Effects of Diets Supplemented with Ensiled Mulberry Leaves and Sun-Dried Mulberry Fruit Pomace on the Ruminal Bacterial and Archaeal Community Composition of Finishing Steers

RESEARCH ARTICLE Effects of Diets Supplemented with Ensiled Mulberry Leaves and Sun-Dried Mulberry Fruit Pomace on the Ruminal Bacterial and Archaeal Community Composition of Finishing Steers a11111 Yuhong Niu1,2,3, Qingxiang Meng1,2, Shengli Li1,2, Liping Ren1,2, Bo Zhou1,2, Thomas Schonewille4, Zhenming Zhou1,2* 1 State Key Laboratory of Animal Nutrition, Beijing, 100193, P. R. China, 2 College of Animal Science and Technology, China Agricultural University, Beijing, 100193, P. R. China, 3 College of Agronomy and Biotechnology, China Agricultural University, Beijing, 100193, P. R. China, 4 Department of Farm Animal Health, Utrecht University, Utrecht, 3584 CL, The Netherlands * OPEN ACCESS Citation: Niu Y, Meng Q, Li S, Ren L, Zhou B, Schonewille T, et al. (2016) Effects of Diets Supplemented with Ensiled Mulberry Leaves and Sun-Dried Mulberry Fruit Pomace on the Ruminal Bacterial and Archaeal Community Composition of Finishing Steers. PLoS ONE 11(6): e0156836. doi:10.1371/journal.pone.0156836 Editor: Abhishek Deshpande, Cleveland Clinic, UNITED STATES Received: November 11, 2015 Accepted: May 17, 2016 Published: June 3, 2016 Copyright: © 2016 Niu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the China Agricultural Research System (CARS-38), the National Natural Science Foundation of China (31372335), and Special Fund for Agro-scientific Research in the Public Interest (201503134). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract This study investigated the effects of ensiled mulberry leaves (EML) and sun-dried mulberry fruit pomace (SMFP) on the ruminal bacterial and archaeal community composition of finishing steers. Corn grain- and cotton meal-based concentrate was partially replaced with EML or SMFP. The diets had similar crude protein (CP), neutral detergent fiber (NDF), and metabolizable energy. Following the feeding trial, the steers were slaughtered and ruminal liquid samples were collected to study the ruminal microbiome. Extraction of DNA, amplification of the V4 region of the 16S rRNA gene, and Illumina MiSeq pyrosequencing were performed for each sample. Following sequence de-noising, chimera checking, and quality trimming, an average of 209,610 sequences were generated per sample. Quantitative realtime PCR was performed to examine the selected bacterial species in the rumen. Our results showed that the predominant phyla were Bacteroidetes (43.90%), Firmicutes (39.06%), Proteobacteria (4.31%), and Tenericutes (2.04%), and the predominant genera included Prevotella (13.82%), Ruminococcus (2.51%), Butyrivibrio (2.38%), and Succiniclasticum (2.26%). Compared to the control group, EML and SMFP groups had a higher abundance of total bacteria (p < 0.001); however, the bacterial community composition was similar among the three groups. At the phylum level, there were no significant differences in Firmicutes (p = 0.7932), Bacteroidetes (p = 0.2330), Tenericutes (p = 0.2811), or Proteobacteria (p = 0.0680) levels among the three groups; however, Fibrobacteres decreased in EML (p = 0.0431). At the genus level, there were no differences in Prevotella (p = 0.4280), Ruminococcus (p = 0.2639), Butyrivibrio (p = 0.4433), or Succiniclasticum (p = 0.0431) levels among the groups. Additionally, the dietary treatments had no significant effects on the archaeal community composition in the rumen. Therefore, EML and SMFP supplementation PLOS ONE | DOI:10.1371/journal.pone.0156836 June 3, 2016 1 / 14 Effect of Mulberry on Ruminal Bacterial and Archaeal Community Composition Competing Interests: The authors have declared that no competing interests exist. had no significant effects on the ruminal bacterial or archaeal community composition of finishing steers. Introduction Mulberry (Morus spp., family Moraceae), a fast-growing deciduous tree, thrives under variable climatic conditions ranging from temperate to tropical. Mulberry is a multipurpose tree that produces fruits for human consumption, foliage for rearing silkworm, medicine for patients, and fodder for animal feed [1]. Mulberry leaves are succulent, characterized by high crude protein (CP; 19.4%) and low neutral detergent fiber (NDF; 36.1%) [2]; mulberry fruit pomace is rich in carbohydrates (20.85%), CP (21.86%) and low in NDF (49.06%) [3]. These mulberry by-products represent potential feed sources for herbivores and monogastric animals. Studies have shown that the addition of mulberry leaves to ruminant feed reduces the need for expensive protein supplements [4,5]. Therefore, researchers have evaluated the use of mulberry leaves and fruit pomace in animal feeding [4–9]. Our previous study has shown that ensiled mulberry leaves (EML) and sun-dried mulberry fruit pomace (SMFP) can be used in finishing steer diets without impairing their productive performance or carcass characteristics. Our results revealed that the SMFP-fed group had lower ruminal ammonia and total volatile fatty acid (VFA) concentrations than the EML-fed group [10], probably due to differences in the ruminal microbiome between the two groups. It has been reported that there is a correlation between host physiology and genus abundance. Dietary changes affect the ruminal microbiome (i.e., bacteria, protozoa, and fungi) [11–13], and changes in the ruminal microbiome affect the digestive capacity of the animal (e.g., improved fiber utilization and/or decreased methane production) [14–16]. We hypothesize that the partial replacement of concentrate with 8% EML or 6.3% SMFP in the diet affects the ruminal microbiome. Therefore, the objective of this study was to assess the effects of diets supplemented with EML or SMFP on the ruminal bacterial and archaeal community composition of finishing steers. Materials and Methods Experiments were conducted at the Beef Cattle Research Station of China Agricultural University in Daxing, Beijing. The protocol was approved by the China Agricultural University’s Animal Welfare and Ethical Committee (Permit No. DK1008). Animals, diets, and samples This study was part of a larger experimental trial investigating the effects of EML and SMFP on growth performance, ruminal fermentation, blood biochemical parameters, and carcass characteristics of finishing steers [10]. In which, medium-frame crossbred Simmental steers (357.06 ± 16.5 kg; 15 months of age) were divided into three groups. The control group (CON) received a typical total mixed ration (TMR); the EML group received a typical TMR supplemented with 8% EML; and the SMFP group received a typical TMR supplemented with 6.3% SMF (...truncated)