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Anim Biosci > Volume 32(6); 2019 > Article
Sun, Wang, Gentu, Jia, Hou, and Cai: Changes in microbial population and chemical composition of corn stover during field exposure and effects on silage fermentation and in vitro digestibility

Abstract

Objective

To effectively use corn stover resources as animal feed, the changes in microbial population and chemical composition of corn stover during field exposure, and their silage fermentation and in vitro digestibility were studied.

Methods

Corn cultivars (Jintian, Jinnuo, and Xianyu) stovers from 4 random sections of the field were harvested at the preliminary dough stage of maturity on September 2, 2015. The corn stover exposed in the field for 0, 7, 15, 30, 60, 90, and 180 d, and their silages at 60 d of ensiling were used for the analysis of microbial population, chemical composition, fermentation quality, and in vitro digestibility. Data were analyzed with a completely randomized 3×6 [corn stover cultivar (C)×exposure d (D)] factorial treatment design. Analysis of variance was performed using SAS ver. 9.0 software (SAS Institute Inc., Cary, NC, USA).

Results

Aerobic bacteria were dominant population in fresh corn stover. After ensiling, the lactic acid bacteria (LAB) became the dominant bacteria, while other microbes decreased or dropped below the detection level. The crude protein (CP) and water-soluble carbohydrate (WSC) for fresh stover were 6.74% to 9.51% and 11.75% to 13.21% on a dry matter basis, respectively. After exposure, the CP and WSC contents decreased greatly. Fresh stover had a relatively low dry matter while high WSC content and LAB counts, producing silage of good quality, but the dry stover did not. Silage fermentation inhibited nutrient loss and improved the fermentation quality and in vitro digestibility.

Conclusion

The results confirm that fresh corn stover has good ensiling characteristics and that it can produce silage of good quality.

INTRODUCTION

Corn (Zea mays L.) is an important crop for food production in China and worldwide. Corn stover includes the leaves, stalks, husks, and cobs, and is often reported as a ratio of dry matter (DM) of corn grain to residue of 1:1. Corn stover is the main crop residue in most of China, with an annual yield of approximately 640 million t/yr [1]. Using corn stover as animal feed has been proven economically viable, not only as a method of disposing of corn stover residue, but also as an alternative livestock feed in regions where corn is the main crop [1]. Corn stover is sometimes incinerated in the field [2], raising concerns about air pollution, fine particle generation, and global warming. Moreover, corn stover is considered an important local resource that can be used by farmers to prepare silage as an animal feed and as a rural fuel [3]. Not only fresh corn stover but also its silage is used as an animal feed; large amounts of dried stover are fed to dairy or beef cattle in some localities, China. However, the dry corn stover that has been left exposed in the field for a long time is very fibrous, low in nutrients and difficult to digest for animals [1]. Feeding livestock low-quality roughage can result in low production. The major constraint for livestock production in some cold climates, such as Inner Mongolia, is a shortage of good-quality feed, where corn stover or hay is the major source of roughage for livestock in winter. Fresh corn stover has good ensiling characteristics due to its relatively high DM content at harvest, low buffering capacity, and adequate water-soluble carbohydrate (WSC) content [4,5]. Corn stover silage has become the major component of forage for dairy cows under most dietary regimes in China [2,6], and has greater potential to improve ruminant production than the more conventional preserved forages used in corn production areas [7].
Silage preparation and storage are the most effective prepa ration techniques for fresh stover [8,9]. Corn stover silage can be beneficial to dairy cattle, beef cattle, or sheep producers because it can provide a locally available feed source at a low cost for animal production [1], and silage is a commonly preserved feed in many countries, including China. The preservation of forage crops as high-quality silage depends on the production of enough acids to inhibit the activity of undesirable epiphytic microorganisms under anaerobic conditions, especially lactic acid bacteria (LAB), which are naturally present on forage crops [10,11].
However, most studies in silage research have analyzed silage fermentation of fresh corn stover, and limited information is available on the effects of field drying corn stover on feed composition, silage fermentation, and digestibility. In this study, the changes in the microbial population and chemical composition of corn stover during field exposure for up to 180 d and their silage fermentation characteristics were studied. In order to evaluate the nutritional value of the corn stover and silages, we also studied the in vitro digestibility of the stover and silage in a corn production area of Inner Mongolia, China.

MATERIALS AND METHODS

Animal care

Animal experiments were approved by the Committee of Animal Experimentation and were performed under the institutional guidelines for animal experiments of the College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, China. The experiments were performed according to recommendations proposed by the European Commission (1997) to minimize the suffering of animals.

Corn stover and silage preparation

Local corn (Zea mays L.) cultivars (Jintian, Jinnuo, and Xianyu) were obtained from an experimental field at the Inner Mongolia Agricultural University (Huhhot, China). Corn stovers from 4 random sections of the field were harvested at the preliminary dough stage of maturity on September 2, 2015. Silage was prepared from stover exposed in the field for 0, 7, 15, 30, 60, 90, and 180 d. The ensiling material in triplicate for each cultivar was cut into 20-mm segments with a chopping machine (TS420; Qufu Machinery Equipment Co., Ltd., Qufu, China), and adequate water added to get a moisture content of approximately 60%. Silage was fermented in laboratory-scale polyethylene silos (1-L volume, 10-cm diameter, 20-cm length; Shenzhen Guanruilong Chemical Co., Ltd., Shenzhen, China), at approximately 760 g (fresh weight). The silos were sealed with a screw top and plastic tape and stored at ambient temperature (20°C to 25°C). The silos were opened after 60 d of ensiling, and the microbial composition, chemical composition, fermentation quality, and in vitro digestibility were analyzed.

Microbiological and chemical analysis

Corn stover and silage samples (10 g) were blended with 90 mL of sterilized water, and serially diluted in sterilized saline solution (8.50 g/L NaCl) from 10−1 to 10−5 [12]. The number of LAB was determined using the plate-counting method on MRS agar (Difco Laboratories, Inc., Detroit, MI, USA) incubated at 30°C for 48 h in an anaerobic box (MGC C-31; Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). Yeasts and molds were counted on potato dextrose agar (Nissui Ltd., Tokyo, Japan) after incubation at 30°C for 96 h, and aerobic bacteria were counted on nutrient agar medium (Qingdao Hope Bio-technology Co., Ltd., Qingdao, China). All microbial data were transformed to log10 colony-forming units (cfu) based on fresh matter (FM).
The DM contents of corn stover and silage was determined by weighing samples that had been oven-dried at 65°C for 48 h, and the data were corrected for residual moisture at 105°C [13]. The crude ash (CA) content was determined after placing samples in a muffle oven for 3 h at 550°C according to the method of No.942.05 of the AOAC [14]. The organic matter (OM) was calculated as weight loss upon ashing. Total nitrogen was measured using a Kjeldahl apparatus (KDY-9830, Shanghai, China) using the procedure of the AOAC [14]. Crude protein (CP) was calculated as follows: total nitrogen×6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined using the ANKOM A200i fiber analyzer (ANKOM Technology, Macedon, NY, USA) [15]. The ether extract (EE) content was determined according to procedure No.963.15 of the AOAC [14]. The WSC concentration of fresh materials and silages were determined using the method of Kim and Adesogan [16].
For the silage fermentation analysis, 10 g of sample was homogenized with 90 mL of deionized water for 5 min [17]. The extract was filtered through two layers of cheesecloth and a filter paper (8 cm; Aoke Co., Ltd., Taizhou, China), and the pH was measured using a glass electrode pH meter (STARTER 100/B; OHAUS, Shanghai, China). The ammonia nitrogen (N) content was analyzed using a steam distillation method of the filtrates, and lactic, acetic, propionic, and butyric acids were analyzed by high-performance liquid chromatography, as described by Cai [17].

In vitro incubation and degradability measurements

In vitro fermentation was performed in serum bottles following the method described by Contreras-Govea et al [18] and Kowalski et al [19]. Rumen fluid was obtained before morning feeding from four Inner Mongolia semi-fine-wool sheep (wethers) through stomach tube. Animals were fed 40 g of alfalfa hay, 400 g of guinea-grass hay, and 240 g of concentrate containing 10% wheat bran, 25% soybean meal, 65% corn grain, and supplemental vitamins and minerals on a DM basis.
Approximately 1 g ground sample (through 1 mm sieve) was placed in 130-mL serum bottles. Rumen fluid was filtered through four layers of gauze and mixed 1:2 (v/v) to buffer; 60 mL of the mixture was transferred into each serum bottle. The buffer was prepared by the method described by Menke [20] and consisted of (added in order) 400 mL H2O, 0.1 mL solution A (13.2 g CaCl2 ·2H2O, 10.0 g MnCl2·4H2O, 1.0 g CoCl2·6H2O, 8.0 g FeCl3·6H2O and made up to 100 mL with H2O), 200 mL solution B (39 g NaHCO3/L, H2O), 200 mL solution C (5.7 g Na2HPO4, 6.2 g KH2PO4, 0.6 g MgSO4·7H2O and made up to 1,000 mL with H2O), 1 mL resazurine (0.1%, w/v) and 40 mL reduction solution (95 mL H2O, 4 mL l N NaOH and 625 mg Na2S·9H2O). Each serum bottle was kept under CO2 in a water bath at 39°C, after being capped with a butyl rubber stopper and sealed with an aluminum crimp. In the experiment, the gas production (GP) was released and measured during in vitro incubation at every 4 hours’ point intervals. The cumulative 48 h GP data was the total amount of gas produced every 4 hours. Immediately after 48 h incubation, the undigested solids were precipitated by centrifugation at 1,000×g for 10 min at room temperature and dried in an aerated oven at 65°C for 48 h, and were then assayed for DM and CA. The in vitro degradability of OM (OM-D) was calculated as change in its respective weight. All corn stover and their silages were performed in two separate in vitro experimental runs, and each run consisted of 132 bottles: three cultivars×seven exposure d×three replicates×2 (corn stover+its silage), plus six blanks. The blanks were the same bottle with no materials. The cumulative 48 h GP was corrected by subtracting GP from blank bottles.
Ruminal pH was measured immediately after collection using a digital pH meter (STARTER 100/B; OHAUS, China), and a 20-mL sample was preserved with 0.5 mL of 9 M sulfuric acid and stored at −20°C for subsequent analysis of total volatile fatty acids (VFAs). The total VFA concentration was measured using an automated gas chromatograph [21].

Statistical analysis

Data on the microorganism population, chemical composition, and fermentation quality after 60 d of ensiling and in vitro digestibility were analyzed with a completely randomized 3×6 (corn stover cultivar [C]×exposure d [D]) factorial treatment design. Analysis of variance was performed using SAS ver. 9.0 software (SAS Institute Inc., Cary, NC, USA), and the statistical model is as follows:
Yijk=μ+αi+βj+αβij+ɛijk
Where Y ijk is the observation, μ is the overall mean, αi is the effect of corn stover cultivar (i = Jintian, Jinnuo, and Xianyu), βj is the effect of exposure d (j = 0, 7, 15, 30, 60, 90, and 180), αβij is the effect of corn stover cultivar×exposure d, and ɛijk is the error. The mean values were compared using Duncan’s test [22].

RESULTS

Table 1 presents the chemical composition of corn stover during the field exposure. At 0 d of exposure, the three cultivars had similar DM contents (30.21% to 30.67%), which increased by 4% to 25% after 7 to 180 d of exposure. The CP contents of the three cultivars were 6.74% to 9.51% of DM. After 180 d of exposure, the CP content decreased to 3.28% to 4.41% of DM. The NDF and ADF contents in the three cultivars were 43% to 61% and 27% to 38% of DM, respectively. The exposure d means in the three cultivars were similar at 7 and 30 d, and differed significantly (p<0.05) for the other exposure d. With the increase of exposure time, the WSC content decreased markedly, and the mean WSC in the three cultivars decreased sequentially with increasing exposure time (p<0.05). C, D, and C×D influenced (p<0.0001) the DM, OM, CP, NDF, ADF, and WSC contents. C and D influenced (p<0.0001, p = 0.0006) the EE content, but C×D did not (p = 0.999).
Table 2 presents the microbial population of corn stover over the course of the field exposure. Fresh (0 d of exposure) Jinnuo, Jintian, and Xianyu corn stover contained 104 to 108 cfu/g of FM of LAB, aerobic bacteria, yeasts, and molds. From 7 to 180 d of exposure, these microorganism counts of the three cultivars were 104 to 107 cfu/g of FM. The mean aerobic bacteria and molds counts of Jinnuo and Jintian at 180 d of exposure were significantly (p<0.05) lower than other exposure days. C, D, and C×D influenced (p<0.0001) the counts of all microorganisms.
Table 3 presents the microbial population of corn stover silage after 60 d of ensiling. The LAB counts in the silages of the three cultivars were 104 to 107 cfu/g FM. The mean LAB counts of the silages of the three cultivars from the first few d of exposure (0, 7, and 15 d) were significantly (p<0.05) higher than those of silages after 30, 60, 90, and 180 d of exposure. During exposure, the aerobic bacteria and yeasts remained around <103 to 107 cfu/g of FM, and molds in all silages were below the detection level (<103 cfu/g of FM). C, D, and C×D influenced (p<0.0001) the counts of LAB, aerobic bacteria, and yeasts in corn stover silage.
Table 4 presents the chemical composition of corn stover silage after 60 d of ensiling. The OM contents of the three cultivar silages after 0 to 180 d of exposure were similar, ranging from 93% to 95% of DM. With the increase of exposure time, the CP contents of all corn stover silages significantly (p<0.05) decreased. The highest (p<0.05) and lowest (p<0.05) CP contents in the three cultivars were obtained at the initial (0 d) and final (180 d) exposure times. During exposure, the mean NDF and ADF contents in the silages of the three cultivars were 46% to 51% and 26% to 33% of DM, respectively; the EE content decreased gradually (p<0.05). With the increase of exposure time, the WSC content of the silages of the three cultivars, as well as the mean decreased (p<0.05). The C, D, and C×D influenced (p<0.0001 or p = 0.0017) the OM, CP, EE, NDF, ADF, and WSC contents. C and D influenced (p<0.0001, p = 0.0017, respectively) EE, but C×D did not (p = 1.0000).
Table 5 presents the fermentation quality of the corn stover silages after 60 d of ensiling. The silages of the three cultivars from corn stover exposed for 0 and 7 d were well preserved, with lower (p<0.05) pH and ammonia-N contents and higher (p<0.05) lactic acid content than those of the silages from stover exposed for 15 to 180 d. The silages prepared from corn stover exposed for 180 d yielded the worst fermentation quality (p< 0.05). Jintian stover had a significantly higher mean of lactic acid content (p<0.05) and lower mean ammonia-N content (p<0.05) than the silage from the other two cultivars. The C, D, and C×D influenced (p<0.0001) the pH and contents of lactic acid, acetic acid, propionic acid, and ammonia-N. The C did not influence (p = 0.2579) the butyric acid content of corn stover silage, but the D (p = 0.025) and C×D did (p = 0.0038).
Table 6 presents the ruminal pH, GP, total VFA content, and OM-D of corn stover and silage. The ruminal pH of the three cultivars at different exposure times were similar (6.12 to 6.30). With the increase of exposure time, the GP, total VFA content, and OM-D of the three cultivars decreased. The highest (p<0.05) and lowest (p<0.05) GP, total VFA content, and OM-D in the three cultivars were observed in stover on the initial (0 d) and final (180 d) d of exposure, respectively. The C and D influenced (p<0.0001) GP, total VFA content, and OM-D. C×D influenced (p = 0.0028) GP but did not influence (p = 0.0816) VFA content or OM-D (p = 0.1884). The A comparison of the three cultivars showed that the GP and total VFA content were highest (p<0.05) in Jintian corn stover. The OM-D of Jinnuo corn stover was higher (p<0.05) than the other cultivars.
The ruminal pH of the stover silages of the three cultivars at different exposure times ranged from 6.61 to 6.86. With the increase of exposure time, the GP, total VFA, and OM-D of the three cultivar silages decreased. Compared to the data from stover collected on day 0, the mean GP, total VFA content, and OM-D of corn stover silage after 180 d of exposure decreased significantly (p<0.05) by more than 5%, 11%, and 6%, respectively. The C and D influenced (p<0.0001) GP, total VFA content, and OM-D. C×D influenced (p = 0.0004) OM-D but did not influence (p = 0.7809) GP or total VFA (p = 0.0647) content. From the cultivar means, the OM-D of Jintian corn stover silage was significantly (p<0.05) higher than those of Xianyu and Jinnuo corn stover silage.

DISCUSSION

Corn stover is an abundant byproduct of corn grain harvesting. Due to economic and environmental concerns, there is an increasing demand for the efficient use of crop residues, including corn stover. Farmers use fresh or dry corn stover as forage to feed ruminants. However, dry corn stover is highly fibrous and difficult to digest when stored for long periods on fields. Therefore, it is important to identify techniques to effectively use corn stover. After drying on fields, the moisture and CP contents of corn stover decrease and the NDF content increases [23]. In this study, the DM of the three cultivars increased by more than 4% to 25% after 7 to 180 d of exposure. Generally, CP includes true protein and non-protein nitrogen such as urea nitrogen and ammonia nitrogen. The CP content is influenced by exposure condition and activity of the protein-degrading microorganisms. In the present study, after 180 d of exposure, the CP contents of corn stover were decreased by 34% to 66% compared to fresh stover or their silages. The reason is that the rain, exposure, and microbial populations may result in the CP loss, and that the good quality of silage could effectively preserve these chemical compositions of stover during ensiling. Aerobic bacteria, yeasts, and molds were distributed in fresh corn stover at concentrations of 104 to 108 cfu FM (Table 1). The mean aerobic bacteria and molds counts of Jinnuo and Jintian at 180 d of exposure were significantly (p<0.05) lower than other exposure days. The reason for this difference is probably due rain before sampling, and epiphytic microorganisms may have consumed some of the chemical components of the corn stover, such as CP and WSCs.
Meanwhile, the NDF and ADF contents of dry corn stover did not show specific trends during field exposure, which differed from the results of previous studies, where the crude fiber content was greater in dry corn stover than fresh stover [23]. The reason for this difference is unclear; however, exposure to rain and natural conditions may have caused a loss in some water-soluble chemical composition, resulting in the increasing rates of fiber on DM basis. Additional studies are necessary to consider the loss ratios of various chemical components in stover during field exposure.
Corn is a high-energy-value crop with good ensiling characteristics due to its relatively high DM content at harvest, low buffering capacity, and adequate WSC content [4,5]. Forage corn silage has the potential to support higher animal performance in ruminant production systems than more conventional conserved forages used in many countries [7]. Fresh corn stover has similar ensiling characteristics as forage corn and can be used to prepare high-quality silage. Corn stover silage has become the major forage component in the diets of dairy cows under most dietary regimes [2,6]. In this study, the fresh corn stover silages of three cultivars were preserved well and of good quality. Conversely, silage prepared from stover that had been exposed for 180 d exhibited poor fermentation quality. The factors involved in assessing fermentation include the chemical composition of the stover materials and the microbial population of epiphytic microorganisms. Fresh stover has a relatively high WSC content and LAB count, and LAB fermented enough sugar to produce lactic acid (Tables 3, 4). In addition, the pH of the silage dropped below 4.0, which inhibited butyric fermentation and ammonia-N production by clostridia. Conversely, silage prepared with stover that had been exposed for 180 d had relatively high pH values and low lactic acid contents. Reductions in sugar and LAB were factors contributing to poor fermentation, as evidenced by the fact that the fresh stover contained abundant sugars as a substrate for LAB to produce lactic acid. Furthermore, as dominant microbes, natural LAB could ferment silage well. Therefore, fresh stover with sufficient sugar content and epiphytic LAB counts, even without LAB inoculation, can be used to make good-quality silage. In a previous study, the natural strains Lactobacillus casei and Lactobacillus plantarum were the species most frequently isolated from corn stover [24] and can produce relatively high amounts of lactic acid under high WSC conditions compared to other strains.
The CP contents of fresh stover and the resulting silage did not differ greatly, indicating that no protein loss occurred during ensiling, and showed that fresh stover silage could effectively preserve the chemical composition of stover during fermentation. In this study, the natural LAB and ensiling characteristics were compatible with corn stover fermentation and were suitable for producing silage. This indicates that when sufficient natural LAB are present on stover materials, it is unnecessary to employ LAB inoculation to produce silage, and future experiments should study the relationships between stover, LAB characteristics, and silage fermentation. Overall, preparing silage from fresh stover has the benefit of promoting the propagation of natural LAB and inhibiting clostridia growth, as well as inhibiting CP loss during fermentation. From the perspective of microbial populations, chemical composition, and silage fermentation, corn stover silage should be prepared immediately after harvesting corn. Ensilage provides an effective means of conserving summer-grown green forage to supply as winter feed for ruminants [25].
Corn stover can provide some of the metabolizable energy and CP required by ruminants but has the disadvantage of poor intake by ruminants when supplied in a hard, dry state due to the low moisture content and large particle size, making it difficult for ruminants to chew. In addition, low intake and subsequent weight gain have indicated that dry stover is not a preferred forage [1]. In this study, the best and worst results for GP, total VFA content, and OM-D in the stover and silages of the three cultivars were observed for the initial (0 d) and final (180 d) exposure times, respectively. This may have been due to a combined effect of LAB and WSC in fresh stover in improving the fermentation quality, reducing protein loss, and providing more digestible substrates for fermentation by rumen microbes, which could facilitate ruminal digestion. Meanwhile, the microbial population varied among the stover and silage from the three cultivars. Epiphytic microbes include both harmful and beneficial microbes. Consuming such microbes with silage could change the microbial diversity and activity in rumen, affecting rumen digestion. Therefore, good fermentation can inhibit molds growth in silage, and improve the nutrient digestibility of fresh corn stover. Generally, corn stover is beneficial to cattle or sheep producers because it can provide a cheap, locally available feed source [1]. Therefore, further experiments are needed to elucidate alternative feeding strategies for cattle and sheep producers by comparing the nutritional values of diets based on corn stover silage with conventional hay-based diets and assessing the effect on local livestock production systems.
The results confirmed that the silage prepared from fresh corn stover can have beneficial synergistic effects by improving the fermentation quality and promoting ruminal degradation compared to dry corn stover.

CONCLUSION

Fresh corn stover contained a relatively high LAB count and WSC content, and the resulting silage fermented well, with minimal nutrient loss and improved in vitro digestibility. With increasing field exposure of corn stover, the CP and WSC contents and in vitro digestibility decreased. From the perspective of microbial population, chemical composition, and silage fermentation, fresh corn stover has suitable ensiling characteristics, and silage should be prepared immediately after harvesting corn.

Notes

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

ACKNOWLEDGMENTS

This work was supported by Project “Regulating Mechanisms of Natural Forage Silage Quality in Inner Mongolia Typical Grassland”, National Natural Science Foundation (NSFC 314 60638), and “Research and Demonstration of Forage Silage Technology and Supporting Facilities and Equipment”, Nonprofit Agricultural Project (201303061), Ministry of Agriculture, China.

Table 1
Chemical composition of corn stover during field exposure
Exposure (d) DM (%) OM CP EE NDF ADF WSC

------------------------------------------------------------% of DM------------------------------------------------------------
Jinnuo
 0 30.21f 93.36c 7.99a 2.34a 59.06a 34.70b 13.04a
 7 36.42e 94.31b 7.64b 2.23a 53.29c 27.44g 12.84b
 15 47.46b 93.99b 7.43bc 2.11a 45.46f 27.74f 12.21c
 30 55.73a 96.02a 7.22cd 1.98a 43.54g 29.17e 12.32c
 60 42.41d 94.20b 7.09ed 1.94a 58.02b 33.53c 10.87d
 90 47.73b 93.91b 6.85e 1.90a 49.72e 33.05d 9.57e
 180 44.61c 93.87b 4.41f 1.87a 50.42d 35.21a 7.13f
Jintian
 0 30.67g 94.46d 9.51a 2.16a 45.59f 30.50c 13.21a
 7 34.46f 95.31b 8.28b 2.04ab 46.00ef 32.08b 13.05b
 15 35.25e 96.77a 8.18b 1.90ab 46.32e 29.09d 12.78c
 30 46.73a 95.14bc 7.94c 1.79ab 51.66c 26.26e 12.24d
 60 39.42d 94.91c 7.13d 1.75ab 49.63d 34.64a 10.44e
 90 45.54b 95.37b 6.33e 1.65ab 52.50b 29.19d 9.43f
 180 43.68c 94.58d 3.28f 1.60b 54.65a 34.74a 7.21g
Xianyu
 0 30.43e 94.68e 6.74a 1.55a 60.69a 34.80c 11.75a
 7 44.78d 95.83b 6.28b 1.46ab 51.99f 31.55e 11.18b
 15 44.62e 95.19d 5.58c 1.30ab 54.57d 29.86f 10.52c
 30 54.45a 96.21a 5.40c 1.23abc 56.15b 35.88b 10.10d
 60 48.52c 94.71e 4.59d 1.18abc 54.86cd 36.15b 8.82e
 90 49.74b 95.49c 4.43d 1.12bc 53.63e 32.97d 7.47f
 180 48.42c 95.38cd 3.63e 0.85c 55.21c 37.83a 6.30g
SEM 0.12 0.08 0.16 0.16 0.11 0.11 0.04
Cultivar (C) means
 Jinnuo 43.51b 94.24c 6.95b 2.05a 51.36b 31.55b 11.14b
 Jintian 39.39c 95.22a 7.24a 1.84b 49.48c 30.93c 11.19a
 Xianyu 45.85a 94.95b 5.24c 1.24c 55.30a 34.15a 9.45c
Exposure (d, D) means
 0 30.44g 93.21e 8.08a 2.02a 55.11a 33.33c 12.67a
 7 38.55f 95.15b 7.40b 1.91ab 50.43e 30.36e 12.36b
 15 42.44e 95.32b 7.06c 1.77abc 48.78f 28.9f 11.84c
 30 52.30a 95.79a 6.85d 1.67bcd 50.45e 30.44e 11.55d
 60 43.45d 94.61d 6.27e 1.62cd 54.17b 34.77b 10.04e
 90 47.67b 94.93c 5.87f 1.56cd 51.95d 31.74d 8.82f
 180 45.57c 94.61d 3.77g 1.44d 53.43c 35.93a 6.88g
Significance of main effects and interaction
 C <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
 D <0.0001 <0.0001 <0.0001 0.0006 <0.0001 <0.0001 <0.0001
 C×D <0.0001 <0.0001 <0.0001 0.9999 <0.0001 <0.0001 <0.0001

DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; WSC, water-soluble carbohydrate; SEM, standard error of the mean.

a–g Means within a column with different superscripts differ (p<0.05).

Table 2
Microbial population of corn stover during field exposure
Exposure (d) LAB Aerobic bacteria Yeasts Molds

---------------------- log10 cfu/g of FM -----------------
Jinnuo
 0 5.25a 6.81cb 5.76c 5.83a
 7 5.15ab 7.03b 5.63cd 5.42bc
 15 5.03b 7.56a 5.45d 5.35c
 30 4.85c 6.73c 5.46d 5.21c
 60 4.76cd 6.45d 5.23e 5.78a
 90 4.31e 5.31e 6.23b 5.61ab
 180 4.62d 4.56f 6.72a 4.57d
Jintian
 0 5.13b 7.21b 5.41b 5.62a
 7 4.82c 6.83c 5.67a 5.26b
 15 4.53d 7.57a 3.58e 5.12c
 30 4.42d 6.58d 4.52c 5.05c
 60 5.24b 5.73e 5.72a 5.62a
 90 4.52d 5.72e 4.13d 5.37b
 180 5.41a 2.65f 3.52e 4.57d
Xianyu
 0 4.52b 8.84a 4.67c 5.59a
 7 4.31c 6.43d 4.31d 5.31b
 15 5.21a 6.73c 5.23b 5.28b
 30 5.16a 7.47b 3.52e 5.27b
 60 3.65d 6.35d 5.32b 5.57a
 90 3.37e 5.42e 5.93a 5.28b
 180 3.42e 6.42d 5.73a 5.14b
SEM 0.05 0.07 0.07 0.06
Cultivar (C) means
 Jinnuo 4.85a 6.35b 5.78a 5.40a
 Jintian 4.87a 6.04c 4.65c 5.23b
 Xianyu 4.23b 6.81a 4.96b 5.35a
Exposure (d, D) means
 0 4.97a 7.62a 5.28bc 5.68a
 7 4.76b 6.76d 5.20c 5.33bc
 15 4.92a 7.29b 4.75d 5.25cd
 30 4.81b 6.93c 4.50e 5.18d
 60 4.55c 6.18e 5.42a 5.66a
 90 4.07d 5.48f 5.43a 5.42b
 180 4.48c 4.54g 5.32ab 4.76e
Significance of main effects and interaction
 C <0.0001 <0.0001 <0.0001 <0.0001
 D <0.0001 <0.0001 <0.0001 <0.0001
 C×D <0.0001 <0.0001 <0.0001 <0.0001

The samples stored for 60 d and 180 d was soaked by rain before sampling.

LAB, lactic acid bacteria; cfu, colony-forming unit; FM, fresh matter; SEM, standard error of the mean.

a–g Means within a column with different superscripts differ (p<0.05).

Table 3
Microbial population of corn stover silages at 60 d of ensiling
Exposure (d) LAB Aerobic bacteria Yeasts Molds

----------------------log10 cfu/g of FM-----------------
Jinnuo
 0 7.26b <103 4.81c ND
 7 7.86a <103 4.53d ND
 15 7.21b <103 3.13e ND
 30 7.02c 3.74b 4.82c ND
 60 6.73d 5.64a 4.38d ND
 90 6.85d <103 7.10b ND
 180 6.75d <103 7.83a ND
Jintian
 0 7.16b 7.68a 3.47d ND
 7 7.42a 6.47b 4.21b ND
 15 6.52d 6.35b <103 ND
 30 6.41ed <103 3.82c ND
 60 6.31e 3.16d 4.46a ND
 90 6.50d 4.73c 4.23b ND
 180 6.73c <103 <103 ND
Xianyu
 0 6.52b 7.68a 3.82e ND
 7 7.46a 3.15f 6.20b ND
 15 7.53a 5.36b 4.56d ND
 30 6.02c <103 <103 ND
 60 5.28d 3.36e 6.78a ND
 90 4.46e 3.78d 5.41c ND
 180 4.32e 4.65c 5.38c ND
SEM 0.09 0.09 0.05 -
Cultivar (C) means
 Jinnuo 7.10a <103 5.23a -
 Jintian 6.72b 4.63a 3.58c -
 Xianyu 5.94c 4.41b 4.88b -
Exposure (d, D) means
 0 6.98b 5.79a 4.03d -
 7 7.58a 3.59d 4.98c -
 15 7.09b 4.36b 3.39f -
 30 6.48c 3.09f 3.55e -
 60 6.11d 4.05c 5.21b -
 90 5.94e 3.23e 5.58a -
 180 5.93e <103 5.19b -
Significance of main effects and interaction
 C <0.0001 <0.0001 <0.0001 -
 D <0.0001 <0.0001 <0.0001 -
 C×D <0.0001 <0.0001 <0.0001 -

LAB, lactic acid bacteria; cfu, colony-forming unit; FM, fresh matter; ND, not detection; SEM, standard error of the mean.

a–e Means within a column with different superscripts differ (p<0.05).

Table 4
Chemical composition of corn stover silages at 60 d of ensiling
Exposure (d) DM OM CP EE NDF ADF WSC

----------------------------------------------------------------- % DM --------------------------------------------------
Jinnuo
 0 33.43a 94.11bc 7.89a 2.36a 54.62b 31.63b 5.87a
 7 33.89a 93.97c 7.66b 2.21a 50.31c 24.75g 5.21b
 15 33.32a 93.92c 7.61b 2.13a 43.57f 25.21f 5.12b
 30 33.48a 94.46b 7.55b 2.01a 40.62g 26.83e 5.15b
 60 33.74a 95.23a 7.02c 1.96a 55.48a 30.42c 4.65c
 90 33.27a 94.53b 7.04c 1.93a 46.72e 30.02d 4.13d
 180 33.89a 94.28bc 4.35d 1.91a 47.41d 32.57a 4.06d
Jintian
 0 33.75a 94.39c 8.58a 2.15a 40.32g 27.63d 6.43a
 7 33.31a 94.28c 8.13b 2.06ab 43.64f 30.02c 6.21b
 15 33.42a 95.57a 7.83c 1.92ab 45.13e 26.35e 6.14b
 30 33.35a 95.74a 7.66c 1.81b 48.47c 23.41f 5.79c
 60 33.74a 94.83b 7.46d 1.76ab 46.78d 31.68b 5.65d
 90 33.21a 94.91b 6.72e 1.68ab 50.24b 27.48d 5.07e
 180 33.85a 94.57bc 3.74f 1.59b 51.69a 32.31a 4.43f
Xianyu
 0 33.00a 93.25e 6.10a 1.56a 54.68a 32.52c 5.15a
 7 33.65a 93.53ed 5.88a 1.42a 47.52f 29.46d 5.07a
 15 33.53a 94.36c 5.61b 1.28ab 50.68e 26.73e 4.32b
 30 33.63a 95.28b 5.30c 1.22b 53.76b 32.75c 4.12c
 60 33.52a 95.95a 5.27c 1.17ab 52.94c 33.82b 4.11c
 90 33.21a 94.37c 5.17c 1.15ab 51.32d 29.73d 4.04c
 180 33.68a 93.59d 3.58d 0.89b 54.51a 35.25a 4.02c
SEM 0.35 0.07 0.14 0.16 0.1 0.12 0.04
Cultivar(C) means
 Jinnuo 33.57a 94.36b 7.02b 2.07a 48.39b 28.78b 4.88b
 Jintian 33.52a 94.90a 7.16a 1.85b 46.61c 28.41c 5.67a
 Xianyu 33.46a 94.33b 5.27c 1.24c 52.20a 31.47a 4.40c
Exposure (d, D) means
 0 33.39a 93.92d 7.52a 2.02a 49.87c 30.59c 5.82a
 7 33.62a 93.93d 7.22b 1.90ab 47.16f 28.08e 5.50b
 15 33.42a 94.62b 7.02c 1.78abc 46.46g 26.10g 5.19c
 30 33.49a 95.16a 6.84d 1.68bcd 47.62e 27.66f 5.02d
 60 33.67a 95.342a 6.58e 1.63bcd 51.73a 31.97b 4.80e
 90 33.23a 94.60b 6.31f 1.59cd 49.43d 29.08d 4.41f
 180 33.81a 94.15c 3.89g 1.46d 51.20b 33.38a 4.17g
Significance of main effects and interaction
 C 0.8274 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
 D 0.483 <0.0001 <0.0001 0.0017 <0.0001 <0.0001 <0.0001
 C×D 0.9683 <0.0001 <0.0001 0.9999 <0.0001 <0.0001 <0.0001

DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; WSC, water-soluble carbohydrate; SEM, standard error of the mean.

a–g Means within a column with different superscripts differ (p<0.05).

Table 5
Fermentation quality of corn stover silages at 60 d of ensiling
Exposure (d) pH Lactic acid (% of FM) Acetic acid (% of FM) Propionic acid (% of FM) Butyric acid (% of FM) Ammonia-N (g/kg of FM)
Jinnuo
 0 3.76d 1.83a 0.58c 0.08c ND 0.60f
 7 3.90d 1.07bc 0.30d 0.07c ND 0.55f
 15 4.29c 0.89c 0.97b 0.07c 0.02a 1.42e
 30 4.38bc 1.79a 0.98b 0.11c 0.02a 3.56a
 60 4.47b 1.11bc 1.50a 0.34b 0.01ab 2.95c
 90 4.35bc 1.23b 0.81b 0.15c ND 2.52d
 180 6.63a 0.94c 0.42cd 0.46a 0.01ab 3.37b
Jintian
 0 3.84d 2.53a 0.42c 0.09d 0.01ab 0.37d
 7 4.07c 2.43a 0.86b 0.11d 0.02a 0.42d
 15 4.25b 0.83d 0.42c 0.37b 0.02a 0.67c
 30 4.35b 0.58e 0.53c 0.20c ND 1.41b
 60 4.28b 1.32c 1.17a 0.53a 0.01ab 1.30b
 90 4.32b 1.52b 1.20a 0.09d 0.01ab 2.33a
 180 5.64a 0.85d 0.41c 0.51a ND 2.40a
Xianyu
 0 4.07c 1.06b 0.68bc ND ND 0.67e
 7 4.04c 1.36a 0.64bc ND ND 0.62e
 15 4.28b 0.89cd 0.55c 0.07cd 0.01ab 1.52d
 30 4.43b 0.79de 0.74ab 0.13bc ND 2.43c
 60 4.36b 0.64f 0.87a 0.30a 0.01ab 2.65b
 90 4.39b 0.95c 0.56c 0.06cd ND 2.70b
 180 6.31a 0.73ef 0.61bc 0.20b 0.02a 3.25a
SEM 0.05 0.05 0.05 0.03 0.0049 0.06
Cultivar (C) means
 Jinnuo 4.54a 1.27b 0.79a 0.18b 0.01a 2.14a
 Jintian 4.39b 1.44a 0.72b 0.27a 0.01a 1.27c
 Xianyu 4.55a 0.92c 0.66b 0.11c 0.01a 1.98a
Exposure (d, D) means
 0 3.89e 1.81a 0.56ed 0.06d 0.00b 0.55e
 7 4.00d 1.62b 0.6d 0.06d 0.01b 0.53e
 15 4.27c 0.87e 0.65d 0.17b 0.02a 1.20d
 30 4.39b 1.05d 0.75c 0.15bc 0.01b 2.47b
 60 4.37b 1.02d 1.18a 0.39a 0.01ab 2.30c
 90 4.35bc 1.23c 0.86b 0.10cd 0.00b 2.52b
 180 6.19a 0.84e 0.48e 0.39a 0.01ab 3.01a
Significance of main effects and interaction
 C <0.0001 <0.0001 0.0002 <0.0001 0.2579 <0.0001
 D <0.0001 <0.0001 <0.0001 <0.0001 0.0247 <0.0001
 C×D <0.0001 <0.0001 <0.0001 <0.0001 0.0038 <0.0001

FM, fresh matter; ND, not detection; SEM, standard error of the mean.

a–e Means within a column with different superscripts differ (p<0.05).

Table 6
Ruminal pH, gas production, total VFA content and OM-D of corn stover and silages at 60 d of ensiling
Exposure (d) Corn stover Corn stover silage


Ruminal pH GP (mL/g DM) Total VFA (mmol/L) OM-D (%) Ruminal pH GP (mL/g DM) Total VFA (mmol/L) OM-D (%)
Jinnuo
 0 6.15c 44.12a 40.47a 40.10a 6.53d 49.32a 51.21a 46.42a
 7 6.18bc 43.95a 40.05ab 39.84a 6.61cd 49.03a 50.14ab 45.34a
 15 6.19bc 44.02a 39.58ab 38.43b 6.65bc 47.22b 49.24bc 44.76ab
 30 6.23abc 42.56a 38.42b 38.15b 6.70bc 46.52bc 49.05bc 43.25bc
 60 6.25ab 40.23b 36.14c 36.43c 6.73b 46.02bcd 48.46c 43.06c
 90 6.28a 39.21b 35.46c 35.21d 6.84a 45.21cd 44.47d 43.02c
 180 6.30a 35.46c 32.10d 33.16e 6.88a 44.28d 41.10e 41.16d
Jintian
 0 6.12b 45.21a 41.25a 40.19a 6.64e 50.04a 53.86a 47.25a
 7 6.18ab 45.16a 40.56ab 39.86a 6.67de 49.61a 52.61ab 47.14a
 15 6.21ab 44.68a 40.21ab 38.43b 6.71cde 48.72ab 52.46b 47.06a
 30 6.23a 43.27b 39.72b 36.72c 6.74bcd 47.81b 50.72c 46.84a
 60 6.26a 42.81b 37.62c 34.16d 6.80bc 46.26c 48.25d 45.23b
 90 6.25a 41.53c 36.41d 33.71d 6.83b 45.87c 45.65e 43.64c
 180 6.27a 38.24d 34.71e 31.72e 6.93a 44.18d 42.19f 41.30d
Xianyu
 0 6.19a 43.51a 38.54a 39.82a 6.50e 47.26a 50.12a 46.29a
 7 6.20a 43.16ab 38.69a 38.43ab 6.56de 47.03a 49.26ab 46.11a
 15 6.21a 42.28b 36.27b 37.13bc 6.64cd 46.14ab 49.01ab 46.08a
 30 6.22a 39.84c 35.43b 35.41cd 6.71bc 45.67abc 47.83b 44.26b
 60 6.24a 39.27cd 33.76c 34.35de 6.76ab 45.24bc 45.76c 41.34c
 90 6.25a 38.46d 31.84d 32.85e 6.81ab 44.29c 42.08d 40.62c
 180 6.26a 36.73e 30.73d 30.73f 6.86a 42.16d 38.65e 38.86d
SEM 0.03 0.40 0.46 0.48 0.03 0.53 0.46 0.47
Cultivar (C) means
 Jinnuo 6.23a 41.36b 37.46b 37.33a 6.71b 46.80b 47.67b 43.86b
 Jintian 6.22a 42.99a 38.64a 36.40b 6.76a 47.50a 49.39a 45.49a
 Xianyu 6.22a 40.46c 35.04c 35.53c 6.69b 45.40c 46.10c 43.37b
Exposure (d, D) means
 0 6.15d 44.28a 40.09a 40.04a 6.56f 48.87a 51.73a 46.65a
 7 6.19cd 44.09a 39.77a 39.38a 6.61e 48.56a 50.67b 46.20a
 15 6.20bcd 43.66a 38.69b 38.00b 6.67d 47.36b 50.24b 45.97a
 30 6.23abc 41.89b 37.86c 36.76c 6.72cd 46.67bc 49.2c 44.78b
 60 6.25ab 40.77c 35.84d 34.98d 6.76c 45.84cd 47.49d 43.21c
 90 6.26a 39.73d 34.57e 33.92e 6.83b 45.12d 44.07e 42.43d
 180 6.27a 36.81e 32.51f 31.87f 6.89a 43.54e 40.65f 40.44e
Significance of main effects and interaction
 C 0.8004 <0.0001 <0.0001 <0.0001 0.0004 <0.0001 <0.0001 <0.0001
 D <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
 C×D 0.927 0.0028 0.0816 0.1884 0.7522 0.7809 0.0647 0.0004

VFA, volatile fatty acid; OM-D, in vitro OM degradability for 48h incubation; GP, gas production for 48h incubation; SEM, standard error of the mean.

a–f Means within a column with different superscripts differ (p<0.05).

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