Changes in fermentation pattern and quality of Italian ryegrass (Lolium multiflorum Lam.) silage by wilting and inoculant treatments

Objective This study was conducted to investigate the effects of wilting and microbial inoculant treatment on the fermentation pattern and quality of Italian ryegrass silage. Methods Italian ryegrass was harvested at heading stage and ensiled into vinyl bags (20 cm×30 cm) for 60d. Italian ryegrass was ensiled with 4 treatments (NWNA, no-wilting no-additive; NWA, no-wilting with additive; WNA, wilting no-additive; WA, wilting with additive) in 3 replications, wilting time was 5 hours and additives were treated with 106 cfu/g of Lactobacillus plantarum. The silages samples were collected at 1, 2, 3, 5, 10, 20, 30, 45, and 60 days after ensiling and analyzed for the ensiling quality and characteristics of fermentation patterns. Results Wilting treatment resulted in lower crude protein and in vitro dry matter digestibility and there were no significant differences in acid detergent fiber (ADF), total digestible nutrient (TDN), water-soluble carbohydrate (WSC), ammonia content, and pH (p>0.05). However, wilting treatment resulted in higher ADF and neutral detergent fiber content of Italian ryegrass silage (p<0.05), and the WNA treatment showed the lowest TDN and in vitro dry matter digestibility. The pH of the silage was higher in the wilting group (WNA and WA) and lower in the additive treatment group. Meanwhile, the decrease in pH occurred sharply between the 3–5th day of storage. The ammonia nitrogen content was significantly lower in the additive treatment (p<0.05), and wilting had no effect. As fermentation progressed, the lactic and acetic acid contents were increased and showed the highest content at 30 days of storage. Conclusion The wilting treatment did not significantly improve the silage fermentation, but the inoculant treatment improved the fermentation patterns and quality of the silage. So, inoculation before ensiling is recommended when preparing high quality of Italian ryegrass silage, and when wilting, it is recommended to combine inoculation for making high quality silage.


INTRODUCTION
Two types of ryegrass, perennial and annual, are being cultivated in worldwide. Italian ryegrass (IRG, Lolium multiflorum Lam.) is an herbaceous annual or biennial grass that is grown for silage, and as a cover crop. In the United States, Lolium multiflorum is sometimes used as a winter cover crop to prevent erosion, build soil structure, and suppress weeds. It grows on about 1 million ha in the humid, southern United States, being used primarily for winter pasture in clear seeding and in dormant bermudagrass sods [1].
Korea has four distinct seasons, with crop cultivation during winter being extremely limited. Also, cultivation using paddy fields is dominant because the forage production bases are weak [2]. IRG is one of the wintering crops that is grown after rice cultivation. About 135 thousand ha of IRG were cultivated in 2015 and being approximately 52% of to tal forage cultivation areas [3]. IRG is one of the fastest growing grasses available to farmers. It is widely distributed throughout temperate and tropical or subtropical regions of the world and is one of China's major forage crop used either fresh greenchop, hay, or silage [4].
Unfortunately, rains come often at the proper harvest time of the Italian ryegrass in Korea (early May to mid May), which restrict the storage methods. Most farmers stored IRG in the form of silage, and some farmers are trying to store as hay. Produced silage (round bale) is wrapped in plastic vinyl and distributed throughout the country [2]. However, due to the lack of silage preparation technology, the quality of the sold silage is uneven and there is a distrust between producers and consumers.
Ensiling forage crop is well known method of conserva tion for a shortage season. Lactic acid bacteria (LAB) convert water soluble carbohydrates (WSC) under anaerobic condi tions into lactic acid. As a result of the pH decline, the silage is well preserved. Acidification of well fermented silage in hibits undesirable microorganisms. During the fermentation process, competition takes place between LAB and undesir able microorganism, and fermentation quality always depend on the result of the competition [5].
Dry matter (DM) content of raw materials has a great in fluence on silage fermentation, which affects all fermentation characteristics, pH level and quality parameter of silage. En siling with low DM content around 25% could cause inferior fermentation and high pH level deducing serious DM loss, compared with higher DM content. At less than 300 g/DM kg may also generate an increase in seepage loss and expe dite clostridial fermentation reducing voluntary intake [6]. But wilting reduces the amount of fermentable carbohydrate required to properly preserve the silage and restricts the growth of undesirable microorganism.
The application of silage additives is normally recommend ed to ensure and improve silage fermentation. At present, LAB inoculants are the main additives in many parts of the world [7]. LAB additives usually increase the rate of lactic acid production, thereby accelerating the pH decline and reduc ing postharvest proteolysis. In addition, rapid acidification results in the inhibition of detrimental microorganisms [8].
Generally, wilting results in lower WSC content, extensive protein breakdown and sometime higher total volatile fatty acid (VFA) during ensiling. Wilting also affects the chemical composition, DM losses, silage fermentation and animal per formance [9].
This study evaluated the effect of wilting and inoculant on fermentation dynamics and qualities of IRG silage.

Silage preparation
Italian ryegrass (IRG) was cultivated in an experimental field of Pyeongchang campus, Seoul National University (37.32°N, 128.26°E, 550 m ASL). "Kogreen" variety, developed by the National Institute of Animal Science, was seeded on about 1 ha area on September 27, 2016 at a seeding rate of 40 kg/ha. At seeding date, 40 kg/ha of nitrogen, 150 kg/ha of phosphate, and 75 kg/ha of potassium were applied as fertilizer. An ad ditional 100 kg/ha of nitrogen and 75 kg/ha of potassium fertilizer were applied in early March 2017.
IRG was harvested at 16 May 2017 using mower condi tioner (Novacat 301, Pöttinger, Harvest width 3.04) and chopped into about 2 to 3 cm pieces using a forage cutter (Richi Machinery Co., Ltd, Henan, China). The ryegrass harvested for wilting treatment was dried in the field for 5 hours. After manual mixing, chopped IRG was treated with silage inoculant ("ChungmiLacto", Chungmi Co., Lactoba cillus plantarum). Recommended level (10 6 cfu/g fresh matter) of inoculant was dissolved in tap water (1 L per 1 g of inocu lant) and sprayed (Air spray gun, Newstar Co., China) into mixed samples. Thereafter, approximately 600 g treated ma terial was packed into vinyl bag (28 cm×36 cm, Korea), air was taken out, sealed (vacuum sealer, Zhejiang Hongzhan Packing Machinery Co., Ltd, China) and stored at the ambient temperature (22°C to 28°C) in the shade.

Laboratory analysis
Three vinyl bag silos per treatment were randomly opened on 1, 2, 3, 5, 10, 20, 30, 45, and 60 days after ensiling, respec tively. Two subsamples per vinyl bag silo were retained for further analysis. One subsample (about 300 g) was dried at 65°C in a forcedair drying oven for 72 h and then used to determine DM content and other chemical compositions, including acid detergent fiber (ADF), neutral detergent fiber (NDF), crude protein (CP), in vitro dry matter digestibility (IVDMD), and WSC. Another subsample (about 300 g) was stored at -80°C in a refrigerator and subsequently used for sequential determination of silage acidity (pH), organic ac ids, microorganisms, and ammonia nitrogen.
Dried samples were ground to pass through a 1mm screen and kept in doubleplug type plastic bottles for analysis. The CP was determined using the Kjeldahl method [10]; ADF and NDF were measured following the method of Goering and Van Soest [11] using an Ankom200 Fiber Analyzer (Ankom Technology, Macedon, NY, USA). We determined the IVDMD of the IRG silage using the twostage technique described by Tilley and Terry [12] for a period of 72 h using Ankom II Daisy Incubators (Ankom Technology, USA). The total digestible nutrient (TDN) content was estimated as TDN (%) = 88.9-(0.79×ADF) following the method of Holland et al [13].
A frozen 10 g sample of each silage (three replications per treatment) was macerated with 90 mL of distilled water for 30 min. in a shaker and filtered, and the filtrates were used to measure pH with a pH meter (HI 9024; Hanna Instruments Ltd., Leighton Buzzard, UK). 10 g sample of each silage was macerated with 90 mL of distilled water for 24 h and filtered through filter paper (#6). The filtrates were analyzed for VFA and lactic acid contents. The VFAs were analyzed by Kim et al [14] using gas chromatography (Model 3400; Varian Co., Harbor City, CA, USA), and lactic acid was analyzed by high performance liquid chromatography (HP1100; Hewlett Packard Co., Palo Alto, CA, USA). WSC was determined using the anthrone method of Thomas [15] and NH 3 N con centrations were analyzed by the method of Chaney and Marbach [16] using a spectrophotometer (UVIDEC610; Jasco Co., Tokyo, Japan).

Statistical analysis
Data on fermentation dynamics and chemical composition were subjected to twoway analysis of variance with the fixed effects of treatments, ensiling days and interaction (treatment ×ensiling days) using the general linear model procedure of SAS ver 9.1 (SAS Institute, Cary, NC, USA) [17]. Least signifi cant difference tests were used to determine specific differences among means. The level of statistical significance was p<0.05.

RESULTS
The chemical composition, pH, WSC, and ammonia nitro gen contents of preensiled IRG are given in Table 1. The CP and IVDMD contents of wilted materials were lower than those of no wilted (p<0.05). The NDF content of NWA treat ment was lower than WNA treatment (p<0.05). There was no significant difference in ADF, TDN, WSC, ammonia ni trogen content and pH among treatments (p>0.05).
As presented in Table 2, inoculant increased the CP con tent of IRG silage, but there was no significant difference (p> 0.05) and wilting increased the NDF content. This result seems to be due to proteolytic degradation of raw materials with wilting. TDN and IVDMD of WNA treated silage was the lowest among treatments (p<0.05). Table 3 presents the results for silage fermentation charac teristics. The DM content of wilted silages were significantly higher than those of nowilted (p<0.05), but inoculation had no effect on DM. The pH of silage was significantly lowered by inoculation and the pH of wilted silage was higher (4.12 and 4.66). Inoculation increased the lactic acid content of  both wilted and nonwilted silages but, wilting lowered the lactic acid content of IRG silage. Acetic and butyric acid con tents were lower in the silages treated with both additive and wilting and the lowest in NWA silage. The residual contents of WSC after the silage fermentation were significantly higher (60.4 and 57.8 g/kg) in the additive treatment (p<0.05). The ammonia nitrogen content was significantly higher in the noninoculated silage (p<0.05). Table 4 shows the change in forage quality from 1 to 45 days after silage preservation. The CP content was increased in NWA and WA treated silages during fermentation periods and there was not a significant difference between inoculated silages (NWNA and WNA silage). But CP content was slightly increased in inoculated silage during fermentation. There was significant difference among treatment and number of fermentation days. NWNA silages were significantly higher in each elapsed day and lower in WNA and WA silage. In addition, CP in IVDMD was not different at the early stage of fermentation but decreased after 30 days. However, there was no significant difference in NWNA treatment. The ADF and NDF contents tended to increase with fermentation and wilted silages were higher. However, there was no significant difference in the ADF and NDF content among fermenta tion periods of NWNA silage (p>0.05). On the other hand,  the TDN content, estimated as ADF content, of NWNA silage showed no significant difference according to the fermenta tion period. Figure 1 shows the DM content, pH, WSC and ammonia nitrogen content of the silage. The DM content increased continuously as fermentation proceeded but decreased slightly after 20 days. Wilted silages showed the higher DM content among treatments. The pH of silages was significantly de creased from 3 to 5 days after fermentation, and the inoculated silages were significantly lower (p<0.05). In addition, wilting treatment generally delayed the pH decrement. WSC con tent increased until the 2nd day of storage and then decreased sharply again, while WNA silage was highest at 3 days and decreased. Overall, the WSC content of 45th days' silage was significantly lower in no inoculated silage (p<0.05). Change in ammonia nitrogen content was significantly lower in the additive treated silage (p<0.05) and tended to increase dur ing the fermentation periods. In particular, the silage treated with additive remained near 50 g/kg, but the silage without additive continued to increase. Table 5 shows the changes of organic acid content during silage fermentation. Lactic acid content increased with fer mentation and decreased at 30 days after conservation at the highest level in all treatments. In addition, lactic acid content was significantly increased by inoculant treatment and highest in NWA silage (p<0.05). Acetic acid content increased for all treatments and then decreased after day 30 but for WA it kept on increasing. Butyric acid content was not detected in the inoculant treated silage or slightly differentiated by 45 days. In NWNA silage, the butyric acid content was detected after 10 days and the content was continuously increased. On the other hand, the lactic/acetic acid content ratio was signifi cantly higher in the additive treated silage, indicating that homo type fermentation was dominant.

Analysis of fresh Italian ryegrass
Wilting has a big impact on the fermentation pattern of si lage. In this experiment, wilting decreased the CP, NDF, and IVDMD content of raw materials. Kim et al [2] reported that IVDMD decreased with increasing wilting period in rye si lage, but increased fiber content. Fitzgerald [18] observed that wilting decreased CP content, but Cottyn et al [19] re ported that there was no significant difference.
On the other hand, the WSC content of the material was  not significantly different among treatments, and the aver age level was 153.4 g/kg. Parker [20] stated that the minimum WSC content required for silage fermentation should be 25 to 30 g/kg and the wilted silage should be higher than 38 g/kg. In this experiment, the WSC levels were high. McDonald et al [6] observed that the WSC content varies depending on the species, growth, daily time, light intensity, temperature, and fertilization level and reported that the range recorded in the major five grasses was between 5 to 315 g/kg.

Forage quality of Italian ryegrass silage
After 60 days of fermentation, the CP content of IRG silage was low in the wilting group, and the inoculant treatment increased CP content. This was judged to be the result of lowering protein degradation by improving fermentation pattern. Kennedy [21] also reported that CP content was significantly higher in the LAB treatment.
On the other hand, ADF and NDF contents were higher and IVDMD contents were lower in WNA treatment silage. The wilting treatment increased the NDF content of the raw material, which is believed to have increased since the high level was maintained even after fermentation.
Comparing the IVDMD before and after silage prepara tion, the IVDMD of silage was lowered (763.4 vs 735.0 g/kg). Kim et al [2] also showed that the IVDMD of rye silage was lowered by the silage fermentation, which is consistent with this test. Wilkins [22] reported that digestibility tended to decrease by wilting.
After 60 days of storage, the DM content of IRG silage tend ed to increase in wilted silage but the addition of the inoculant had no significant effect on DM. Keady and Murphy [23] also reported that LAB additives did not affect the dry mat ter content of silage.

Silage quality analysis
The most important changes in silage quality was in pH.
Wilting resulted in higher silage pH and inoculant was lower.
Many studies have reported that LAB treatment lowers the final pH of silage. Wilting increased final pH of silage by increasing the DM content of the raw material. Wilting has negative effect on silage acidity due to increasing DM con tent and restricted fermentation [24]. The inoculant treatment increased the lactic acid content of the silage, but the wilting treatment resulted in a decrease. This is the result of limited fermentation due to the decrease in moisture content. Kim et al [2] also found that lactic acid content decreased with prolonged wilting period in rye si lage. On the other hand, acetic acid and butyric acid contents were decreased by treatment with inoculant. In general, it was reported that LAB additives increased lactic acid and decreased butyric acid contents [25].
The NH 3 N/total nitrogen (TN) ratio, which indicates the degree of proteolytic degradation, was reduced by LAB treat ment, and according to Haigh [25], when the NH 3 N content was less than 10% of the TN, it was classified as high quality silage. So, the silage of this experiment can be classified as good quality. Sharp et al [26] also found that the NH 3 N con tent produced by proteolysis is reduced by the treatment of LAB additives. According to the report of Dawson et al [27], wilting grass before ensiling increased silage pH and ammonia nitrogen concentration, results that agree with many previous studies [21]. The concentration of many of the fermentation products in the silage were also reduced because of wilting, indicating a more restricted fermentation in the wilted silage.

Analysis of fermentation pattern
The contents of CP, IVDMD, ADF, and NDF in IRG silage showed a significant difference with wilting, LAB treatment and number of fermentation days. The LAB treatment did not show any difference in CP content during fermentation, but CP showed a tendency to increase slightly in control (NWNA). IVDMD of silage showed a tendency to decrease with fermentation, but there was no significant difference in NWNA treatments. The contents of ADF and NDF increased with the fermentation progress, but there was no significant difference in NWNA treatment. Keady and Murphy [23] re ported that LAB treatment reduced ADF and NDF content of silage but was not significant. But Patterson et al [9] showed a tendency to increase in ADF and NDF content. During silage fermentation, the pH changes gradually de creased over time and significantly decreased from 3rd day. However, in the WNA treatment, the pH decrease sharply occurred from slightly late 5th day. However, Zhao et al [28] reported that the pH change was stabilized after the sharpest decrease by day 3 in analysis of rice straw silage fermentation pattern.
Wilting increased ammonia nitrogen content. Derbyshire et al [29] and Haigh [25] also reported that ammonia nitro gen of silage increased by wilting. LAB treatment promoted the production of lactic acid in silage and reduced the pro duction of acetic acid and butyric acid. On the other hand, lactic acid/acetic acid ratio showed a tendency to increase by the treatment of LAB additive, which shows that Homotype fermentation was predominant.

CONCLUSION
Wilting resulted in lower CP and IVDMD, but no significant differences in ADF, TDN, WSC, ammonia content and pH (p>0.05) of raw materials. However, wilting treatment re sulted in higher ADF and NDF content of IRG silage (p<0.05). The pH of the silage was higher in the wilting group (WNA and WA) and lower in the additive treatment group. The decrement in pH occurred sharply on the 3th to 5th day of storage. The ammonia nitrogen content was lower in the ad ditive treatment (p<0.05). As fermentation progressed, the lactic and acetic acid contents increased and showed their highest content at 30 days of storage. In conclusion, the wilt ing did not significantly improve the silage fermentation, but the inoculant treatment improves the quality of the si lage. So, inoculation before ensiling is recommended when preparing high quality of IRG silage, and when wilting, it was recommended to combine inoculation to make high quality silage.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.