Effects of Rice Straw Silage with Combining Additives of Lactobacillus Plantarum, Trichoderma Viride and Wheat Bran on the Growth Performance, Digestibility and Rumen Fermentation in Growing Lambs 

In recent years, with grassland changes and degradation, the amount of forage is getting reduced, making the situation unable to meet the increased demand for feed in the domestic animals (Ramirez-Lozano et al., 2018). As the annual production in China ranges between 180 and 270 million tons, rice straw is one of the most abundant and important agriculture residues in China (Xia et al., 2018). However, a large amount of rice straw is burnt or discarded, causing a waste of resources and serious environmental pollution (Zhu et al., 2015). Rice straw rich in crude fibers, cellulose, hemicellulose and lignin, plays an important role in biomass production or feeding ruminants (Sambusiti et al., 2013), whereas these complicated fiber structures, especially the lignin and silica are the major limiting factors for improving the quality of rice straw (Van Soest, 2006). Therefore, several techniques are being explored and utilized to enhance the quality of rice straw in recent years. Rice straw silage with combined additives promotes cellulose degradation, feedstock preservation and palatability, as well as animal feed intake and nutrient digestibility (Li et al., 2018). The additives which are commonly used in the silage process mainly include cellulolytic enzyme, lactic acid bacteria and carbohydrate source (Zhao et al., 2018). We hypothesized that the combination of these three additives can also improve the feed utilization efficiency of rice straw silage. Therefore, the main purpose of this study was to investigate the effects of RSSA on the growth performance, nutrient digestibility and rumen fermentation of growing lambs.

Silage preparation
 
Fresh rice straw harvested in agricultural rice fields in Yangzhou University (Jiangsu, China), contained dry matter (DM) content of 635.5 g/kg of fresh matter (FM), crude protein, NDF, ADF and water soluble carbohydrate (WSC) of 58.6 g/kg, 716.3 g/kg, 405.4 g/kg, 46.8 g/kg DM, respectively. The fresh rice straw was chopped into 3-5 cm with a fodder chopper followed by manual mixing.

Rice straw was ensiled with: (i) no additive (RSSW); (ii) L. plantarum (8.0×10⁵ cfu/g FW) + T. viride (6.0×10⁵ cfu/g FW) + wheat bran (20 mg/g FW) (RSSA). Both of L. plantarum and T. viride were purchased from Guangzhou Luhui Biotechnology Co., Ltd (Guangzhou, China). Wheat bran was purchased from China National Medicines Co., Ltd (Beijing, China). All additives were utilized according to the recommendations of the manufacturer. L. plantarum and T. viride were diluted with ultrapure water to an equivalent of 20 mL/kg FW and sprayed evenly on rice straw. The RSSW group evenly sprayed the same amount of ultrapure water then stored at 20±2°C for 45 days.
 
Animals and management
 
The following experimental procedures, including animal ethics and usage, were approved by the Animal Welfare Committee of Yangzhou Veterinarians of the Agriculture Ministry of China (Yangzhou, China).
 
This experiment was performed in Huaian (Jiangsu, China), from April to July of 2017. Twelve male growing lambs of Boer with similar initial age (45 ± 3.8 d) and body weight of 12.8 ± 0.4 kg were selected and randomly assigned into 2 groups. Each lamb was individually housed in a barn stall. The feeding experiment comprised of 14 d adaptation and 60 d experimental periods. The experimental lambs were fed 2 different treatment diets such as RSSW (25% on the DM basis) and RSSA (25% on the DM basis). These diets were formulated to meet the nutrient requirements for lambs, growing at 80 g/d (NRC 2007). The nutrition contents of the diet are listed in Table 1. Feed was offered twice daily (at 06:00 h and 18:00 h) within individual pens. The amount of diet refused by individual lamb was weighed before feeding, and the feed intake was calculated by the quantity offered minus refused each day, then the amount of supplementation was adjusted daily on the basis of intake on the previous day. The ad libitum water access was provided for each pen. The DMI was calculated as the mean of feed intake on daily basis. The body weight (BW) of growing lambs was recorded in early morning on every 7d before feeding. The average daily gain (ADG) of each lamb was calculated as the regression of BW on day of measurement during the growing period. Additionally, the feed conversion ratio (FCR) was calculated as the ratio of DMI to ADG.
 

 
Sample collections and chemical analysis
 
Feed samples and refusals samples were collected weekly. Fecal samples were collected through the total fecal collection from each lamb using fecal bags from 31 to 34 d and from 51 to 54 d of the experimental period and all samples were immediately frozen at -20°C for determination of apparent digestibility of the nutrients. All the samples were ground with a Wiley mill and passed through 1 mm screen after drying at 65°C to a constant mass in a forced air oven. The DM, Ether extract (EE), Ca and P content in these samples were analyzed according to the Association of Official Analytical Chemists (AOAC 2006). The crude protein (CP) content was determined with a Kjeldahl 2300 analyzer (FOSS Analytical AB, Hoganas, Sweden). The NDF and ADF were determined by the filter bag technique (ANKOM 2000; Ankom Technology Co., Fairport, NY) as described by Van Soest et al., (1991).
 
Rumen fluid samples were collected from 4 randomly selected lambs in both groups 2 hours post feeding in the morning at 15, 30, 45, 60 d of the experimental period. These samples were then filtered through four layers of gauze and dispensed in 5 mL centrifuge tubes. The pH of the rumen fluid was immediately measured using an electrode pH meter (model pHS-3C, Shanghai Chemical Company, Shanghai, China). The rumen fluid was immediately stored at -20°C to determine the concentrations of ammonia nitrogen (NH₃-N), MCP and volatile fatty acids (VFA). The concentration of NH₃-N in rumen fluid was measured according to Rhine et al., (1998) and MCP concentration in rumen fluid was determined according to Wang et al., (2008). Rumen fluid was thawed to determine the VFA concentration using gas chromatography according to Wang et al., (2017).
 
Statistical analysis
 
The statistical analysis of the data was performed using PROC MIXED procedure of SAS version 9.2 software (SAS Institute Inc, Cary, NC, USA). The following model was used: Y_ijk = μ + T_i + S_j + G_k + C_ijk, where Y = observation value, μ = overall means, T_i = the effect of treatment, S_j = the effects of sampling time, G_k = the random effect of goat, C_ijk = the error term. The significance of differences among treatments was evaluated by Tukey’s multiple range test. The effects of the treatments were declared significant at P<0.05.

Effect of RSSA on growth performance in growing lambs
 
As shown in Table 2, no significant difference was observed in final BW and DMI between treatments RSSW and RSSA (P> 0.05), the ADG was significantly increased by the RSSA supplementation as compared with RSSW supplementation (P< 0.05). Whereas the FCR was significantly higher with RSSW supplementation as compared with the RSSA (P< 0.05).
 

 
The component of feed plays an important role in DMI. The CP content in feed could enhance DMI, whereas the fiber fractions such as ADF and NDF depress DMI (Riaz et al., 2014). Good quality and palatability of feed also promote the DMI (Brown et al., 2016). The silage process not only softens the hard carbohydrate structure of feed, but also produces lots of aromatic and sweet organic acid, so that it could promote the quality and palatability of feed (Muck et al., 2018). Moreover, lactic acid bacteria ferment WSC into lactic acid, accordingly reduce the pH value and inhibit the aerobic microorganisms which consume quantities of nutrients in silage feed (Borreani et al., 2018). Bernardes et al., (2012) reported that dry matter losses could reach up to 70% in the feed stored in the peripheral area of silage silos due to improper processing. This study showed that the DMI was enhanced due to the RSSA supplementation, which shows that RSSA fermentation quality is better than the RSSW fermentation quality. That is why the ADG was increased while the FCR was decreased with RSSA supplementation.
 
Effect of RSSA on the apparent digestibility of nutrients in growing lambs
 
Total tract apparent digestibility of nutrients was significantly affected by the RSSA supplementation (Table 3). The apparent digestibility of DM, as well as NDF and ADF were significantly increased by RSSA supplementation compared to the RSSW supplementation (P < 0.05). However, the digestibility of CP in RSSA was higher than that in RSSW, but there was no significant difference observed.
 

 
Feed efficiency, as well as DMI, have a strong relationship with the nutrient digestibility, as high digestibility of nutrients can promote the feed efficiency (Mertens et al., 2010). This experiment showed that the feed efficiency and apparent digestibility of nutrients was enhanced by the RSSA supplementation. L. plantarum reduced silage pH rapidly and inhibited the spoilage and pathogenic microbes, thereby preserving the nutritional value of ensiled forage and increasing DM recovery as well as silage digestibility (Borreani et al., 2018). In addition, rice straw is rich in structural carbohydrate, which is the most difficult parts of feed to be digested (Xia et al., 2018). However, cellulase or cellulolytic enzymes or bacteria could promote the degradation of crude fiber to dietary degradable carbohydrates such as glucose and organic acid (Tamboli et al., 2017). Therefore, RSSA supplementation enhanced the nutrients digestibility and similar to this in vivo study, the application of bacterial inoculant also increased nutrients digestibility of rice straw silage in vitro (Zhao et al., 2019). Thereby, ruminal microorganisms can easily degrade the carbohydrate to produce volatile fatty acids, which promote the nutrient digestibility.
 
Effect of RSSA on rumen fermentation parameter in growing lambs
 
As it can be seen in Table 4 that the pH and NH₃-N concentrations in the rumen were not affected overall (P > 0.05), however the MCP concentration was enhanced by the RSSA supplementation (P < 0.05). Moreover, the TVFA, acetate, butyrate and valeric acid concentrations were increased by the RSSA supplementation (P < 0.05). Additionally, the ratio of acetate to propionate was also not affected by the treatments.
 

 
Silage bacteria not only protect the protein from decomposition by proteolytic bacteria, but also utilize the nitrogenous substances in silage feed to synthesis bacterial protein (Solati et al., 2018). Therefore, the RSSA contained higher protein content than RSSW, accordingly enhancing the MCP synthesis by microorganisms in rumen. VFA plays a central role in lipids, glucose and energy metabolism for ruminants (Luna-Palomera et al., 2018). Oskoueian et al., (2019) found that inoculated silage rice straw increased the TVFA in vitro. In this study, the TVFA, acetate, butyrate and valeric concentration were increased by the RSSA supplementation. The main reason might be the increased fiber contents especially the NDF digestibility of RSSA supplementation, as the fiber could be fermented into acetate and butyrate in rumen (Baldwin and Connor, 2017). All of these results indicate that RSSA supplementation could improve the rumen fermentation parameters of growing lambs.

Based on the results of this experiment, rice straw silage with L. plantarum, T. viride and wheat bran supplementation could enhance the average daily gain and feed efficiency, dry matter, neutral and acid detergent fiber apparent digestibility. The microbial protein concentration as well as acetate and butyrate acid concentrations are also increased in rumen fermentation of growing lambs. Therefore it can be concluded that rice straw silage with L. plantarum, T. viride and wheat bran helps to promote the growth performance, rumen fermentation and apparent digestibility of nutrients of growing lambs.

We gratefully acknowledge the support of the National Fundamental Research Program of China (2018YFD0502100), Natural Science Foundation of China (31672446) and the Huaian Li Wang Boer goat farm for providing lambs for this experiment.