Changes in microbial population and chemical composition of corn stover during field exposure and effects on silage fermentation and in vitro digestibility.

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:

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 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.

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

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 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.

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

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 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.

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

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 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.

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

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 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.

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

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.

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.

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

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.