Effects of Addition of Lactobacillus plantarum and Enterococcus faecium Inoculants to High-Nitrogen Fertilized Timothy (Phleum pratense L.) on Fermentation, Nutritive Value, and Feed Intake of Silage

The objective of this study was to examine the effects of addition of inoculant to high-nitrogen (N) fertilized timothy on fermentation, nutritive value, and feed intake of silage. The silage of timothy cultivated with a N fertilizer rate at two levels (high level (H), standard level (S)) was prepared with (SI, HI) or without (SC, HC) an inoculants. The CP content of H increased by 38 g·kg−1 DM compared with that of S, and the WSC and ADF contents decreased compared with those of S. Regarding the fermentation of silage, the pH and NH3-N ratio was significantly lower in the silage with inoculant (SI and HI), showing improvement of the fermentation compared with those of the silage without the addition (SC and HC). The CP content was significantly higher in HC and HI than in SC and SI, and the NDF content was the lowest in HI among the four treatments. The CP digestibility of HC and HI was significantly higher than those of SC and SI. The EE digestibility of SI was significantly higher than that of SC, and that of HI was significantly higher than that of HC. The DCP Corresponding authors.


Introduction
In Japan, roughage given to dairy cattle is mainly grass, and it is generally stored in silage.The milk yield per cow has recently increased markedly, in association with which the amount of concentrates given to them has increased to meet the nutrient intake, elevating the dependence on concentrates.Since concentrates are mostly imported, the feed cost ratio to the operating expenses can be reduced by increasing the self-supplied feed rate.
The crude protein (CP) content of temperate grass silage produced in Japan is low (8.8 -13.9 g•kg −1 DM) [1] and insufficient to play the role of a protein source, which is a factor increasing the amount of concentrates required.Methods to increase the CP content of grass silage, such as increasing nitrogen (N) fertilizer rate [2] and the ratio of legumes in mixed seeding [3], have been developed into practical techniques and their use has spread.Many research accomplishments have been achieved for N fertilize rate-based grass cultivation methods.It has been reported that, for grass cultivation in Hokkaido, fertilizer application of N at a level exceeding the standard increased the CP content of the grass but reduced the increase efficiency of the dry matter yield [4].Moreover, inappropriate and excess N fertilizer rate decreases the water-soluble carbohydrate (WSC) content of grass [5] [6], increases the nitrate N content [7] [8], increases residual N in soil [9]- [11], and induces loss of soil N [12].
On the other hand, it has been reported that the fermentation deteriorates in silage prepared with high-CP orchardgrass cultivated with high N fertilizer rate [13].Reduction of fermentation is due to increased ammonia production through degradation of CP in the fermentation process [14].Accordingly, a storage technique that does not reduce the fermentation of high-CP grass silage is required.The promotion of lactic acid fermentation is the best approach to prevent reduction of the fermentation of silage.Inoculant plays the major role in the promotion of lactic acid fermentation, and improvement of the fermentation of grass silage by adding inoculant has been reported by many researchers [15].However, only a few studies on the fermentation of silage prepared with high-CP grass have been performed.If high-N fertilizer rate increases the CP content of grass materials and the fermentation of silage is improved, the silage-derived CP intake of dairy cattle increases, with which the concentrates-derived CP intake can be decreased.
In this study, we cultivated timothy with a N fertilizer rate at two levels to prepare grass materials with two levels CP contents.Silage of these grass materials was prepared with the addition of an inoculant, and the fermentation was compared.In addition, a silage feeding trial was performed, in which sheep were fed with the silage, and the feeding value was compared with regard to the digestibility, nutritive value, and feed intake of the silage.

Grass Materials and Fertilizer Rate
For the grass material, a late variety of timothy, "Hokusyu", was used.It was seeded on July 26, 2006, in a field of Tenpoku Substation of Kamikawa Agricultural Experiment Station, Hokkaido Research Organization.The seeding density was 20 kg•ha −1 .The N fertilizer rate was 160 and 240 kg•ha −1 per year, being designated as standard (S) and high (H) level applications, respectively.The fertilizer rate distribution was two-thirds on May 7, 2007, and one-thirds on June 25, 2007.The field area for each fertilizer rate was 100 m 2 (10 m × 10 m), and three replicates each of S and H applications were randomly set.The samples of grass material in the three replicates were mixed and subjected to grass material analysis.For the N fertilization, urea was used.The total annual phosphoric acid and potassium fertilizer rate were 60 and 150 kg•ha −1 , respectively, in all treatments.For the phosphoric acid and potassium fertilizations, superphosphate of lime and potassium sulfate were used, respectively.The distribution and timing of fertilization were the same as for the N fertilization.Grass was harvested on June 22, 2007.

Silage Preparation
Silage was prepared with (SI and HI) and without (SC and HC) an inoculant.In each treatment, the unwilted grass material was cut into 1 -4 cm lengths and ensiled in 220 L FRP silos.Three replicates were prepared.For the inoculant, 11F25 (Lactobacillus plantarum and Enterococcus faecium) of Pioneer Hi-Bred Japan Co. Ltd. (Tokyo, Japan) was used.The preparation was added at 1 mg•kg −1 fresh grass, and the number of viable microorganisms was 1.0 × 10 5 cfu•g −1 fresh grass.The silage was subjected to evaluation of the fermentative quality and feed and digestion trials after storage.

Feed and Digestion Trials
Feed and digestion trials were performed using four male Corriedale cross sheep at the Tokyo University of Agriculture (Abashiri, Hokkaido, Japan).The sheep weighed 77.3 ± 9.3 kg (mean ± standard deviation).The trials were performed between August 2, 2007, and September 18, 2007.Both trials were performed by maintaining sheep in a metabolic cage capable of separating feces and urine, employing the total feces collection method.Four sheep were assigned to each treatment, and the 4 × 4 Latin-square method was employed for analysis.One trial period was comprised of a 7-day adaptation period and 5 day collection period, lasting 12 days in total.The feed and digestion trials were performed after approval by the Tokyo University of Agriculture Animal Care and Use Committee.The animals were given free access to feed, water, and mineral blocks throughout the trial period.

Chemical Analysis
Air-dried samples of the grass materials, silage, and feces were prepared by draught drying at 60˚C for 48 hours and then analyzed.Silage extract was prepared by homogenizing 50 g of fresh silage with 100 mL of distilled water.The dry matter (DM), CP, ether extract (EE), and gross energy (GE) contents of the grass materials, silage, and feces were measured using the AOAC method [16], and acid detergent fiber (ADF), neutral detergent fiber (NDF) content were measured using the method reported by Van Soest et al. [17].The water-soluble carbohydrate (WSC) and nitrate nitrogen (NO 3 -N) contents of the grass materials were measured using the methods reported by Deriaz [18] and Morimoto et al. [19], respectively.
The pH of silage was measured using a glass electrode pH meter (HM-25G; Toa DKK Co. Ltd., Tokyo, Japan), the lactic acid content was measured using the Barker-Summerson method [20], the volatile fatty acid (VFA) content was measured using gas chromatography (GC-14A; Shimadzu Co. Ltd., Kyoto, Japan) [21], and the ammonia nitrogen (NH 3 -N) content was measured using the steam distillation method [22].The V-score was calculated from the acetic acid, butyric acid contents, and the ratio of the NH 3 -N content to the total nitrogen content (NH 3 -N ratio) using the score table of a new evaluation method [23].

Statistical Analysis
All data were subjected to analysis using SAS statistical software (SAS Institute Japan Ltd., Tokyo, Japan).Twoway ANOVA was used to test the effects of the main factors (N fertilizer rate and addition of inoculant) and their interactions on fermentation, chemical composition, digestibility, nutritive value, and feed intake of silage.

Chemical Compositions of Grass Materials
The chemical compositions of the grass materials are shown in Table 1.The CP content was higher by 38 g•kg −1 DM in H (174 g•kg −1 DM) than in S (136 g•kg −1 DM), and the ADF content was lower by 37 g•kg −1 DM in H (323 g•kg −1 DM) than in S (360 g•kg −1 DM).The WSC content was lower by 22 g•kg −1 DM in H (61 g•kg −1 DM) than in S (83 g•kg −1 DM).In WSC, the glucose and fructose contents were high and the sucrose content was low in both grass materials.The NO 3 -N content of H was 2.2 g•kg −1 DM, being higher than that of S (1.6 g•kg −1 DM).
No significant difference was noted in the DM content among the four treatments, and it ranged from 134 to 138 g•kg −1 .No significant difference was noted in the CP content between SC (138 g•kg −1 DM) and SI (134 g•kg −1 DM) and between HC (171 g•kg −1 DM) and HI (165 g•kg −1 DM).The CP contents of HC and HI were significantly higher than those of SC and SI (P < 0.01).The NDF content was lowest in HI (564 g•kg −1 DM) among the treatments (P < 0.05).No significant difference was noted in the EE, WSC, and GE content among the treatments.

Digestibility, Nutritive Value, and Feed Intake of Silage
The digestibility, nutritive value, and feed intake of silage are shown in Table 3.The DM and OM digestibility of HI were the highest (0.721 and 0.723, respectively) among the four treatments (P < 0.05).The CP digestibility of HC (0.763) and HI (0.821) were higher than those of SC (0.722) and SI (0.729) (P < 0.01).The EE digestibility of SI (0.743) was significantly higher than that of SC (0.668) (P < 0.05), and that of HI (0.737) was significantly higher than that of HC (0.704) (P < 0.05).The ADF and NDF digestibility of HI were high and those of SC were low.The DCP contents of HC (130.1 g•kg −1 DM) and HI (135.1 g•kg −1 DM) were significantly higher than those of SC (99.6 g•kg −1 DM) and SI (97.4 g•kg −1 DM) (P < 0.01).No significant difference was noted between HC and HI.The TDN content of SC (618 g•kg −1 DM) was the lowest (P < 0.05), and that of HI (709 g•kg −1 DM) was the highest (P < 0.05).The DE content of SC (13.3 MJ•kg −1 DM) was the lowest and that of HI (15.1 MJ•kg −1 DM) tended to be high.No significant difference was noted in the DM, TDN, and DE intake among the four treatments, but that in the HI-fed treatment was the highest.The DCP intakes of HC (4.3 g•kg −1 DM) and HI (4.8 g•kg −1 DM) were significantly higher than those of SC (3.5 g•kg −1 DM) and SI (3.2 g•kg −1 DM) (P < 0.01).

Discussion
The CP content of grass material H was high, as previously reported [8] [24], suggesting that high-N fertilization promoted N metabolism of the plant, and protein was synthesized from absorbed N and carbohydrate produced by photosynthesis [25].The low WSC content of grass material H may have been due to promotion of vegetative growth by high-N fertilization, which increased the yield consuming sugars [14] [26].The glucose, fructose, and sucrose contents were not markedly different between grass materials S and H, suggesting that the N fertilizer rate did not have a marked influence on mono-and disaccharides.Accordingly, the decrease in the WSC content of the grass materials was assumed to be due to a decrease in the fructosan content, which is a polysaccharide [26].
The pH of HC was 4.2 or higher and the NH 3 -N ratio was 100 g•kg −1 DM or higher, showing poor fermentation.The fermentation of SC was also poor, being only slightly different from that of HC.It is unlikely that only the high CP content was involved in the poor fermentation of HC.The fermentation of silage of high-N-fertilized grass was described as being poor in many reports [13] [27] [28].The fermentation depends on pH elevation, reduction of the lactic acid content, and an increase in the NH 3 -N content, and the causes of these include high CP content of grass materials and degradation products of fermentation [29] [30] and low WSC content of grass materials [31].On the basis of the findings of our study, there may be another factor influencing fermentation other than high CP content and low WSC content.
Lactic acid fermentation was promoted and the NH 3 -N ratio decreased in HI compared with those in HC, which improved the fermentation.The addition of an inoculant improved the fermentation of grass silage in many studies [32]- [35].A marked improvement was also noted in SI, strongly suggesting that the influence of the inoculant was exhibited because the number of lactic acid bacteria adhering to the grass material was markedly low.A marked effect of inoculant was frequently noted when the WSC content of the grass material was 50 -60 g•kg −1 DM or higher [36] [37], and this may be another factor causing the effect.
The CP content differed by 33 g•kg −1 DM between SC and HC, and the CP digestibility of HC tended to be higher.Regarding the chemical composition and digestibility of high-N-fertilized silage, Izumi et al. [27] reported that, when the N fertilizer rate was set to the range of 30 -90 kg•ha −1 , the CP content of timothy silage increased from 143 to 173 g•kg −1 DM with an increase in the N fertilizer rate, and the CP digestibility improved from 0.681 to 0.768.Poulton & Woelfelalso [2] reported similar findings, suggesting that changes in the CP digestibility with an increase in N fertilizer rate are influenced by the CP content.In contrast, the CP digestibility of HI was not significantly different from that of HC, and this was similar to findings of the addition of inoculant to timothy silage reported by Souma et al. [38], alfalfa silage reported by Cao et al. [39], and corn silage reported by Aksu et al. [40], showing that inoculant has only a small influence on CP digestibility.
The DCP content and DCP intake of HC were higher than those of SC, and the DCP intake of the sheep increased upon feeding on HC.However, the differences in the TDN and DE contents, and DM, TDN and DE intakes were small.Regarding the DCP and TDN contents, and DM, DCP and TDN intakes of high-N-fertilized silage, Poulton & Woelfel [2] and Izumi et al. [27] [41] reported that the DCP content and the DCP intake of silage increased with an increase in the N fertilizer rate, but no influence was noted on the TDN content and TDN intake, and Ataku & Narasaki [42] reported that an increase in the N fertilizer rate did not influence the DM and TDN intakes of the silage, similarly to our findings.However, the TDN content increased in HI compared with that in HC, showing the effect of the inoculant.The improvement of CP and EE digestibility may have been associated with the increase in TDN content.No difference was noted in the DM and TDN intake between HI and HC, which may have been due to the absence of an increase in the DM intake of HI, not leading to an increase in the TDN intake, although the TDN content increased.Regarding the DM intake of silage with the addition of inoculant, Winters et al. [43] reported an increase in the intake of Italian ryegrass silage, whereas no increases in the intake of perennial ryegrass, alfalfa, and mixed-seeded grass and legume silage were reported by Patterson et al. [44], Kung et al. [45], and Stokes [46], respectively, showing no consistent tendency.Since the DCP and TDN contents, and DCP intake of HI were higher than those of SC and SI, silage with the high DCP and TDN contents, and DCP intake may have been prepared by the addition of inoculant to the high-CP grass material.

Conclusion
The high-N fertilizer rate increased the CP content of the grass material by 38 g•kg −1 DM, and the CP content of silage (HC and HI) also increased.The CP digestibility was high in sheep fed HC and HI, and the DCP content and DCP intake increased.The addition of the inoculant improved the silage fermentation and increased the TDN content.On the basis of these findings, silage with high CP content could be prepared by increasing the N fertilizer rate, and high fermentation could be achieved by the addition of the inoculant, which increased the DCP intake of sheep.

Table 1 .
Chemical composition of grass materials.

Table 2 .
Fermentation quality and chemical composition of silages.

Table 3 .
Digestibility, nutritive value and feed intake of silages.