1. Introduction
The shortage of high-quality roughage is a limitation for the effective management of Chinese ruminant livestock industries. Conserved forages are required to feed the livestock when pasture grazing is not available. The growth of livestock and human populations in China means that there is limited land available for the production of conventional forages, such as maize silage. Ensiling a crop reduces any loss of nutrients, compared with making hay, and it is widely used in animal husbandry. Alternative sources of roughages offer the potential to improve resource utilization and reduce environmental pollution. The mulberry tree, a native of Korea and China, is widely distributed in China due to its strong drought and temperature resistance [
1]. It is deep-rooted and when harvested as an animal feed has a high dry matter (DM) production in a wide range of conditions, up to 20–30 tonnes/ha/year when cut at 9–10 week intervals [
2,
3]. Mulberry trees are also recognized internationally as an important bioenergy and biofuel crop. They do not require irrigation, except sometimes during establishment in dry conditions.
Although it is estimated to be grown on 1.67 million ha in China, maize silage has several limitations for use as a forage for ruminants, with the short growing season a significant limiting factor in the north of the country [
4]. Although yields of 20–25 t DM/ha/year are achievable [
5], irrigation is often required. Maize grown for feeding to livestock has a high nitrogen demand [
4], as about 300 kg/ha are harvested and 80% of this has to be applied externally [
6], either in the form of artificial fertilizer or manure nitrogen. This results in large emissions of ammonia, which is responsible for major adverse effects on atmospheric pollution and human health. In the US, maize accounts for approximately 18% of total ammonia emissions, which contribute to global climate change and increase PM2.5 (particulate matter with a diameter of less than or equal to 2.5 microns in the air) to adversely affect human respiratory health. The significant fertilizer nitrogen requirements of maize also contribute to carbon dioxide emissions from fertilizer manufacture, and they are therefore important in global climate change.
Mulberry trees, by contrast, are important carbon dioxide sequestrators and relatively unresponsive to supplementary N, P and K, although good yield responses to vermicompost have been reported [
7]. By developing multipurpose cultivated mulberry varieties, its utilization has been extended over 5000 years of its cultivation to fruits for Chinese medicine, cosmetics in the form of skin and hair tonics, and leaves for sericulture and livestock. Leftover branches and leaves after silkworm feeding are also sometimes fed to livestock. The nutritional value of mulberry leaves is high. Its crude protein (CP) content can be as high as 34%, with balanced amino acid composition. Its fiber content is low, and it is rich in flavonoids, sterols, polysaccharides, alkaloids and other biologically active substances [
8,
9,
10]. Therefore it is a high-quality protein feed resource with broad application prospects [
11]. Mulberry leaves are more digestible by goats [
12] and sheep [
13] than alfalfa hay and oat hay by sheep [
14]. However, fresh mulberry leaves have a low DM content and are not easy to preserve. Dried mulberry leaves lose many nutrients, limiting their application in animal production [
15]. Mulberry silage retains more nutrients while reducing the content of anti-nutritional factors in mulberry [
16]. Mulberry silage may therefore have great application prospects in livestock production; however, its high content of water-soluble carbohydrates promotes clostridial activity to generate butyric acid and silage with low palatability. Therefore, ensiling should include the application of inoculants, such as cellulase and Lactobacillus casei LC [
17].
There are no reports on the effects of mulberry silage on the rumen microflora of sheep, which is now possible to investigate with the use of high-throughput sequencing technology. We hypothesized that when used as a livestock feed, mulberry silage may have a positive effect on rumen fermentation and nutrient digestion and absorption due to its high content of polyphenols and flavonoids [
18,
19]. To investigate this further, we offered mulberry silage as a partial replacement for maize silage and soyabean meal in the diet of Hu lambs. In particular, we explored the effects of replacing maize silage in diets with mulberry silage on growth performance, gastrointestinal health, rumen fermentation parameters, and microbial diversity in Hu lambs, a sheep breed that originates from Mongolian sheep and is well adapted to the hot, humid conditions that prevail in central China.
2. Materials and Methods
2.1. Experimental Design
This study was conducted in Yuemeihe Agriculture and Animal Husbandry Co, Ltd., Lin Ying County, Luohe, Henan Province, China, from April to June 2021. The feeding trial was performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agriculture University (Permit Number: 12-1328; Date: 05-2021). Sheep were housed in a semi-closed portal-framed building, with a transparent roof allowing good lighting of the sheep’s environment. The temperature was between 18 and 28 °C. A total of 96 healthy 3-month-old fattening Hu lambs with similar body weight (27.59 ± 3.03 kg) and physiological state were selected and divided into 4 treatment groups (6 replicates in each group, 4 animals in each replicate) according to a completely randomized design. Mulberry silage was used to replace 0% (CON group), 20% (L group), 40% (M group) and 60% (H group) of the maize silage in the Hu lambs’ diets, which were fed as a total mixed ration (TMR). To ensure that both diets were formulated to meet a 120 g/d growth rate for fattening sheep (NRC, 2007), the input of soybean meal was progressively decreased as mulberry silage replaced maize silage (
Table 1) because of the higher crude protein (CP) concentration in mulberry silage. Thus, in the treatment CON, 12% (L), 24% (M) and 35% (H) of the soybean allocation was replaced by the mulberry silage. Despite this, there was an increase in ether extract, CP and Ca at high mulberry silage concentrations and a reduction in NDF and acid detergent fiber (ADF). In designing the feed composition, we aimed for a crude protein content of 14.5% (DM), but the crude protein and other nutrients in the feed changed somewhat in the different feed batches.
The feeding trial was 75 days, including a 15-day acclimation period and a 60-day formal trial, this being the normal length of the fattening period. The lambs were provided with ad libitum TMR feed twice a day at 07:00 a.m. and 17:00 p.m. Feed residues were weighed daily and the ration supply was adjusted so that 5% of the feed remained as orts each day. Each group of lambs had a separate drinking bowl providing for ad libitum intake. The body weight of each lamb was recorded (Guandong Senssun Weighing Apparatus, Zhongshan, China, error ± 0.01 kg) for two consecutive days before the morning feeding every month, and the average daily weight gain was calculated. Before the end of the experiment, 12 animals in each group were randomly selected for blood sampling, of which 6 were used for slaughter. These 6 lambs were fasted for 24 h and not given access to water for 2 h before transportation for approximately 30 min to a local slaughterhouse, complying with Regulation (EC) No. 1099/2009, where they were killed after stunning with high voltage electricity.
2.2. Feeding and Management of Experimental Animals
Before the experiment, the pens were sterilized, and the flocks were dewormed and sheared. Pens were disinfected with 3% Lysol twice a week. The health of the flock was checked daily, and any animals with suspected illness were treated promptly. No animals died during the test period.
2.3. Preparation of Mulberry Silage
Mulberry was obtained from Yuemeihe Agriculture and Animal Husbandry Development Co., Ltd, Xuchang, China. When the mulberry had grown to 1.5 m, we used a silage harvester (Claas jaguar 800, Wister, Germany) to collect the whole mulberry trees to a stubble height of 15–20 cm. The trees were harvested 3–4 times a year, with regrowth apparent 3–7 days after harvest. One gram of Chr Hansen lactococcus lactis and lactobacillus brucei powder was added to each ton of mulberry silage (lactic acid bacteria >1.3 × 10
11 CFU/g). An automatic silage wrapping machine (Qufuxinlian Heavy Industry XL-5552, Shandong, China) was used to exclude air from the harvested material, and the samples were stored for 60 days after harvesting. After fermentation, dry matter (DM) (Association of Official Analytical Chemists AOAC, 1990: Method 934.01), ether extract (EE) (AOAC, 1990: Method 920.39), ash (AOAC, 1990: Method 942.05), calcium (Ca) (AOAC, 1990: Method 985.35), phosphorus (P) (AOAC, 1990: Method 986.24) and CP contents were determined using a Kjeldahl analyzer (Kjeltec 2300; FOSS Analytical AB, Hoganas, Sweden). Neutral and acidic detergent fibers were determined using an Ankom fiber analyzer (Ankom Technology, Fairport, NY, USA) as described by Van Soest [
20]. The nutrient composition of the mulberry and maize silages is shown in
Table 2.
2.4. Collection and Analysis of Samples
2.4.1. Feed and Rumen Sample Collection and Chemical Analysis
Samples of the total mixed rations (TMR) fed for each group were collected, and their DM, EE, NDF, ADF, Ash, CP, Ca, P and gross energy (GE) were determined by the methods described above. On the 75th day of the experiment, 6 lambs in each group were randomly selected, and approximately 50 mL rumen content samples were taken by a gastric tube rumen fluid sampler (Shanghai Silidi Scientific Instrument Co., LTD., Shanghai, China) 3 h after feeding in the morning. This method has been used in previous experiments (Xue [
21] and Sun [
22]), which proved that the sampler and intubation method had no effect on animal health and test results. The rumen fluid samples were filtered by 4 layers of gauze and divided into 2 centrifuge tubes with 5 mL (stored in liquid nitrogen and sent to Shanghai Meiji Biotechnology Co., Ltd. (Shanghai, China) for 16S rDNA sequencing for bacterial community structure analysis). A 10 mL sample in the centrifuge tube was stored at −20 °C for NH
3-N and volatile fatty acids (VFA) analysis. The pH of the remaining rumen fluid was immediately determined using a pH S-3C precision acidity meter (Shanghai Lei Ci Instrument Factory, Shanghai, China). The concentrations of VFA were detected using an ion chromatography system (S-150; Sykam, Munich, Germany) equipped with an chromatographic column (NaOH EGCS, Thermo Finsher, Waltham, MA, USA). The NH
3-N concentration was analysed using the phenol hypochlorite colorimetric method [
23]. Immediately after slaughter, 2 cm lengths of the jejunum and ileum and 2 cm
2 of the rumen compartments were excised, washed with phosphate buffer and immediately fixed with 4% formaldehyde solution for observation of tissue morphology.
2.4.2. qPCR Amplification of 16S rDNA Genes
All collected rumen fluid samples were subjected to high-throughput sequencing of the V3 + V4 region of the 16SrDNA gene to analyze the microbial diversity. The sequencing was performed at Shanghai Meiji Biotechnology Co, Ltd. (Shanghai, China) DNA was extracted from the samples using the Fast DNA Soil Rotation Kit (MPBio, Santa Ana, CA, USA) and high-throughput sequencing analysis was performed on the Majorbio cloud platform (
www.majorbio.com, accessed on 18 March 2022). Forward primer 338F, 5′ACTCCTACGGGAGgCAGCAGCAGcag3′ and reverse primer 806R-5′-GGACTachVGGGTWTCTAAT3′ were used to amplify the 16SrDNA gene in V3 and V4 regions. Polymerase linked reaction was carried out using the methods of Wang [
24]. A DNA library was constructed and run on the I11nmina MISeq instrument, and an amplification library was completed on the I11nmina MISeq PE300 platform. The NCBI Sequence Read Archive (SRA) database was used to deposit raw reads (Accession Number: PRJNA819287).
2.4.3. Analysis of Gastrointestinal Tissue Morphology
Morphological examination of the intestinal tract and rumen was based on Nosworthy [
25] hematoxylin-eosin staining. The tissues fixed in formaldehyde were dehydrated in ethanol (50%, 70%, 80%, 90%, 100%) solution, rinsed with xylene, and embedded in paraffin. Four 5 µm slices were cut from each well-embedded sample and stained with hematoxylin-eosin. The height of the intestinal villi, the depth of the crypt, and the length and width of the rumen papilla were measured under the microscope at 10 points each (Olympus Corporation CKX53, Tokyo, Japan).
2.5. Statistical Analysis
All data were initially processed using Excel software and then SPSS software (v. 26, IBM, Armonk, NY, USA). The results of the data were analysed using the ANOVA procedure. Statistically significant differences between pairs of groups were assessed using Duncan’s test. Differences were considered significant at p < 0.05.
5. Conclusions
Compared with maize silage, mulberry silage had some nutritional advantages, even though these were not reflected in body weight gain. The replacement of maize silage and soyabean meal with mulberry silage improved the rumen fermentation pattern, and possibly increased the utilization rate of nitrogen. Its reduced fiber content appears to have retarded rumen papillae development, which may explain why the improved nutrient content did not improve weight gain. Although mulberry silage can cause changes in the rumen histological morphology and the rumen microbial composition, the bacterial diversity indices did not change significantly, indicating that the rumen fermentation status remained stable after adding mulberry silage to the diets. Therefore, replacing maize silage with mulberry silage offers great potential in reducing the environmental impact of livestock production without sacrificing production capacity.