
5.57abA
5.69abA
5.04bcA
6.28aA
4.19cA
0.17
**,L
**
**
300
6.25aA
2.74cB
6.08aA
2.48cB
3.88bB
3.13bcB
Items
Density
Treatments
kg/m3
Control
LAB, log10 CFU/g
200
Yeasts, log10 CFU/g
Moulds, log10 CFU/g
400
4.95aB
2.98bB
3.65bB
2.60bB
3.44bB
3.25bAB
500
3.46aC
3.36abB
2.59abC
2.38bB
3.46aB
2.38bB
LAB,lactic acid bacteria; CFU, Colony Forming Units; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-CValues show significant (P<0.05) differences in the same column of each item; *, Significant at P<0.05;**, Significant at P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 3 Contents of dry matter (DM), dry matter losses (DM losses) and components of nitrogen and sugar in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
28.06
28.25
26.42
25.16
25.26
27.26
0.23
**, L
**
NS
300
29.07
28.55
28.28
27.13
25.25
28.82
400
30.38
30.59
28.43
28.10
29.30
30.37
500
31.02
30.67
30.58
29.73
29.29
31.37
200
21.85bA
19.68bA
26.32aA
27.24aA
27.38aA
21.14bA
0.69
**, L
**
**
300
15.91cB
15.02cB
20.00bB
21.37bB
24.35aB
16.10cB
400
15.09bB
10.71cC
17.47aC
18.57aC
12.74bcC
11.88cC
500
11.04aC
8.10bD
10.45abD
12.11aD
11.01aC
8.20bD
200
6.02bcA
5.85bcdA
6.05bA
5.74cdA
6.40aA
5.71d
0.04
**, L
**
*
Items
Density
Treatments
kg/m3
Control
DM, %
200
DM loss, %DM
CP,%DM
AN, %TN
WSC,%DM
Starch,%DM
300
5.64aB
5.34bcB
5.17cC
5.08cB
5.64aB
5.47ab
400
5.32bcC
5.46bB
5.56abB
5.05cB
5.78aB
5.60ab
500
5.26bC
5.46bB
5.47bB
5.26bB
5.79aB
5.51ab
200
5.02bcB
4.08cB
6.38aB
5.80abB
5.76abAB
4.82bc
300
4.76cB
5.77bcA
11.16aA
10.74aA
6.98bA
5.50c
400
8.95bA
5.06cAB
10.98aA
10.83aA
6.11cAB
5.04c
500
8.51bA
5.46cA
10.02aA
10.06aA
5.42cB
5.05c
200
1.86
1.31
1.65
1.51
1.16
1.58
300
1.69
1.13
1.46
1.12
1.13
1.27
400
1.61
1.26
1.40
1.29
1.10
1.23
500
1.43
1.07
1.45
1.09
1.19
1.19
200
5.43dB
8.03ab
6.08cdB
7.64abcC
6.53bcdC
8.73aB
300
7.49bcA
9.23ab
7.37cAB
9.71aB
6.98cBC
8.59abcB
0.29
**, L
**
**
0.03
**, L
**
NS
0.25
**, L
**
*
400
8.95A
9.60
8.27A
9.82B
8.39B
9.69B
500
8.58bA
9.14b
8.23bA
12.21aA
11.77aA
12.07aA
DM, dry matter; CP, crude protein; AN, ammoniacal nitrogen; WSC, water-soluble carbohydrates; LAB, lactic acid bacteria additive; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-DValues show significant (P<0.05) differences in the same column of each item; *, Significant at P<0.05;**, Significant at P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 4 Contents of structural carbohydrates in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM significance
LAB
M
E
M+LAB
E+LAB
D
D*A
56.46abc
55.55bcB
60.15aA
57.59abcC
59.35abAB
54.22cAB
0.48
**, Q **
**
300
59.40bc
58.24cAB
57.5cAB
65.09aA
63.26aA
55.62cA
400
59.04ab
62.39aA
57.21bAB
63.04aAB
55.65bBC
51.35cB
500
59.26a
59.92aA
54.07bB
59.52aBC
54.77bC
51.89bAB
200
32.93ab
33.23abB
35.37aA
33.46abB
35.72aA
31.13b
0.36
*, Q
**
**
300
34.00b
34.02bAB
33.61bAB
37.36aA
37.61aA
32.91b
400
34.22abc
36.37abA
33.97bcAB
37.02aA
31.61cdB
29.92d
500
34.55abc
34.84abAB
31.63cdB
35.40aAB
31.92bcdB
30.36d
200
4.10
4.72
4.17
4.21
5.25
3.97
0.14
NS
NS
NS
Items
Density
Treatment
kg/m3
Control
NDF ,%DM
200
ADF ,%DM
ADL ,%DM
A
Hemicellulose ,%DM
Cellulose ,%DM
300
3.72
4.05
3.38
5.38
5.93
4.34
400
3.37
4.29
4.02
5.65
4.78
3.80
500
4.29
3.98
3.08
4.49
3.45
5.06
200
23.53ab
22.31bB
24.79a
24.13abB
24.95aAB
23.09ab
300
25.40ab
24.22bcAB
23.90bc
27.73aA
25.64abA
22.72c
400
24.82ab
26.11aA
23.24bc
26.02aAB
24.03abAB
21.43c
500
24.70ab
25.08aA
22.44bc
24.12abB
22.86abcB
21.54c
200
28.83
28.52
31.19
29.25
30.47
27.34
300
30.29
29.97
30.23
31.97
31.69
28.57
400
30.85
32.02
29.95
31.37
26.83
26.12
500
30.26
30.85
28.55
30.91
28.47
25.30
0.26
*, Q
**
*
0.33
NS
**
NS
DM, dry matter; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; LAB, lactic acid bacteria additive; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex
additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-CValues show significant (P<0.05) differences in the same column of each item; *, Significant at P<0.05;**, Significant at P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 5 In vitro digestibility of rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
44.41ab
45.74abB
41.80bB
46.08aA
43.40abC
45.15abB
0.52
**, L
**
**
300
44.66bc
49.81aA
46.62bA
40.63cB
41.00cC
48.01abB
400
44.58cd
43.89dB
48.64bcA
42.79dAB
49.49bB
55.10aA
500
44.20d
45.61cdB
49.75bcA
45.61cdA
54.04aA
52.51abA
200
16.87b
15.69bC
15.91bB
24.70a
16.37bC
17.84bB
0.53
**,L
*
**
300
18.58b
28.55aA
18.73bAB
20.37b
19.23bBC
22.44bAB
400
19.45b
20.92abBC
23.33abA
21.00ab
23.15abB
26.03aA
500
17.87c
22.03bcB
22.11bcA
21.55bc
28.83aA
23.50bA
200
11.29B
13.98B
12.38B
14.03B
13.9B
13.48B
0.69
**,L
*
*
300
13.07bAB
24.04aA
15.48bAB
16.24bAB
16.33bB
15.42bB
Items
Density
treatment
kg/m3
Control
IVDMD ,%
200
IVNDFD ,%
IVADFD, %
400
15.43cAB
15.61bcB
20.89abA
18.85abcAB 16.56bcB
23.12aA
500
17.71bA
18.82bAB
19.07bA
21.16abA
24.55aA
24.87aA
IVDMD, in vitro dry matter digestibility; IVNDFD, in vitro neutral detergent fibre digestibility; IVADFD, in vitro acid detergent fibre digestibility; LAB, lactic acid bacteria additives; M, molosses additives; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB. a-d Values
show significant (P<0.05) differences on the same line; A-CValues show significant (P<0.05) differences in the same column of each
item; *, Significant at P<0.05; **, Significant at P<0.01; L, linear effect (P<0.05); Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Jipeng Tian: Methodology, Investigation, Formal analysis, Writing - Original Draft Nengxiang Xu: Methodology, Data Curation, Resources, Investigation Beiyi Liu: Resources, Investigation Hailin Huan: Resources, Investigation, Validation Hongru Gu: Methodology, Writing - Review & Editing Chenfei Dong: Methodology, Writing - Review & Editing Chenglong Ding: Conceptualization, Methodology, Project administration, Funding acquisition
As the silo density increased, the quality of rice straw silages improved. Lactic acid bacteria (LAB) improved fermentation quality of rice straw silages. Combination of enzymes and LAB and high silo density (>400kg/m3) reached the best.
S0960-8524(19)31642-6 https://doi.org/10.1016/j.biortech.2019.122412 BITE 122412
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2019 6 November 2019 8 November 2019
Please cite this article as: Tian, J., Xu, N., Liu, B., Huan, H., Gu, H., Dong, C., Ding, C., Interaction effect of silo density and additives on the fermentation quality, microbial counts, chemical composition and in vitro degradability of rice straw silage, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122412
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Interaction effect of silo density and additives on the fermentation quality, microbial counts, chemical composition and in vitro degradability of rice straw silage Jipeng Tiana,b, Nengxiang Xua,b, Beiyi Liua,b, Hailin Huana,b, Hongru Gua,b, Chenfei Donga,b, Chenglong Dinga,b aInstitute
of Animal Science, Jiangsu Academy of Agricultural Science, Nangjing
210014, China bKey
Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture,
Jiangsu Academy of Agricultural Science, Nangjing 210014, China
Corresponding author at: Institute of Animal Science, Jiangsu Academy of Agricultural Science, No. 50, ZhongLingJie Road, Nangjing 210014, JiangSu, China. E-mail address: [email protected] (C. Ding).
Abstract: This research evaluated the effect of molasses (M), cellulosic enzymes (E) and lactic acid bacteria (LAB) alone or in combination (M+LAB and E+LAB) on the fermentation quality, microbial counts, chemical composition and in vitro digestibility of rice straw silages in different silo densities (200, 300, 400 and 500 kg/m3). The M or E groups alone increased the dry matter (DM) losses at low silo densities. Acetic acid produced by LAB-related groups significantly inhibited yeast and mould at the silo density of 300 kg/m3. Under high silo densities (> 400 kg/m3), LAB-related additives significantly improved the fermentation quality and reduced the DM losses. The use of E+LAB further improved the in vitro degradability of rice straw silages at high silo densities. In conclusion, higher silo density and appropriate complex additives were of great significance to improve the quality of rice straw silage. Keywords: silo density, lactic acid bacteria, cellulosic enzymes, rice straw silage, in vitro digestibility
1. Introduction Rice cultivation is the main form of agricultural production in the plain area of South China (Chen et al., 2011). Much of the rice straw is used for biofuel or roughage. In view of the humid and rainy climate in Southern China, ensiling is a feasible processing method for the storage of rice straw. The low water-soluble carbohydrate (WSC) content and lactic acid bacteria (LAB) counts in rice straw prevent the pH from decreasing rapidly, and the pH will not be low enough to inhibit harmful microorganisms (Oladosu et al., 2016). As a result, it is difficult to obtain high-quality silage from rice straw alone (Li et al., 2010). In a previous study, the use of exogenous LAB was indicated to have good effects on rice straw silage (Gao et al., 2008). At present, most LAB additives used have been mixtures of homofermentative LAB and heterofermentative LAB (Oliveira et al., 2017). Complex LAB additives used in this study were shown to be effective for gramineous crops or grass silages (Liu et al., 2019). There are two ways to improve the WSC content in rice straw. One method is to add cheap sources of exogenous WSC, such as molasses and distiller grains (Yuan et al., 2016). The other method is to degrade cellulose into monosaccharides or oligosaccharides, which can be used by LAB with the addition of cellulose degradation enzymes (Tian et al., 2014). Sufficient WSC not only provides sufficient substrates for lactic acid bacteria fermentation but also increases the nutritional quality of rice straw silage. The hollow stalk of rice straw is difficult to compact, resulting in a large amount of
oxygen remaining in silage during early ensiling which made Rice straw silages more susceptible to the negative effects of low silo densities in the actual production process than corn silages (Oladosu et al., 2016). Insufficient compacting density during silage production may lead to air permeability and aerobic deterioration of silage and then lead to the proliferation of yeasts, moulds, and other undesirable microorganisms, thereby increasing the loss of dry matter (DM) and nutrition (Anesio et al., 2017; Sucu et al., 2016). Yeast, mould and mycotoxins produced by moulds in rice also have potentially negative effects on animal and human health (Sun et al., 2017). The studies mentioned above about the effect of additives on rice straw silages were mostly conducted under the condition of high silo density, while few studies have been conducted on whether different additives have effects under the condition of low silo density. On the basis of high silo density, higher density may not have any significant effect on the silage (Yildiz, 2017). It is of great practical significance to select the right additive combination for rice straw silage under several different silo density conditions, and more research is needed on the interaction between silo density and different additives. In this study, the effects of single and combinations of LAB, molasses and enzymes on the fermentation quality, nutritional value, microbial counts and in vitro digestibility of rice straw silage with different silo densities were analysed. 2. Materials and methods 2.1 Preparation of rice straw silages
The rice (Nanjing 46, hybrid rice supplied by the Institute of Food Crops, Jiangsu Academy of Agricultural Science, Jiangsu, China) was harvested at the full ripening stage at the experimental station of the Jiangsu Academy of Agricultural Science in November. The rice straw was obtained after threshing and cut to 1 cm using a guillotine cutter. The additive was evenly sprayed onto the rice straw, and the DM content was adjusted to 33.75% fresh matter (FM) (original DM content of rice straw was 36.58% FM). The rice straw materials contained WSC of 6.86% DM, starch content of 17.71% DM, crude protein (CP) of 5.09% DM, neutral detergent fibre (NDF) of 57.46% DM, acid detergent fibre (ADF) of 32.68% DM, acid detergent lignin (ADL) of 3.58% DM, hemicellulose of 24.78% DM and cellulose of 28.90% DM. The counts of LAB, yeasts and moulds was 4.95, 4.78 and 6.39 log10 colony-forming units per gram (log10 CFU/g) in rice straw materials, respectively. The additives were as follows: (1) LAB inoculants of our own consisted of Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus buchneri, the details of which can be found in our previous study (Liu et al., 2019); (2) molasses (purchased in the market of Nanjing, China); and (3) complex enzyme preparations (Guangdong VTR Bio-Tech Co., Ltd., guangdong, China) containing cellulase, xylanase, glucanase, and pectinase (overall activity > 800,000 U with a proportion of 9:9:2.4:0.7 was used in this study). The five treatments were as follows: LAB group (added at 5 × 105 CFU/g of fresh material), M group (molasses, added at 1% of fresh material), E group (added at 21.1 U/g of fresh material with Tween 80 at 0.1% of fresh material), M+LAB group (mixture of
molasses and LAB), E+LAB group (mixture of complex enzyme and LAB) and control group (supplemented with the same volume of distilled water). The rice straw was ensiled in triplicate for each treatment in 1 L silos. All forages were ensiled for 90 days before opening. 2.2 Fermentation quality Twenty grams of the silage was homogenized at room temperature (25°C) with 180 mL of sterilized distilled water for 30 pulses of 2 s and then filtered through four layers of cheesecloth. The pH of the filtrate was measured using a glass electrode pH metre (Mettler Toledo, Zurich, Switzerland). The lactic acid (LA), acetic acid (AA), propionic acid (PA), iso-butyric acid (ISOBA) and butyric acid (BA) were determined by an Agilent 1260 HPLC system equipped with a UV detector (Agilent Technologies, California, America). The analytical conditions were as follows according to the our previous study Tian et al. (2014): column, Shodex RSpak KC-811S-DVB gel C (8.0 mm × 30 cm, Shimadzu, Tokyo, Japan); oven temperature, 50°C; mobile phase, 3 mM HClO4; flow rate, 1.0 mL/min; injection volume, 5 μL. 2.3 Microbial Counts Twenty grams of each sample was homogenized in 180 mL of sterilized normal saline for 30 min in an orbital shaker. The LAB, yeast and mould counts of samples were performed according to the study of Liu et al. (2019) while the media selected were respectively de Man, Rogosa, Sharpe (MRS) agar (Beijing Aoboxing Bio-tech Co., Ltd., Beijing, China)
for LAB count and Dichloran Rose Bengal Chloramphenicol Agar (DRBC, Beijing Aoboxing Bio-tech Co., Ltd., Beijing, China) for the counts of Yeast and Mould. The results were expressed as log10 CFU/g. 2.4 Chemical analysis Approximately 100 g of samples in the silo were dried at 65°C for approximately 48 h and weighed to determine the DM content. The weight and DM content before and after ensiling were weighted to calculate the rate of DM loss. The rice straw forages and silages were ground to pass a 1 mm screen with a Wiley mill for compositional analysis. Kjeldahl Nitrogen (i.e., total nitrogen) was analysed with method 954.01 of the Association of Official Analytical Chemists (AOAC, 1990). CP was calculated as Kjeldahl N × 6.25. The ammonia nitrogen (AN) content was determined according to the method of of Broderick and Kang (1980). The NDF (Van Soest et al., 1991), ADF and ADL (973.18 of AOAC, 1990) contents were determined by using a semi-automatic fibre analyser (Ankom 200i, Ankom Tech Co. USA). The hemicellulose and cellulose contents were calculated by the difference between NDF, ADF and ADL. The WSC content was estimated by the method described by Mcdonald and Henderson (1964). The starch content was determined using hydrolysis with 30% perchloric acid (Rose et al., 1991). 2.5 In vitro degradability The in vitro digestibility of DM (IVDMD) was estimated by the method of Goto and Minson (1977) by using the pepsin-cellulase assay. Ground and dried samples (1g, passing
a 1-mm-screen) of rice straw silages were incubated in pepsin–HCl solution for 16 h, followed by hydrolysis with a cellulase-acetate buffer (pH 4.6) solution for another 48 h. After 30 minutes of inactivation at 90℃, dried and weighed the residue. The residue of enzymatic hydrolysis was used to determine the residual NDF and residual ADF, and then the in vitro digestibility of NDF (IVNDFD) and in vitro digestibility of ADF (IVADFD) were calculated. 2.6 Statistical analysis Analysis of variance was used to test the statistical significance of the silo density effect (D), additive effect (A), and the silo density × additive effect interaction (D × A) using the general linear model of SPSS 20.0 for Windows. A simple effects test was conducted when the interaction was significant (P<0.05). Polynomial contrasts were used to test the effects of silo density on rice straw silages (P<0.05). Correlation analyses were used to test the correlations among the fermentation quality, nutritional value, microbial counts and in vitro digestibility of rice straw silage. 3. Results and discussion 3.1 Raw materials of rice straw The WSC (6.86% DM) content of rice straw materials in our study was suitable for silage fermentation (Woolford and Pahlow, 1998) and was much higher than that reported (1.59% DM) in the study of Li et al. (2017), but similar to that reported (6.38% DM) in the study of Zhao et al. (2019). Study of Dong et al. (2012) have shown that there were
significant differences of non-structural carbohydrates including soluble sugars and starches in rice straw among different varieties. The combination of WSC and starch content of rice straw used in this experiment exceeded 20%, which may be related to the variety character and the delay of harvest. The counts of LAB attached to the rice straw raw material were insufficient (4.95 log10 CFU/g). In contrast, fungi, especially moulds (6.39 log10 CFU/g), tend to consume more WSC in the aerobic state at the beginning of ensiling. 3.2 Fermentation quality The silo density, additives and their interactions significantly affected the pH value and organic acid contents of the rice straw silage (P<0.01, Table 1). With increasing silo density, the contents of LA, PA, ISOBA and BA showed a linear increasing trend (P<0.05), while the pH decreased linearly (P<0.05). The content of AA changed quadratically (P<0.05) with increasing silo density (P<0.05), and the highest AA reached in 300 kg/m3. A study of Sucu et al. (2016) showed a decrease in AA but no change in LA with an increase in the silo density of corn and sorghum silages. This may be because the heterofermentative LAB, such as L. buchneri, used in the compound LAB additives in this study were inhibited to some extent with increasing silo density. There was no significant difference between the M group and the control group in pH value, or LA and AA contents (P>0.05). The classic explanation is that molasses does not decrease the final pH value unless the WSC content is too low (Leibeinsperger and Pitt, 1988). Except for the silo density of 200 kg/m3 and a few exceptions (the difference between M+LAB and the control for AA content at 500 kg/m3 was not significant), the use
of LAB, M+LAB, E+LAB significantly increased the LA, AA and ISOBA contents and decreased the pH value compared to the control group at the same density (P<0.05), which is similar to the study of Fang et al. (2012). The final pH values of rice straw silages at high silo density (>400 kg/m3) and treated with LAB, M+LAB, E+LAB were lower than 4.2, which is required for high quality silages. L. plantarum and L. paracasei were the main strains of the LAB group used in our study. The study of Gao et al. (2008) showed that L. plantarum, L. paracasei and L. fermentum were the dominant species during the fermentation of rice straw silages and could tolerate the high acidity prevalent after 30 days of ensiling. At high silo densities (400 and 500 kg/m3), the PA and BA contents of the LAB-, M+LAB- and E+LAB-treated groups were lower (P<0.05) than the control group under the same density conditions. At a silo density of 500 kg/m3, the PA and BA contents of the M and E groups were significantly higher than those of the control group (P<0.05). BA can be produced by Clostridium butyrate (Szymanowska-Powalowska et al., 2014). Under anaerobic conditions, the inhibition of C. butyrate requires a rapid decrease in pH and a very low final pH value (Emerstorfer et al., 2011). In this experiment, groups M and E failed to reduce the pH value of rice straw silage to an appropriate level, and the content of BA increased. The pH value of the LAB-related groups (LAB, M+LAB, E+LAB) was low enough to inhibit C. butyrate in rice straw silage. 3.3 Microbial counts The silo density, additives and their interactions significantly affected the counts of LAB,
yeast and mould (P<0.01, shown in Table 2). The count of LAB changed quadratically (P<0.05) and was similar to the variation in AA. The use of additives significantly increased the LAB counts (P<0.05) compared with the control group at the same density, with a few exceptions in the M group at silo densities of 400 and 500 kg/m3. At higher densities, the rice straw itself retained sufficient soluble sugars for anaerobic fermentation, and it did not improve the LAB counts as well as the M did in soybean silage (Ni et al., 2017). Yeast and mould counts showed a linear decrease with increasing silo density (P<0.05). The use of the LAB (P<0.05, at a silo density of 400 kg/m3), M+LAB (P<0.05 for mould, at a silo density of 400 kg/m3) and E+LAB (P<0.05, at a silo density of 400 and 500 kg/m3) groups in high silo densities can be expected to inhibit the counts of yeast and mould to obtain the low final pH value (Alonso et al., 2013). At a silo density of 300 kg/m3, the use of three LAB-related additives can significantly reduce the counts of yeast and mould, which is attributed to the activity of L. buchneri contained in LAB, and the AA produced has a significant inhibitory effect on yeast and mould (Weinberg et al., 2011). The E group also inhibited the counts of yeast (at silo densities of 300 and 500 kg/m3) and moulds (P<0.05, at all silo densities). Studies have shown that the use of cellulose degradation enzymes can promote the increase in yeast counts (He et al., 2018). A possible explanation is that AA at low density (300 kg/m3) and BA at high density (500 kg/m3) have the ability to inhibit yeast counts. 3.4 DM content, DM losses, nitrogen and non-structural carbohydrate components of rice straw silages
Silo density and additives have significant effects on DM, DM losses, nitrogen and non-structural carbohydrate components of rice straw silages (P<0.01, shown in Table 3). As the silo density increased, the DM, AN and starch contents showed a linear increasing trend (P<0.05), while the DM losses, CP and WSC contents showed a linear decreasing trend (P<0.05). DM losses of more than 10% in the control groups predicted that aerobic bacteria and fungi consumed large amounts of WSC (6.86% to lower than 2%) and starch (17.7% to lower than 9%) in the early stages of silage. The increase in silo density led to a decrease in oxygen content in rice straw silage. In the control, M and E groups, there were not enough LAB to make the pH low enough to inhibit the Clostridium spp. which could increase the AN content (Mcdonald, 1981). The interactions between the silo density and additives had effects on the DM losses (P<0.01), CP (P<0.05), AN (P<0.01) and starch (P<0.05) contents. The DM losses and AN contents of the LAB and E+LAB treatment groups at silo densities of 400 and 500 kg/m3 were significantly lower (P<0.05) than those of the control group. At a silo density of 500 kg/m3, the use of M+LAB and E+LAB significantly (P<0.05) increased the starch content of rice straw silage. This result was similar to the study of Zhang et al. (2010), which showed that the LAB (containing L. buchneri and Pediococcus pentosaceus) additives could decrease the DM losses and AN contents. The M and E groups resulted in a significant increase in the DM losses (P<0.05, except at the silo density of 500 kg/m3) and AN contents (P<0.05, except for the E groups at a silo density of 200 kg/m3) but a significant decrease in the CP (P<0.05 at a silo density
of 300 kg/m3) contents compared to the control group. At low silo densities (200 and 300 kg/m3), DM losses in the M+LAB group were also significantly higher (P<0.05) than those in the control group. This is similar to the study of Lynch et al. (2014), which showed that the use of exogenous fibrolytic enzymes could increase the DM losses of silages, but contrary to the result of Chen et al. (2014) and Guney et al. (2018), which showed that the use of molasses reduced the DM losses of silage. In this experiment, large nutrient consumption by yeast and mould occurred in the M group at low silo densities. At high silo densities, groups M and E consumed more nutrients due to the higher final pH and the active C. butyrate, which could use cellulose and exogenous molasses for butyric fermentation (Sushkova et al., 2013). 3.5 Structural carbohydrate components of rice straw silages For NDF, ADF and hemicellulose contents, silo density (P<0.05, P<0.01 for NDF), additives (P<0.01) and their interactions (P<0.01, P<0.05 for hemicellulose) have significant effects (as shown in Table 4). Only the E+LAB group significantly decreased (P<0.01) and reached the lowest (P<0.05) cellulose content. With increasing silo density, the NDF, ADF and hemicellulose contents of rice straw silage showed a quadratic trend (P<0.05), and rice straw silage at a silo density of 300 kg/m3 reached the peak (P<0.05). At high silo densities (400 and 500 kg/m3), the E+LAB group significantly reduced the levels of NDF, ADF and hemicellulose compared to the control group. The NDF and ADF contents of the M and M+LAB treatment groups were also lower (P<0.05) than the control group at a silo density of 500 kg/m3. These results were similar to the study of
Chen et al. (2017). However, a previous study (Li et al., 2014) has also shown that the addition of cellulase can reduce the content of NDF and ADF in king grass silages. The positive effect of E+LAB on NDF and ADF was similar to that in L. chinensis silage (Tian et al., 2014; Zhang et al., 2016). Further analysis on the effect of composite additives of LAB and cellulolytic enzymes is necessary in the future. 3.6 In vitro digestibility of rice straw silages The silo density (P<0.01), additives (P<0.05, P<0.01 for IVDMD) and their interactions (P<0.01, P<0.05 for IVADFD) significantly affected the IVDMD, IVNDFD and IVADFD of rice straw silage (shown in Table 5). As the silo density increased, the IVDMD, IVNDFD and IVADFD showed a linear increase (P<0.05). At a silo density of 500 kg/m3, the M group showed a significant increase (p<0.05) in IVDMD compared with the control groups, but the LAB and E treatment groups had no significant effect on IVDMD. At a silo density of 500 kg/m3, the M+LAB and E+LAB groups showed significant increases (p<0.05) in IVDMD, IVNDFD and IVADFD, while E+LAB exhibited higher (P<0.05) IVDMD, IVNDFD and IVADFD than the M+LAB group at a silo density of 400 kg/m3. The effect of M was similar to the study of Chen et al. (2016). However, M was considered to have no effect on the IVDMD of rice straw silage in the study of Zhao et al. (2019), while the cellulase had a positive effect on the IVDMD, IVNDFD and IVADFD of silages (Li et al., 2018; Nawaz et al., 2016). LAB can also be considered to have a positive effect on the IVDMD of fresh rice straw silages in the study of Kim et al. (2017). The use of a single type of additive is highly uncertain and needs to
be carefully selected according to the characteristics of the raw material. The use of complex additives with different types would be better. 3.7 Correlations among the fermentation quality, DM losses, chemical composition, microbial counts and in vitro digestibility of rice straw silages The correlations among fermentation quality, DM losses, chemical composition, microbial counts and in vitro digestibility of rice straw silages are shown in Figure 1. In the present study, the increase in DM losses of rice straw silages was mainly due to the increase in yeast (r=0.569, P<0.01) and mould (r=0.573, P<0.01) counts, large consumption of starch (r=-0.603, P<0.01) and the deficiency of LA (r=-0.697, P<0.01), AA (r=-0.294, P<0.05) and BA (r=-0.294, P<0.05). In this process, starch was consumed by yeast (r=0.543, P<0.01) and mould (r=-0.632, P<0.01). As a result, the in vitro digestibility of DM (r=-0.496, P<0.01), NDF (r=-0.377, P<0.01) and ADF (r=-0.544, P<0.01) in the rice straw silage decreased with the change of DM losses. Although starch is beneficial to improving the nutritional quality of rice straw silage, given that the current collection and processing level of rice straw is more difficult than that of corn silage, the retention of more starch in rice straw without targeted measures may be more conducive to aerobic deterioration caused by yeast and mould in the silage (Wilkinson and Davies, 2013). AA was mainly produced by LAB (r=0.739, P<0.01) that consume WSCs (r=-0.482, P<0.01) and have a significant inhibitory effect on yeast (r=-0.622, P<0.01) and mould (r=-0.620, P<0.01). LA is also produced by LAB (r=0.449, P<0.01), but in addition to
WSC (r=-0.469, P<0.01), the degradation of hemicellulose (r=-0.260, P<0.05) and cellulose (r=-0.327, P<0.01) could also be related to the production of LA. Corresponding, IVDMD (r=0.563, P<0.01), IVNDFD (r=0.500, P<0.01) and IVADFD (r=0.540, P<0.01) could also be enhanced by LA. The count of LAB itself is only significantly negatively correlated with WSC (r=-0.455, P<0.01). This suggests that the differences in LAB counts after ensiling may be mainly due to the differences in L. buchneri (Weinberg et al., 1999). On the other hand, the degradation of hemicellulose and cellulose is beneficial to improve fermentation by L. plantarum in rice straw silage (Zhao et al., 2018). The emergence of BA can significantly inhibit the counts of LAB (r=-0.391, P<0.01), yeast (r=-0.287, P<0.05) and mould (r=-0.354, P<0.01), while the increase in AN had a significant inhibitory effect on LAB (r=-0.309, P<0.01) and mould (r=-0.260, P<0.05). The increases in BA (r=-0.396, P<0.01) and AN (r=-0.573, P<0.01) are mainly due to the consumption of CP in the rice straw silage. More attention should be paid to the BA content in legume forages (Zhang et al., 2018) and gramineous forages with an earlier growth period (Zhang et al., 2016). 4. Conclusions The quality of rice straw silage was determined by the characteristics of raw materials, silo density and additives. The use of the M or E group alone is risky for rice straw silages. L. buchneri is active at 300 kg/m3, while L. plantarum and L. paracasei gradually take the lead with increasing silo density. The use of complex additives, E+LAB, further improved the IVDMD, IVNDFD and IVADFD of rice straw silages. It is important to select
compound additives according to the raw material and silo density to improve the quality of rice straw silage. Acknowledgments This work was financially supported by the Agricultural Innovation Fund of Jiangsu Province (grant no. CX[17]3037). Conflict of interest Authors declare that they have no competing interests. Ethical approval This article does not contain any study with human participants or animals reported by other authors. References 1.
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Bioresour. Technol. 266, 158–165. Figure Captions Figure 1 Correlations among the fermentation quality, DM losses, chemical composition, microbial counts and in vitro digestibility of rice straw silages. DM, dry matter; CP, crude protein; AN, ammoniacal nitrogen; WSC, water-soluble carbohydrates; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; IVDMD, in vitro dry matter digestibility; IVNDFD, in vitro neutral detergent fibre digestibility; IVADFD, in vitro acid detergent fibre digestibility; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; ISO-BA, isobutyric acid; LAB, lactic acid bacteria; *, Significant at P<0.05; **, Significant at P<0.01.
Table 1 The pH values and contents of organic acids in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
7.47abA
6.86bA
7.01abA
6.91bA
7.86aA
6.71bA
0.17
**,L
**
**
300
7.86aA
4.94bcB
7.41aA
5.47bB
4.83bcB
4.51cB
400
5.54aB
4.14bC
5.34aB
5.17aB
4.09bC
4.07bB
500
5.27aB
4.06cC
5.08aB
4.77bC
3.87cC
4.03cB
200
0.09
0.48C
0.17B
0.18C
0.12D
0.66C
0.20
**,L
**
**
300
0.10d
1.15bC
0.18cdB
0.7bcdBC
0.86bcC
1.90aB
400
0.56b
3.58aB
0.76bAB
0.99bB
3.62aB
4.12aA
500
0.63d
4.48bA
1.21dA
2.10cA
5.79aA
4.40bA
200
0.23B
0.40B
0.59
0.47B
0.32C
0.65B
0.06
**, Q
**
**
Items
Density
treatment
kg/m3
Control
pH value
200
LA,%DM
AA,%DM
PA,%DM
ISO-BA,%DM
BA,%DM
300
0.22dB
1.35bcA
0.56d
1.01cA
2.15aA
1.68bA
400
0.73bA
1.28aA
1.00ab
1.16abA
1.32aB
1.32aA
500
0.62cAB
1.35aA
0.70c
0.96abcA
0.89bcB
1.26abA
200
NDB
ND
0.04C
NDB
0.01
ND
300
NDbB
NDb
0.14aAB
0.03bB
NDb
NDb
400
0.27aA
0.04bc
0.12bBC
0.10bcB
NDc
NDc
500
0.09bB
NDb
0.24aA
0.24aA
NDb
NDb
200
1.35aA
0.69bB
0.80bB
0.49bc
0.27cC
0.67bB
300
0.61bB
1.31aA
0.74bB
0.74b
1.33aB
1.41aA
400
0.74bcB
1.42aA
0.98bAB
0.51c
1.52aAB
1.63aA
500
0.78cB
1.54abA
1.20bA
0.70c
1.72aA
1.64aA
200
NDC
NDB
0.03D
NDC
0.01
ND
300
NDdC
0.07bcA
0.11bC
0.17aB
NDd
0.03cd
0.01
**,L
**
**
0.06
**,L
**
**
0.02
**,L
**
**
400
0.29aA
0.05cAB
0.34aB
0.21bB
0.05c
NDc
500
0.18cB
0.08dA
0.65aA
0.32bA
0.02de
0.02e
LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; ISO-BA, isobutyric acid; LAB, lactic acid bacteria additive; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-CValues
show significant (P<0.05) differences in the same column of each item; ND, not detected; *, Significant at P<0.05;**, Significant at
P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 2 Microbial counts in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
7.61c
8.16abB
8.07abA
7.87bcB
7.68cC
8.37aB
0.05
**,Q
**
**
300
7.43c
8.78aA
7.94bA
7.99bAB
8.74aA
8.74aA
400
7.55b
8.43aB
7.45bB
8.26aA
8.41aB
8.46aAB
500
7.44b
8.40aB
7.54bB
8.25aA
8.20aB
8.37aB
200
6.87AB
6.50A
7.38A
6.98A
7.23A
6.56A
0.14
**,L
**
**
300
7.19aA
5.96bAB
7.42aA
4.41dC
5.32bcC
4.81cdB
400
6.17aBC
4.14cC
5.34abB
5.43aB
5.85aBC
4.50bcB
500
5.42bC
5.48bB
4.96bcB
4.43cC
6.39aAB
4.39cB
200
6.21aA
5.57abA
5.69abA
5.04bcA
6.28aA
4.19cA
0.17
**,L
**
**
300
6.25aA
2.74cB
6.08aA
2.48cB
3.88bB
3.13bcB
Items
Density
Treatments
kg/m3
Control
LAB, log10 CFU/g
200
Yeasts, log10 CFU/g
Moulds, log10 CFU/g
400
4.95aB
2.98bB
3.65bB
2.60bB
3.44bB
3.25bAB
500
3.46aC
3.36abB
2.59abC
2.38bB
3.46aB
2.38bB
LAB,lactic acid bacteria; CFU, Colony Forming Units; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-CValues show significant (P<0.05) differences in the same column of each item; *, Significant at P<0.05;**, Significant at P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 3 Contents of dry matter (DM), dry matter losses (DM losses) and components of nitrogen and sugar in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
28.06
28.25
26.42
25.16
25.26
27.26
0.23
**, L
**
NS
300
29.07
28.55
28.28
27.13
25.25
28.82
400
30.38
30.59
28.43
28.10
29.30
30.37
500
31.02
30.67
30.58
29.73
29.29
31.37
200
21.85bA
19.68bA
26.32aA
27.24aA
27.38aA
21.14bA
0.69
**, L
**
**
300
15.91cB
15.02cB
20.00bB
21.37bB
24.35aB
16.10cB
400
15.09bB
10.71cC
17.47aC
18.57aC
12.74bcC
11.88cC
500
11.04aC
8.10bD
10.45abD
12.11aD
11.01aC
8.20bD
200
6.02bcA
5.85bcdA
6.05bA
5.74cdA
6.40aA
5.71d
0.04
**, L
**
*
Items
Density
Treatments
kg/m3
Control
DM, %
200
DM loss, %DM
CP,%DM
AN, %TN
WSC,%DM
Starch,%DM
300
5.64aB
5.34bcB
5.17cC
5.08cB
5.64aB
5.47ab
400
5.32bcC
5.46bB
5.56abB
5.05cB
5.78aB
5.60ab
500
5.26bC
5.46bB
5.47bB
5.26bB
5.79aB
5.51ab
200
5.02bcB
4.08cB
6.38aB
5.80abB
5.76abAB
4.82bc
300
4.76cB
5.77bcA
11.16aA
10.74aA
6.98bA
5.50c
400
8.95bA
5.06cAB
10.98aA
10.83aA
6.11cAB
5.04c
500
8.51bA
5.46cA
10.02aA
10.06aA
5.42cB
5.05c
200
1.86
1.31
1.65
1.51
1.16
1.58
300
1.69
1.13
1.46
1.12
1.13
1.27
400
1.61
1.26
1.40
1.29
1.10
1.23
500
1.43
1.07
1.45
1.09
1.19
1.19
200
5.43dB
8.03ab
6.08cdB
7.64abcC
6.53bcdC
8.73aB
300
7.49bcA
9.23ab
7.37cAB
9.71aB
6.98cBC
8.59abcB
0.29
**, L
**
**
0.03
**, L
**
NS
0.25
**, L
**
*
400
8.95A
9.60
8.27A
9.82B
8.39B
9.69B
500
8.58bA
9.14b
8.23bA
12.21aA
11.77aA
12.07aA
DM, dry matter; CP, crude protein; AN, ammoniacal nitrogen; WSC, water-soluble carbohydrates; LAB, lactic acid bacteria additive; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-DValues show significant (P<0.05) differences in the same column of each item; *, Significant at P<0.05;**, Significant at P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 4 Contents of structural carbohydrates in rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM significance
LAB
M
E
M+LAB
E+LAB
D
D*A
56.46abc
55.55bcB
60.15aA
57.59abcC
59.35abAB
54.22cAB
0.48
**, Q **
**
300
59.40bc
58.24cAB
57.5cAB
65.09aA
63.26aA
55.62cA
400
59.04ab
62.39aA
57.21bAB
63.04aAB
55.65bBC
51.35cB
500
59.26a
59.92aA
54.07bB
59.52aBC
54.77bC
51.89bAB
200
32.93ab
33.23abB
35.37aA
33.46abB
35.72aA
31.13b
0.36
*, Q
**
**
300
34.00b
34.02bAB
33.61bAB
37.36aA
37.61aA
32.91b
400
34.22abc
36.37abA
33.97bcAB
37.02aA
31.61cdB
29.92d
500
34.55abc
34.84abAB
31.63cdB
35.40aAB
31.92bcdB
30.36d
200
4.10
4.72
4.17
4.21
5.25
3.97
0.14
NS
NS
NS
Items
Density
Treatment
kg/m3
Control
NDF ,%DM
200
ADF ,%DM
ADL ,%DM
A
Hemicellulose ,%DM
Cellulose ,%DM
300
3.72
4.05
3.38
5.38
5.93
4.34
400
3.37
4.29
4.02
5.65
4.78
3.80
500
4.29
3.98
3.08
4.49
3.45
5.06
200
23.53ab
22.31bB
24.79a
24.13abB
24.95aAB
23.09ab
300
25.40ab
24.22bcAB
23.90bc
27.73aA
25.64abA
22.72c
400
24.82ab
26.11aA
23.24bc
26.02aAB
24.03abAB
21.43c
500
24.70ab
25.08aA
22.44bc
24.12abB
22.86abcB
21.54c
200
28.83
28.52
31.19
29.25
30.47
27.34
300
30.29
29.97
30.23
31.97
31.69
28.57
400
30.85
32.02
29.95
31.37
26.83
26.12
500
30.26
30.85
28.55
30.91
28.47
25.30
0.26
*, Q
**
*
0.33
NS
**
NS
DM, dry matter; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; LAB, lactic acid bacteria additive; M, molosses additive; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex
additives containing M and LAB; E+LAB, complex additives containing E and LAB; a-d Values show significant (P<0.05) differences on the same line; A-CValues show significant (P<0.05) differences in the same column of each item; *, Significant at P<0.05;**, Significant at P<0.01; NS, not significant; L, linear effect (P<0.05), Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Table 5 In vitro digestibility of rice straw silages treated with individual or compound additives and stored in different silo densities.
SEM
significance
LAB
M
E
M+LAB
E+LAB
D
A
D*A
44.41ab
45.74abB
41.80bB
46.08aA
43.40abC
45.15abB
0.52
**, L
**
**
300
44.66bc
49.81aA
46.62bA
40.63cB
41.00cC
48.01abB
400
44.58cd
43.89dB
48.64bcA
42.79dAB
49.49bB
55.10aA
500
44.20d
45.61cdB
49.75bcA
45.61cdA
54.04aA
52.51abA
200
16.87b
15.69bC
15.91bB
24.70a
16.37bC
17.84bB
0.53
**,L
*
**
300
18.58b
28.55aA
18.73bAB
20.37b
19.23bBC
22.44bAB
400
19.45b
20.92abBC
23.33abA
21.00ab
23.15abB
26.03aA
500
17.87c
22.03bcB
22.11bcA
21.55bc
28.83aA
23.50bA
200
11.29B
13.98B
12.38B
14.03B
13.9B
13.48B
0.69
**,L
*
*
300
13.07bAB
24.04aA
15.48bAB
16.24bAB
16.33bB
15.42bB
Items
Density
treatment
kg/m3
Control
IVDMD ,%
200
IVNDFD ,%
IVADFD, %
400
15.43cAB
15.61bcB
20.89abA
18.85abcAB 16.56bcB
23.12aA
500
17.71bA
18.82bAB
19.07bA
21.16abA
24.55aA
24.87aA
IVDMD, in vitro dry matter digestibility; IVNDFD, in vitro neutral detergent fibre digestibility; IVADFD, in vitro acid detergent fibre digestibility; LAB, lactic acid bacteria additives; M, molosses additives; E, complex enzyme preparations containing Cellulase, xylanase, glucanase, pectinase and tween 80; M+LAB, complex additives containing M and LAB; E+LAB, complex additives containing E and LAB. a-d Values
show significant (P<0.05) differences on the same line; A-CValues show significant (P<0.05) differences in the same column of each
item; *, Significant at P<0.05; **, Significant at P<0.01; L, linear effect (P<0.05); Q, quadratic effect (P<0.05); SEM, standard error of means; D, silo densities; A, additives; D×A, interaction between D and A.
Jipeng Tian: Methodology, Investigation, Formal analysis, Writing - Original Draft Nengxiang Xu: Methodology, Data Curation, Resources, Investigation Beiyi Liu: Resources, Investigation Hailin Huan: Resources, Investigation, Validation Hongru Gu: Methodology, Writing - Review & Editing Chenfei Dong: Methodology, Writing - Review & Editing Chenglong Ding: Conceptualization, Methodology, Project administration, Funding acquisition
As the silo density increased, the quality of rice straw silages improved. Lactic acid bacteria (LAB) improved fermentation quality of rice straw silages. Combination of enzymes and LAB and high silo density (>400kg/m3) reached the best.