Effect of substituting Pennisetum sinese with bamboo shoot shell (BSS) on aerobic stability and digestibility of ensiled total mixed ration

Abstract This study aimed to evaluate the effect of substituting Pennisetum sinese with bamboo shoot shell (BSS) on the fermentation quality, chemical composition, in vitro digestibility and aerobic stability of ensiled total mixed ration (TMR). Four ensiled TMRs were designed on fresh matter basis: (i) 45% P. sinese (BSS0); (ii) 15% BSS + 30% P. sinese (BSS15); (iii) 25% BSS + 20% P. sinese (BSS25); (iv) 35% BSS + 10% P. sinese (BSS35). All silages were moderately fermented according to the V-score. Substituting P. sinese with BSS increased acetic acid content and decreased ethanol and ammonia nitrogen contents (p < .05). With the increasing proportion of BSS, neutral detergent fibre decreased, and relative feed value increased (p < .05). Under aerobic exposure, BSS-substituted (BSS15, BSS25 and BSS35) silages were more stable than BSS0 silage with lower silage temperature and yeast population. No significant differences in BSS substitution were observed in in vitro gas production, digestibility, metabolisable energy and net energy for lactation (p > .05). The substitution of P. sinese with BSS had no adverse effect on the fermentation quality and in vitro digestibility while efficiently improving the aerobic stability of ensiled TMR. The BSS35 substitution level is recommended considering the maximum utilisation of BSS. HIGHLIGHTS Bamboo shoot shell (BSS) was explored for ensiled TMR. BSS had no effect on the silage digestibility. BSS improved aerobic stability.


Introduction
With the increasing demand for animal products, the contradiction between food and feed is prominent worldwide and more specifically in China. Exploring local forages or unconventional feedstuffs as alternatives to cereal has become the focus of livestock producers and animal nutritionists (Zhao et al. 2016;Chen et al. 2017). Pennisetum sinese, a hybrid of Pennisetum purpureum and Pennisetum americanum, is widely cultivated as one of the most promising forage grasses in the tropical and subtropical regions due to its high yield and wide adaptability. At present, P. sinese has become the main forage source of local animal husbandry in the Loess Plateau and the Yangtze River Delta of China (Deng et al. 2019). Meanwhile, bamboo (Bambusoideae) shoots as traditional food are popular in many Asian countries with good taste and rich nutrition (Devi and Pamba 2015). China is the largest producer of bamboo shoots in the world, with more than 3 million tons of bamboo shoots per year . However, up to 70% of bamboo shoots, consisting of the nonedible sheaths and the basal part of bamboo shoots, were discarded as waste residues (Lin et al. 2018), resulting in waste of resources and environmental pollution. Making full use of the bamboo shoot shell (BSS) can not only increase the added value of BSS, but also is of great significance to environmental protection. Actually, BSS is rich in protein, amino acids and carbohydrates and can be used as a new source of roughage (Liu et al. 2000). Chen et al. (1979) reported that BSS could be easily digested by cattle, with total digestible nutrients (TDN) as high as 68.5%. Inclusion of BSS to diet improved daily live weight gain of pigs (Zhou et al. 1992) , the growth rate and feed conversion of ruminants (Liu et al. 2000).
Huge quantities of BSS generated seasonally call for effective storage. Ensiling, as a promising technology, is applicable for long-term preservation and yearround availability of BSS. But BSS is seldom used for silage making due to the high moisture content. The traditional method of processing high-moisture materials is wilting or drying, which causes extensive consumption of labour and time. In recent years, total mixed ration (TMR) silage has been widely developed to not only supply a complete formula diet for a long time but also provide the possibility to exploit more local materials as roughage resources (Chen et al. 2015). We assumed that the straw and hay frequently prepared in TMR or ensiled TMR could be worked as moisture absorbents to balance the high moisture content of BSS. In view of the high crude protein (CP) content of BSS, substituting P. sinese with BSS in TMR may be the preferred method to improve the utilisation rate of BSS and reduce the dependence on concentrate. Moreover, BSS was extensively studied due to its polysaccharides having an excellent biological antioxidant activity (Luo et al. 2018;Chen, Fang, et al. 2019). Antioxidants have been broadly applied in food and feed industries to prevent deterioration. It was speculated that the inclusion of BSS might improve the aerobic stability of ensiled TMR. While to our knowledge, little information regarding the correlation between BSS and aerobic stability of silage is available yet.
Thus, the BSS was introduced into a local TMR formula to evaluate its effects on the fermentation quality, chemical composition, in vitro digestibility and aerobic stability of ensiled TMR.

Ensiled TMR preparation
The BSS was obtained from a local bamboo shoots processing factory in Zhejiang, China on 24 August 2014. Pennisetum sinese, rice straw and concentrate were provided by a private beef cattle farm (29.43 N,121.48 E,elevation 4 m,Zhejiang,China). The P. sinese was harvested at the milk ripe stage, leaving the stubble of 30 cm. The concentrate consisted of 45% corn, 20% cottonseed, 15% rapeseed meal, 15% corn distillers' grains, 5% vitamin-mineral premixes. All the roughages were chopped into a length of 2-3 cm by a forage chopper. The chemical compositions of each TMR ingredient were analysed in six replicates, and the data are listed in Table 1.
As shown in Table 2, the partial substitution of P. sinese to BSS was performed to maximise BSS utilisation based on a local TMR formula. Four treatments were designed according to the different substitution proportions (based on fresh matter) of BSS in TMR: (i) no bamboo shoot shell þ 45% P. sinese (BSS 0 ); (ii) 15% bamboo shoot shell þ 30% P. sinese (BSS 15 ); (iii) 25% bamboo shoot shell þ 20% P. sinese (BSS 25 ); (iv) 35% bamboo shoot shell þ 10% P. sinese (BSS 35 ). A batch (180 kg) for each treatment was distributed and thoroughly mixed, then approximately 5.8 kg TMR was tightly packed into a 10 L laboratory silo (polyethylene bottle with a diameter of 27.5 cm and height of 31.6 cm, Lantian biological experimental instrument Co., Ltd., Jiangsu, China) and sealed with screw tops and plastic tape. The packing densities of the BSS 0 , BSS 15 , BSS 25 and BSS 35 were 336, 335, 334 and 333 kg DM/m 3 , respectively. Anaerobic storage was conducted at an ambient temperature ranging from 22 to 28 C for 90 days. Six silos per treatment were sampled for the following analyses.

Chemical and microbial analyses
The fresh ingredients and TMRs were divided into three subsamples and the ensiled TMR was divided into four subsamples. The first subsamples were blended with distilled water at 1:10 w/v ratio and macerated at 4 C for 24 h. After filtering through four layers of gauze, the filtrate was used to determine the buffering capacity (BC) according to the hydrochloric acid-sodium hydroxide method (Playne and McDonald 1966). The second subsamples were immediately oven-dried to determine dry matter (DM) content and then ground with laboratory knife mills (FW100, Taisite Instrument Co., Ltd., Tianjin, China) to pass through a 1-mm screen. The resulting powder samples were used for subsequent analyses of water-soluble carbohydrates (WSC), neutral detergent fibre (NDF), acid detergent fibre (ADF), CP, ether extract (EE) and crude ash (Ash). The contents of DM (934.01), CP (984.13), EE (920.39) and Ash (942.05) were determined following the procedure of the Association of Official Analytical Chemists (AOAC 2019). The WSC was determined via the colorimetric method after reaction with anthrone reagent (Thomas 1977). The NDF and ADF were analysed according to the procedures of Van Soest et al. (1991) and Robertson and Van Soest (1981), respectively, and expressed on a DM basis inclusive of residual ash. Non-fibrous carbohydrate (NFC) was calculated by the following formula: NFC ¼ 100 À CP À NDF À EE À Ash (NRC 2000). The TDN were calculated by the method of Harlan et al. (1991). The third subsample was homogenised with sterilised saline solution (0.85% NaCl) at 1:9 w/v ratio and then serially diluted for 7-fold. The LAB was counted on deMan, Rogosa and Sharp (MRS) agar medium (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) after incubation at 37 C for 2-3 days. Aerobic bacteria (AB) were counted on nutrient agar medium (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and yeasts were counted on potato dextrose agar medium (Sincere Biochemical Technology Co., Ltd., Shanghai, China), agar plates were incubated at 30 C for 2-3 days (Kozaki et al. 1992).
The remaining silage subsample was blended with distilled water at 1:3 w/v ratio and macerated at 4 C for 24 h to obtain the extract. After filtering through two layers of gauze and a filter paper, the filtrate from the extract was used for later determination of pH, organic acids and ammonia nitrogen (NH 3 -N) contents. The pH was measured with a glass electrode pH metre (Hanna HI 2221, Hanna Instruments Italia Srl, Villafranca Padovana, Italy). After centrifugation at 4 C, 10,000 Â g for 10 min, the supernatant fluid was analysed for organic acids (Desta et al. 2016), and NH 3 -N (Broderick and Kang 1980). To evaluate the fermentation quality and digestible energy intake of silage, the V-score (an evaluation method that is calculated from volatile fatty acids and NH 3 -N, Takahashi et al. 2005) and relative feed value (RFV, Rohweder et al. 1978) were adopted, respectively.

In vitro incubation
Rumen fluid was manually collected from the rumens (different positions) of three rumen-cannulated Boer male goats before morning feeding. The goats were fed the diet consisting of alfalfa (6%), guinea grass (59%) and concentrate (35%) at 1.3 times the maintenance level (Feng and Lu 2007). The whole process was carried out under anaerobic conditions with continuous CO 2 flushing. Rumen fluid was homogeneously mixed, immediately filtered and stored at 39 C in a water bath. Before later incubation, rumen fluid was mixed with a buffer solution at a 1:2 v/v ratio as described by Menke (1988).
In vitro incubation was conducted in serum bottles following the method of Contreras-Govea et al. (2011) with some modifications. Approximately 1 g ground samples of ensiled TMR were transferred into filter bags (F57; ANKOM Technology, Macedon, NY, USA) that were previously washed with acetone, dried at 55 C for 24 h and weighed. Each bag was heat-sealed and then placed into each preheated 120 mL serum bottle with 60 mL rumen fluid-buffer mixture under CO 2 at 39 C. A total of 144 silage samples (4 treatments Â 6 silos Â 2 replicates Â 3 runs) were prepared. Meanwhile, six serum bottles with the only inoculum added were served as blank at each run. Gas production was recorded at 4, 8, 12, 24, 36, 48 and 72 h of incubation by injecting gas to a pressure transducer (Yunbao Electrical Equipment Co., Ltd., Nanjing, China) and corrected with gas produced from blank bottles. After incubation, the filter bags were gently rinsed with cold tap water until clean and dried at 65 C for 48 h to determine in vitro digestibility of DM (IVDMD) and NDF (IVNDFD) based on the differences in their respective weight before and after incubation.
Gas production (GP) data were fitted to an exponential model: where y is the cumulative volume of GP at incubation time t (mL), b is the potential GP (mL), and c is the rate constant of GP (Bl€ ummel et al. 2003). The metabolisable energy (ME) and net energy for lactation (NE L ) were calculated using the equations as described by Menke (1988).

Aerobic stability test
After 90 days of ensiling, six silos per treatment on each exposure day were used for a 14-day aerobic stability test. Briefly, silage samples were taken out from each 10 L silo, fully mixed and loosely loaded into a bigger, sterile and open-top polyethylene bottle (capacity 15 L). The bottles were stored at ambient temperature (26 ± 2 C) covering with two layers of gauze to prevent drying and contamination. The thermocouple wires of a data logger (MDL-1048A, Tianhe Automation Instrument Co., Ltd., Shanghai, China) were placed in the geometric centre of the bottle to record silage temperature every 30 min. The ambient temperature near the bottles was recorded as blank. Aerobic stability is defined as the time (h) elapsed when the silage temperature is 2 C above the ambient temperature (Wilkinson and Davies 2013). And the dynamic changes of pH, acetic acid (AA), NH 3 -N, WSC, and microbes counts after 0, 3, 6, 9, and 14 days of aerobic exposure were also analysed as indicators of aerobic deterioration using the same procedures of silage subsamples analyses.

Statistical analyses
Analysis of variance (ANOVA) was performed using the general linear model procedure of SAS rev. 9.2. Data on chemical composition, fermentation quality and in vitro incubation were subjected to one-way ANOVA. And data on aerobic stability were subjected to two- way ANOVA. Statistical difference between means was determined by using Tukey's multiple comparisons and considered as significant at p < .05.

Results
Chemical and microbial compositions of preensiled materials Compared with P. sinese, BSS had higher moisture, NFC and CP contents, and lower WSC, fibre and EE contents. Except for the contents of WSC, EE, BC and NFC, no significant (p > .05) differences were found for DM, CP, NDF, ADF, Ash, RFV and TDN in the TMRs among the treatments ( Table 2). The epiphytic LAB population in all treatments was more than 10 5 cfu/g FM. With the increasing proportion of BSS, the population of aerobic bacteria and yeast significantly decreased (p < .05).

Fermentation quality of ensiled TMR
As presented in Table 3, substitution levels significantly affected the AA, ratio of lactic acid to acetic acid (LA/AA), ethanol and NH 3 -N contents (p < .05). All ensiled TMRs were moderately fermented according to the V-score (>70). The BSS substitution increased the AA content and decreased the ethanol and NH 3 -N contents of resulting silages (p < .05). Negligible contents of propionic acid and butyric acid (<2 g/kg DM) were recorded in all ensiled TMRs. The inclusion of BSS had no effect on the AB population but decreased the LAB and Yeast population of resulting silage.

Chemical compositions of ensiled TMR
The WSC, NDF and NFC contents, as well as RFV of ensiled TMR, were affected by BSS substitution levels (

In vitro parameters of 90-day ensiled TMR
The in vitro measured or estimated parameters of four ensiled TMRs are shown in Table 5. The potential GP (b value) was ranged from 111.32 to 120.88 mL. BSS substitution had no effect on GP 24 , GP 72 , potential GP, GP rate constant (c value), IVDMD, IVNDFD, ME and NE L .

Aerobic stability of ensiled TMR
After aerobic exposure for 14 days, the pH of BSS 0 raised sharply from 4.58 to 5.90 (p < .05), yet no significant (p > .05) change was found among BSS-substituted ensiled TMRs ( Irrespective of the aerobic exposure days, with the increasing proportion of BSS, NH 3 -N content gradually decreases (p < .05). During the 14-day aerobic exposure, the population of LAB decreased while that of AB and Yeasts increased (Table 7). And the yeast population in all ensiled TMRs were always less than 10 6 cfu/ g FM except for BSS 0 . The differences in temperature and aerobic stability among four ensiled TMRs during aerobic exposure are shown in Figure 1. The silage temperature of BSS 0 was 2 C above the ambient temperature after 127 h of    aerobic exposure. But all BSS-substituted silages were no more than 2 C above the ambient temperature under the 14-day aerobic exposure (Figure 1(A)). Namely, the aerobic stability of all BSS-substituted silages exceeded 336 h (Figure 1(B)).

BSS substitution on fermentation quality
The four TMRs had high WSC content (>50 g/kg DM) and sufficient LAB population (>10 5 cfu/g FM), which are conducive for a successful fermentation theoretically (Weinberg 2008). Indeed, all silages were well preserved after 90 days of ensiling according to the Vscore, indicated by low BA and NH 3 -N contents (Catchpoole and Henzell 1971).
In the study, the fermentation products of TMR, especially LA, was lower than that of the forages with similar WSC content, and this could ascribe to its high DM content. High DM content of silage delays the multiplication of microbes including LAB (Chen et al. 2015). The lower WSC content of BSS-substituted TMRs explains the lighter LA production in BSS-substituted ensiled TMR since less substrate is available for LAB. However, the AA content in BSS-substituted ensiled TMR increased with an increasing proportion of BSS, which was difficult to explain but could ascribe to the material specificity of BSS (Yuan et al. 2019). Moreover, when WSC is limited, hetero-fermentative LAB becomes active and several homo-fermentative strains such as Lactobacillus plantarum could turn to the lactate/acetate conversion pathway (McDonald et al. 1991). Conversely, the NH 3 -N content decreased with an increasing proportion of BSS, which corresponded well to the respective pH. The BSS substitution decreased the yeast population, thereby reducing the ethanol content of the resulting silages.

BSS substitution on chemical compositions
The BSS substitution effectively improved the NFC content and RFV without unfavourable effects on the CP, EE and TDN of silages. The numerically higher CP content in BSS-substituted silages than that in BSS 0 is probably due to the high CP content in BSS material. While the obvious lower NDF content in BSS 35 silage could be related to its relatively low pH. Hemicellulose, as a part of NDF, is known to be acidunstable and prone to acidolysis. Moreover, the reduction in NDF content after ensiling was much larger than that in ADF content, indicating that hemicellulose is more susceptible to degradation than cellulose (McDonald et al. 1991). The highest NFC content observed in BSS 35 silage was mainly attributed to its lowest NDF content. The lower NDF and ADF contents observed in BSS-substituted silages than BSS 0 could ascribe to less DM loss, which in turn explained that the inclusion of BSS could enhance the RFV of ensiled TMR. Given above, BSS 35 silage had the best feeding value followed by BSS 25 , BSS 15 and BSS 0 silages.

BSS substitution on in vitro parameters
In vitro GP and digestibility are gained wide acceptance to evaluate the nutritional value of ruminant feeds (Bl€ ummel et al. 2003;Rymer et al. 2005). Meanwhile, the relationship between these two parameters is commonly used to predict the actual DM intake and digestion of ruminants (Getachew et al. Means with different small letters (a, b) show significant difference among treatments at p < .05. BSS 0 , no bamboo shoot shell þ 45% Pennisetum sinese; BSS 15 , 15% bamboo shoot shells þ 30% Pennisetum sinese; BSS 25 , 25% bamboo shoot shells þ 20% Pennisetum sinese; BSS 35 , 35% bamboo shoot shells þ 10% Pennisetum sinese. AT: ambient temperature; AT þ 2: ambient temperature plus 2 C; BSS: bamboo shoot shell. 1998). In the present study, BSS substitution only numerically affected all GP and digestibility parameters as well as estimated ME and NE L , indicating that BSS substitution had no unfavourable effects on rumen utilisation of ensiled TMR.

BSS substitution on aerobic stability
When the air infiltrates, the aerobic microbes begin to proliferate and silages are prone to deterioration: Yeasts, mainly lactate-assimilating yeasts, can metabolise substrates such as lactic acid to increase the pH and temperature of silage; Where after, aerobic bacteria and moulds begin to grow and further aggravate the spoilage (Kleinschmit et al. 2005). Therefore, monitoring the changes in chemical and microbial compositions during aerobic exposure is essential to prevent aerobic deterioration and toxin production resulted from seal damage or silos that are open.
It is well known that silages with lower LA content or those with less residual sugar contents were more stable when exposed to air (Weinberg and Muck 1996), which may partially explain the better aerobic stability (>336 h) of BSS-substituted silages. Furthermore, the high AA content in BSS-substituted silages is also responsible for enhanced aerobic stability as short-chain fatty acids can effectively inhibit spoilage microbes (Kung and Ranjit 2001). Another explanation for the excellent aerobic stability of the BSS-substituted silages might ascribe to the BSS material used in the study. Polysaccharides in BSS have potential antioxidant activities ) and could inhibit the oxidation of substrates induced by aerobic microbes, which explained the low population of AB and yeasts in BSS-substituted silages under aerobic exposure. Yeast population less than 1 Â 10 5 cfu/g FM is required to inhibit the aerobic deterioration of silage as well as keep aerobic stability. Interestingly, BSS 15 silage still maintained aerobic stability even yeast population was above the level of 1 Â 10 5 cfu/g FM at 14 days of aerobic exposure. One possible reason is that BSS substitution favoured the establishment of the WSC-assimilating yeasts rather than lactate-assimilating yeasts. Liu et al. (2018) found that the species of yeasts rather than yeast population was more responsible for aerobic stability. Courtin and Spoelstra (1990) indicated that AB are also related to aerobic deterioration. Unlike yeasts, AB often functioned in the final phase of deterioration with relatively high pH (McDonald et al. 1991). Furthermore, AB were the critical microbes which cause the increase of NH 3 -N under aerobic condition (Nussio 2005), which is consistent with this study that high AB population is accompanied by high NH 3 -N content. Given the above, although the aerobic stability of all BSS-substituted silages exceeded 336 h, it was expected that BSS 35 silage would perform better if the aerobic exposure was further prolonged.

Conclusions
Substituting P. sinese with BSS up to 35% had no adverse effect on the fermentation quality and in vitro digestibility while improving the aerobic stability of ensiled TMR. BSS can be a potential source of roughage for ensiled TMR production, and the BSS 35 substitution level (proportion of BSS with roughages: 35/65) is recommended with the principle of maximum BSS utilisation. Whether BSS polysaccharides are associated with aerobic stability or not is needed for further study.

Ethical approval
All animal experimental protocols were approved by the Animal Care and Use Committee of Nanjing Agriculture University.

Disclosure statement
No potential conflict of interest was reported by the author(s).