Effect of an inoculant and enzymes on fermentation quality and nutritive value of erect milkvetch ( Astragalus adsurgens Pall . ) silages

The objective of this experiment was to determine the ensiling characteristics, microbial changes and in vitro degradation of erect milkvetch (Astragalus adsurgens Pall.) treated with different additives. Erect milkvetch was treated with distilled water (control), inoculant (I, lactic acid bacteria), enzymes (E, fibrolytic enzymes) and inoculant + enzymes (I + E) prior to ensiling. Three bag silos were used and opened after 1, 3, 5, 15, 30 and 45 days for chemical analysis and microbial measurement. For all the silages, there was a rapid decline in pH during the first 5 days of ensiling. Compared with the control, all added treatments (I, E and I + E) resulted in higher (P<0.05) lactic acid concentration at all ensiling periods. The crude protein content was higher, and neutral detergent fibre, acid detergent fibre and sulphuric acid lignin were lower for all treatments compared with the control (P<0.05). Treatments of enzymes (E, I + E) can also significantly improve in vitro dry matter digestibility by 10.9 and 13.6% and in vitro neutral detergent fibre digestibility by 19.8 and 21.7%, respectively. Compared with the control, treatments I and I + E increased in vitro crude protein digestibility (P<0.05). These results indicated that the addition of additives can improve both erect milkvetch silage fermentation quality and in vitro digestibility to some extent.


INTRODUCTION
Erect milkvetch (Astragalus adsurgens Pall.) is used in China as forage and is widely cultivated in diverse environments in arid and semiarid areas of northern China.The planting area has reached 0.6 million hectare (Bai et al., 2009a).In spite of its extensive adaptability, erect milkvetch contains approximately 14 to 17% crude protein (CP), 40% neutral detergent fibre and 2% ether extract on dry matter (DM) basis, respectively (Liu et al., 2000;Bai et al., 2009b).Thus, it is an alternative feed source for animals.
Currently, haymaking is a major conservation method for erect milkvetch.However, this method needs to delay harvest until a mature stage of relatively high DM content is reached, resulting in a reduced digestibility.This fact coupled with an uncertainty of the weather during haymaking often led to a product of variable composition, and usually of low nutritional value (McDonald et al., 1991).Consequently, ensiling may be an appropriate method to preserve the high nutritive value of erect milkvetch.Ensiling is a preservation method that is based on lactic acid bacteria (LAB) converting water-soluble carbohydrates (WSC) into organic acids, mainly lactic acid, under anaerobic conditions.As a result, pH decreases and increase in the concentration of non-dissociated organic acid inhibits the microorganism that causes silage spoilage (McDonald et al., 1991).However, erect milkvetch contains a small amount of WSC and has a high buffering capacity that make it difficult to be ensiled (Bai et al., 2009b).Many studies have indicated that inclusion of inoculants and enzymes can significantly improve fermentation quality of silage (Guan et al., 2002; Hassanat et al., 2007; Xu  et al., 2008; Schmidt et al., 2009).
However, the literature contains little information on the chemical composition and silage fermentation quality of erect milkvetch.Liu et al. (2000) reported the chemical composition of erect milkvetch during different phenophases and Bai et al. (2009b) studied the effect of addition of formic acid and sucrose on fermentation quality, but there is no published study on the nutritive value of erect milkvetch silage.The objective of this study was to assess the effect of inclusion of inoculant, enzymes and their mixture during ensiling on the fermentation quality and in vitro degradation of silage from erect milkvetch forage.

Forage, silage preparation and treatment
Erect milkvetch (Astragalus adsurgens Pall.) for ensiling was harvested at a bud stage from experimental plots at the China Agricultural University, Beijing, in August with a domestic cutter.Triplicate forage samples were taken (200 g each) immediately after harvest and dried by heated air for dry matter (DM) and other chemical analyses.The rest of the harvested forage was chopped into approximately 1 to 2 cm theoretical lengths for ensiling experiment.The chopped erect milkvetch was manually mixed and then divided into four equal portions to subject the following treatments: 1. distilled water (Control); 2. inoculant (LaLsIL Dry, Medipharm, Des Moines, USA) containing Lactobacillus plantarum > 6×10 10 colony forming units (CFU) per gram applied at a rate of 0.01 g/kg of fresh forage (I); 3. fibrolytic enzymes (Acremonium cellulose, Snow Brand Seed Ltd., Sapporo, Japan) applied at a rate of 0.033 g/kg of fresh forage (E); and 4. combination of I and E (I+E).The application rates for I and E followed the recommendations of the manufacturers.Inoculant, E or I+E was first mixed with a certain amount of water and solution was then sprayed on the corresponding chopped forage designated for each treatment using a single nosal sprayer.The silage control was added with the same amount of distilled water as the other three treatments.
Each of the above treated forage was mixed well with hand and then was packed into plastic film bags (Hiryu KN type, 200 × 300 mm; Asahikasei, Tokyo, Japan) with 200 g treated material per bag, which were sealed with a vacuum sealer (BH950, Matsushita, Tokyo, Japan) and stored at ambient temperature (20 to 25°C).According to the experiment design, 15 bag silos were prepared for each treatment and three bag silos per treatment were randomly opened on day 1, 3, 5, 15 and 45 of ensiling and the content was processed for quality assessment and laboratory analysis.

Chemical analysis
Fresh forage.Dried forage harvested for ensiling was ground to pass 1 mm screen with Wiley mill for compositional analysis.The DM and crude protein (CP) were analysed according to methods 934.01 and 990.03, respectively, of AOAC (1990).The neutral detergent fibre was determined with a heat stable amylase and expressed exclusive of residual ash (aNDF; Van Soest et al., 1991), and acid detergent fibre exclusive of residual ash (ADF; Robertson and Van Soest, 1981).The sulphuric acid lignin (Lignin (sa)) was determined by washing samples with 20N H 2 SO 4 for 3 h (AOAC, 1990;method 973.18).The water-soluble carbohydrates (WSC) were estimated by the method of McDonald and Henderson (1964).Glucose, sucrose and fructose were determined by HPLC (Shimadzu, Tokyo, Japan).The analytical conditions were as follows: column, Shodex Sugar SC1011 (8.0 mm × 30 cm, Shoko, Tokyo, Japan); oven temperature, 80°C; mobile phase, water; detector, 1.0 ml/min; detector, Jasco RI-1530.Buffering capacity of erect milkvetch was measured by titration (Playne and McDonald, 1966) using the dried sample.
Silages.Ten grams of silage from each bag at opening was homogenized at room temperature (20-25°C) with 90 ml of sterilized distilled water for 30 pulses of 2 s, and then filtered through four layers of cheesecloth.The pH of the filtrate was measured using a glass electrode pH meter (pHS-3C, Shanghai, China).The organic acid contents were determined by HPLC (Xu et al., 2007).Ammonia-N (NH 3 -N) was determined by steam distillation of the filtrates (Xu et al., 2003) and non-protein nitrogen (NPN) by the Kjeldahl method.
The remaining of erect milkvetch silage in each bag was dried in an oven at 65°C for 48 h, ground to pass a 1 mm screen with a Wiley mill and analysed for DM, CP, aNDF, ADF, Lignin (sa) and WSC using the procedures as described above.

Microbial measurement
The numbers of microbes were counted from the plate count method (Cai et al., 1998).Colonies were counted from the plates at appropriate dilutions and the number of colony forming units (CFU) was expressed per gram of fresh forage.

In vitro digestibility determination
In vitro digestibilities of DM (IVDMD), aNDF (IVNDFD) and CP (IVCPD) were measured according to Tilley and Terry (1963).This is a two-stage in vitro technique in which ground silage samples were digested by mixed rumen microbes for 48 h, followed by hydrolysis with a pepsin-HCl solution (200 mg of pepsin in 2 l of 0.004 mol/l HCl, pH 2.4) for another 48 h.Two ruminally cannulated Holstein cows fed a maintenance energy diet (NRC, 1996) of grass silage without concentrates were used as rumen fluid donor for the in vitro digestibility determination.

Statistical analysis
Analysis of variance was used to test statistical significance of additives, time of ensiling, and the additive × time interaction using the PROC MIXED procedure of SAS (1990) with the model: where: Yij -the dependent variable for additive i and for time j; m -overall mean; Ai -additive effect, i -1, 2, 3, 4 for erect milkvetch silages; Tj -time effect, j -1, 2, 3, 4, 5 for erect milkvetch silages; ATij -additive × time interaction effect; Eij -residual error.
When the F test indicated a significant (i.e.P<0.05) additive effect, means separations were conducted using a least significant difference test.Polynomial contrasts were used to test linear, quadratic and cubic effects of increasing fermentation time.A probability of P<0.05 was used to denote significance unless otherwise indicated.Parameters were plotted when additive × time interactions were significant (P<0.05) to aid in interpretation of results.

Chemical compositions of erect milkvetch
Chemical compositions of fresh erect milkvetch that was harvested for ensiling are present in Table 1.The fresh erect milkvetch had low levels of WSC, glucose, fructose and sucrose, and had a high buffering capacity.

Fermentation quality of silage
Silage pH was affected (P<0.01) by additive, ensiling time and there was an additive × ensiling time interaction (Table 2).Application of I, E and I + E reduced the pH of the silages with the treated silages having a sustained lower pH than control throughout the ensiling period.The pH of treated silages dropped more rapidly compared to that of control at the beginning of ensiling (P<0.01),but all silages were stable after about 5 days, and silages treated with I, E and I + E had lower (P<0.05)pH than control (Figure 1).
All silages treated with I, E and I + E, regardless of the added quantity, were well preserved, with propionic acid, butyric acid and NH 3 -N concentrations being lower (P<0.01),and production of lactic acid higher (P<0.01)than those of the control silage (Table 2).Lactic acid (Figure 1), volatile fatty acid, NH 3 -N and NPN (Table 2) concentrations of treated and control silages increased (P<0.01) between day 1 to day 45 of ensiling.The WSC content in all silages decreased (P<0.01)from 4.73% DM in fresh forage to 0.68 to 0.84% DM by the end of ensiling.

Changes in microbial population during the ensiling period
As shown in Table 3, the number of LAB was affected (P<0.01) by additives and ensiling time with a significant additive × ensiling time interaction.Of all ensiling periods, the application of I, E and I + E significantly increased the number of LAB, especially for treatments I and I+E.For all treatments, the population of LAB increased numerically from days 1 to 5 post-ensiling and then decreased to day 45 post-ensiling.There were no differences in the number of yeast among the treatments.Of all the treatments, yeast tended to have lower numbers for day 5 post-ensiling.No mould was detected in the silages except for the control at day 45 post-ensiling.

Chemical composition and in vitro digestibility of the 45-day silages
Chemical composition of 45-day erect milkvetch silages was presented in Table 4. Similar DM content was seen in all silages ranging from 25.7 to 26.0%.However, CP content was higher, and aNDF, ADF and Lignin (sa) were lower for all treatments as compared with control (P<0.05).Treatments of enzymes (E, I + E) can significantly improve IVDMD by 10.9 and 13.6% and IVNDFD by 19.8 and 21.7%, respectively.In contrast, the treatment I had no effect on IVNDFD (P<0.05).Compared with control silage, treatments I and I + E increased IVCPD (P<0.05),however, treatment E had no effect on IVCPD (P<0.05).844 a 837 a 20.0 <0.001 1 aNDF -neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; ADF -acid detergent fibre expressed exclusive of residual ash; Lignin (sa) -sulphuric acid lignin; WSC -water soluble carbohydrates; IVDMD -in vitro dry matter digestibility; IVNDFDin vitro neutral detergent fibre digestibility; IVCPD -in vitro crude protein digestibility 2 E -enzymes; I -inoculant; I + E -inoculant + enzymes 3 SEM -standard error of the mean; n=3 a-d means in the same row with different superscripts differ (P<0.05)

DISCUSSION
The WSC content (47.3 g/kg DM) of the pre-ensiled erect milkvetch was lower than the theoretical requirement number (60 to 70 g/kg DM) recommended for achieving well-preserved fermentation (Lunden Petterson and Lindgren, 1990), while the buffering capacity (507 meq kg -1 DM) was higher than that of typical forage grass (Woolford, 1984).Thus the erect milkvetch used was typical of legumes with a high buffering capacity and a low WSC content.Therefore, there is a need for better understanding of ensiling characteristics of erect milkvetch so that current technologies can be efficiently applied to the legume ensiling.
Silage pH is one of main criteria reflecting the extent of fermentation and quality of ensiled forages.As shown in Figure 1, for all treated silages, the pH decreased rapidly to below 5.0 during the first 5 days of fermentation except for treatment E that was about 4.3 to 4.8 during the last period of fermentation.However, it was found that the control silage reached a relatively high pH of over 5.3 during the entire periods.Furthermore, all the treated silages had a sustained lower pH than control throughout the ensiling period.Overall, all the treated silages (I, E and I + E) accelerated decline in pH to a lower level than untreated erect milkvetch, especially for the treatments I+E and I.
Addition of LAB inoculants at ensiling ensured rapid and vigorous fermentation which resulted in faster accumulation of lactic acid, lower pH at earlier stages of ensiling and improved forage conservation.Many studies have shown the advantage of such inoculants (Hassanat et al., 2007;Xu et al., 2008;Schmidt et al., 2009).In the present study, treatments of both I and I + E exhibited positive effects on silage fermentation as indicated by lower pH and NH 3 -N, and higher lactic acid concentration, than control silage.The additive 'LaLsIL Dry' used in the present study contained Lactobacillus which can improve silage quality by proliferation of LAB and consequently inhibition of growth of clostridia and aerobic bacteria.
A number of reports showed that the addition of LAB and enzyme to forage at ensiling resulted in reduction in structural polysaccharides and in increase in lactic acid content compared with untreated silages (Weinberg et al., 1993;Xu et al., 2008).In the present study, treatment I + E was well preserved as indicated by lower pH value, NH 3 -N content and higher lactic acid content than others (Table 2; Figure 1).It is expected that other than the addition of LAB, cellulase increased WSC production which could be then used by LAB for primary lactic acid fermentation.Furthermore, treatment I had better effect than the E treated in this study.
Proteolysis is an inevitable consequence of the ensiling process.Up to 75% of forage true protein can be converted to NPN during the first days of ensiling process by the action of plant proteases (Hassanat et al., 2007) and microbial proteases (McDonald et al., 1991).Controlling of proteolysis is based on creating a low pH environment that is unsuitable for the action of plant and microbial proteases (McDonald et al., 1991).In the present study, treatments I and I + E had reduced NH 3 -N content and lowered initial pH, while E treated silage had higher NH 3 -N content and initial pH.These results agree with Mandebvu et al. (1999) who reported a similar result using bermudagrass treated with enzymes.The different trend of decline in pH for control, E, I and I + E silages may help to explain their effect on inhibiting proteolysis.
Of all ensiling periods, population of LAB increased numerically from days 1 to 5 post-ensiling then decreased to day 45 post-ensiling.The decrease in LAB may be attributed to the low pH and lack of fermentable substrates which resulted in death of bacteria (McDonald et al., 1991).In addition, there were no differences for yeast among treatments.
Compared with the control silage, all treatments significantly decreased aNDF and ADF concentration, especially for treatment I + E (Table 4).It is expected that the use of enzymes as silage additives would degrade cell wall and subsequently improve the digestibility of silage fibre (McDonald et al., 1991).However, it is not common that treatment I resulted in a decrease in aNDF and ADF.This may be attributed to degradation of crude fibre by enzymes from microbial secretion during the fermentation (Guan et al., 2002).In addition, Lignin (sa) concentration was decreased in all treated silages and there were no significant differences between treatments.
The IVDMD and IVNDFD were higher in silages treated with enzymes (E and I + E; Table 4).This is consistent with results reported by other researchers (Eun and Beauchemin, 2008).In addition, silages treated with I significantly increased IVDMD and IVCPD (Table 4).However, Filya et al. (2007) reported that the addition of I did not show significant effects on IVDMD of lucerne silage.From more than 60% of trials published between 1990 and 1995, Muck and Kung (1997) summarized that fermentation was improved (i.e.reduced pH, increased lactate to acetate ratio, or both) by inoculants, whereas DM digestibility (in vitro or in vivo) was increased in only 30% of the trials.Weinberg and Muck (1996), in their review, also reported instances in which fermentation was affected by a LAB inoculant, whereas digestibility was not.

CONCLUSIONS
Erect milkvetch is a typical legume with a high buffering capacity and a low water-soluble carbohydrates content.Silages treated with lactic acid bacteria (I), fibrolytic enzyme (E) and I + E were well preserved with high lactic acid content, and low pH, NH 3 -N and NPN content compared with control silages (P<0.05).Treatments of enzymes (E, I + E) significantly improved silage in vitro dry matter and aNDF digestibility concentration of silages.In addition, treatments of I and I + E increased in vitro crude protein digestibility (P<0.05).These results indicated that the additives tested can improve the erect milkvetch silage fermentation quality and in vitro digestibility to some extent.

Table 1 .
Chemical composition (g kg -1 DM) and buffering capacity of erect milkvetch

Table 2 .
Effects of additive treatment and time of ensiling on fermentation characteristics of erect milkvetch silages

Table 3 .
Effects of additive treatment and time of ensiling on microbial population of erect milkvetch silages (log 10 CFU g -1 fresh material)

Table 4 .
Effects of additive treatments on nutrient compositions of 45-d erect milkvetch silages