Influence of cecotrophy on fat metabolism mediated by caecal microorganisms in New Zealand white rabbits
Abstract
Cecotrophy is a special behaviour of rabbits. Eating soft faeces can improve feed efficiency and maintain gut flora in rabbits. In our previous study, we found that fasting from soft faeces significantly reduced growth rate and total cholesterol (TC) in New Zealand white rabbits (NZW rabbits), thereby resulting in lower values for body weight and fat deposition in the soft faeces fasting group than in the control group. However, it has not been demonstrated whether cecotrophy by NZW rabbits can regulate lipid metabolism by changing the diversity of caecal microorganisms. In this study, thirty-six 28-day-old weaned NZW female rabbits were randomly divided into two groups (the soft faeces fasting group and the control group) and fed to 90 days. Rabbits in the experimental group were treated with an Elizabeth circle to prevent them from eating their soft faeces. Then, the caecal contents of three rabbits from the soft faeces fasting group and three rabbits from the control group were collected for metagenomic sequencing. We found that the abundance of Bacteroides increased, while Ruminococcus decreased, compared with the control group after fasting from soft faeces. Relative abundance was depressed for genes related to metabolic pathways such as ascorbate and aldarate metabolism, riboflavin metabolism and bile secretion. Moreover, there was a general correlation between variation in microbial diversity and fat deposition. Bacteroides affects body weight and TC by participating in the riboflavin metabolism pathway. By investigating the effect of cecotrophy on caecal microorganisms of rabbits, we identified the key microorganisms that regulate the rapid growth performance of NZW rabbits, which may provide useful reference for the future research and development of microecological preparations for NZW rabbits.
1 INTRODUCTION
Natural selection plays an important role in the evolution of small- and medium-sized herbivorous mammals. Many medium and small herbivorous mammals are often unable to obtain sufficient nutrients to meet their metabolic needs due to environmental factors. Therefore, animals have evolved a variety of effective physiological mechanisms from less effective precursors over the long-term adaptive evolutionary process (Travers, Eldridge, Dorrough, Val, & Oliver, 2018; Zhang, Lou, Shen, Fu, & Wang, 2017). The colonic separation mechanism involves wriggling the chyme in the caecum to the proximal colon, and small particles of digestive material are returned to the caecum to form soft faeces, while large particles accumulate in the fusus coli to form hard faeces (Schulze, 2015). Cecotrophy refers to the behaviour of animals eating caecal faeces, and the rabbit is one of the most typical small- and medium-sized herbivores that practices cecotrophy (Kuijper, Wieren, & Bakker, 2010).
Rabbits are monogastric animals that, unlike ruminants, have no rumen for microbial fermentation. Over long-term biological evolution, rabbits have gained a caecum with a function similar to that of the rumen along with the behaviour of cecotrophy, so that nutrients are better utilized (Langer, 2002). First, cecotrophy allows small digestive particles to undergo a second fermentation and digestion in the digestive tract, which increases the digestibility of the feed (Sakaguchi, 2015). Second, cecotrophy improves protein utilization efficiency. A previous study reported that preventing an animal from eating its faeces can reduce the animal's absorption of protein and protein digestibility, resulting in slower growth than in faecal-feeding animals (Haichun et al., 2016). Moreover, there are a large number of microorganisms in soft faeces, which produce proteins of high biological value. Fasting from soft faeces led to a one-third reduction in the digesta retention time in the alimentary tract of the rabbit (Gidenne & Lapanouse, 2010). In addition, the intestinal flora plays an important role in the host's physiological functions such as nutrient metabolism, immune system development and disease resistance (Bengmark, 2013; Laparra & Sanz, 2010; Natividad & Verdu, 2013).
Rabbits can produce soft faeces and hard faeces. Hard faeces are usually large, present in large amounts, relatively dry, with a rough surface, and maybe brown to varying degrees due to different ingredients in the feed, while soft faeces are usually soft, mainly discharged at night, covered with a layer of white mucus and smooth with a shiny surface (Zeng et al., 2015). Because the acidity of the rabbit stomach (pH 1.9) is much higher than that of other parts of the digestive tract (pH 4.3–6), the mucous membrane on the surface of the soft stool maintains this high acidity from the stomach (Hirakawa, 2001). Compared with hard faeces, soft faeces contain more water, crude protein, total amino acids, essential amino acids and minerals such as Na, Cl, K and other elements. Moreover, the nutrients of soft faeces and caecal contents are very similar. The crude protein in the caecal contents is approximately 190–340 g/kg, while it is approximately 230–335 g/kg in soft faeces. The protein content in soft faeces can provide 15%–22% of the daily protein intake in rabbits. There are many microorganisms in both types of rabbit faeces, but the microbial content in soft faeces is 4–5 times that of hard faeces (Zeng et al., 2015).
The traditional molecular biological technique for studying intestinal flora is selective culture technology, which is very restrictive for studying the large microecological system of the intestine, because 40%–90% of microorganisms cannot be cultured under laboratory conditions (Zoetendal, Collier, Koike, Mackie, & Gaskins, 2004). With the rapid development of high-throughput sequencing technology, it is possible to use metagenomic sequencing technology to investigate the composition, distribution and functioning of microorganisms in the host. This approach principally investigates the sum of genes of microorganisms such as bacteria and fungi in a specific environment. Metagenomics is the total extraction of microbial DNA from environmental samples such as water, intestines or soil, the use of high-throughput sequencing technology to obtain raw data for all microbial genes and subsequent analysis on the combined database to understand the composition and functioning of the microbial community.
Preventing cecotrophy and concomitant reduced growth and altered lipid metabolism provided the instructive basis for rabbit feeding and production in our previous study (Wang et al., 2019). Briefly, a total of thirty-six 28-day-old New Zealand white rabbits (NZW rabbits) were divided into two groups (the soft faeces fasting group and the control group) and fed to 90 days. We found no significant difference in feed intake between the two groups, but the growth rate of the soft faeces fasting group was slower than that of the control group. There were no significant differences between the two groups in serum indices, triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C) or low-density lipoprotein-cholesterol (LDL-C) (p > .05), while the level of total cholesterol (TC) in the soft faeces fasting group was significantly lower than in the control group (p < .05). However, it has not been proven that cecotrophy affects health and growth of NZW rabbits through alteration of gut microflora. Therefore, the aim of this study was to demonstrate the effect of cecotrophy on lipid metabolism through alteration of caecal microorganisms in NZW rabbits.
2 MATERIALS AND METHODS
2.1 Animals and phenotypic data collection
In our previous study, thirty-six weaned female NZW rabbits were raised in the animal experimental centre of Henan Agriculture University. The temperature was controlled at approximately 23 ± 1℃, and rabbits had free access to food and water. Thirty-six weaned female NZW rabbits were randomly divided into two groups, the soft faeces fasting group and the control group. Growth and blood biochemical indices were measured, and transcriptome sequencing of the liver was conducted. The results showed that growth rate and total cholesterol (TC) were both lower in the experimental group than in the control group (p < .05). See the article by Wang et al. (2019) for details. Here, we examined the caecal contents of 90-day-old rabbits from the study by Wang et al. (2019), three rabbits from the soft faeces fasting group and three rabbits from the control group.
2.2 DNA extraction, library construction and metagenomic sequencing
DNA for metagenomics was extracted from caecal contents by using the E.Z.N.A.® DNA Kit (Omega Bio-Tek), according to manufacturer's protocols. DNA quality was examined with a 1% agarose gel electrophoresis system.
The qualified DNA sample was broken into a fragment of approximately 300 bp using a Covaris M220 ultrasonic generator (Gene Company Limited), and then, the target fragment was selectively recovered using a TruSeq Nano DNA Sample Preparation Kit (Illumina) and the linker was ligated at both ends to construct a sequencing library. After the library was constructed, the Illumina Hiseq2500 platform (Illumina Inc.) was used for double-end sequencing, and the downsampling data were used for subsequent bioinformatics analysis.
2.3 Sequence processing and bioinformatics analysis
To improve the quality and reliability of the subsequent analysis, the original sequence was first optimized using techniques such as splitting, mass shearing and decontamination. The 3’ and 5’ ends were stripped using SeqPrep (https://github.com/jstjohn/SeqPrep). Low-quality reads (length < 50 bp or with a quality value < 20 or having N bases) were removed by Sickle (https://github.com/najoshi/sickle). The optimized sequence was spliced and assembled using the SOAPdenovo v1. 06 splicing software (Li, Li, Kristiansen, & Wang, 2008). Gene prediction was performed for a contig from the splicing results using MetaGene (Hideki, Jungho, & Toshihisa, 2006).
Then, the obtained gene sequences were used to determine relative abundances of species, and functional classification and annotation were performed. The functions and metabolic pathways of genes were estimated using the BLASTP program (e value < le-5) to search the Evolutionary Genealogy of Genes_Non-supervised Orthologous Groups (eggNOG, http://egneg.embl.de/version 3.0) and the Kyoto Encyclopedia of Genes and Genomes (KEGG http://www.genome.jp/kegg/ ) databases. Besides, based on the above analysis, statistical analysis and exploration of similar clustering, group sorting, difference comparisons and other procedures were performed, the results were visualized, and the effective information in the data was mined.
3 RESULTS
3.1 Metagenome sequencing results
The DNA of caecal digesta was extracted, fragmented and sequenced using the Illumina Miseq platform, generating a total of 64.61 Gb of clean reads for six samples, with average sample sizes of 11.36 Gb in the high group and 9.72 Gb in the low group. Genes with nucleic acid length of 100 bp or longer were selected, open reading frames (ORF) were predicted using MetaGene to obtain 4.49 M sequences (Table S1), and the tags were classified taxonomically from genus to species.
3.2 Effect of soft faeces fasting on all sample microorganisms at genus and species levels
Soft faeces fasting affected the microbial diversity in the caeca of NZW rabbits (Figure 1). After fasting from soft faeces, the abundance of Bacteroides was elevated, while Ruminococcus abundance was depressed, compared with the control group (Figure 1a). At the species level, the abundance of norank_g__Bacteroides was higher, while uncultured_Ruminococcus_sp. was lower, than in the control group (Figure 1b).
A linear discriminant analysis (LDA) plot revealed that some genera served as unique caecal microbial biomarkers for the soft faeces fasting group and the control group (Figure 2). Seven genera (Bacteria-k-norank-Bacteria, Bacteria, Lachnospiraceae, Oxalobacteraceae, Cheirogaleidae, Lyophyllaceae and Beloniformes) were significantly more abundant in the soft faeces fasting group than in the control group, while fifty genera (such as Actinobacteria, Clostridiales and Firmicutes-c-norank-Firmicutes) were significantly more abundant in the control group.
Principle component analysis (PCA) was performed to compare the similarities in microbiotas between the soft faeces fasting group and the control group (Figure 3). Microbial composition at the genus and species levels differed between the soft faeces fasting group and the control group.
3.3 Effect of soft faeces fasting on intestinal microbial function
The KEGG annotation revealed that the experimental group enriched 265 pathways, while the control group enriched 279 pathways. Among these pathways, 38 pathways were significantly different, and differences for 7 pathways were extremely significant (Figure 4).
For the main enrichment pathways, aminoacyl-tRNA biosynthesis, mismatch repair, nucleotide excision repair, protein export, carbon fixation in photosynthetic organisms, homologous recombination and glycolysis/ gluconeogenesis were significantly lower in the soft faeces fasting group than in the control group (p < .05), while carbon fixation pathways in prokaryotes, pentose and glucuronate interconversions, and fructose and mannose metabolism were significantly higher than in the control group (p < .05).
Of the pathways with less microbial gene enrichment, there were also pathways related to diseases in NZW rabbits, such as influenza A, hepatitis C and glioma. Moreover, ascorbate and aldarate metabolism, riboflavin metabolism and bile secretion can participate in the metabolic processes of NZW rabbits.
These results showed that soft faeces fasting significantly affected the proliferation of caecal microbes, the utilization of polysaccharides and the metabolism of NZW rabbits.
3.4 Analysis of correlations of environmental factors with caecal genus levels and caecal microbial KEGG function
The heatmap plot shows correlations of environmental factors with microbial genus levels and caecal microbial KEGG function (Figure 5). There were 3 genera significantly associated with TC: norank_d__Bacteria and Oscillibacter were positively correlated with TC, while Akkermansia was negatively correlated; 2 genera were related to weight: norank_o__Clostridiales and Coprococcus were negatively correlated.
Most of the KEGG pathways of caecal microbes were associated with TC. Among them, aminoacyl-tRNA biosynthesis, nucleotide excision repair, protein export, mismatch repair and homologous recombination were significantly different between the soft faeces fasting group and the control group. There were 4 pathways significantly associated with weight: pentose and glucuronate interconversions, cyanoamino acid metabolism, butanoate metabolism and glycerophospholipid metabolism.
4 DISCUSSION
In this study, we investigated and compared the caecal intestinal microbes of the soft faeces fasting group and the control group using metagenome sequencing technology in NZW rabbits. At the genus level, the abundance of Bacteroides increased after soft faeces fasting. Currently, the thick-walled bacteria and the Bacteroides are considered to be related to obesity, and the higher the ratio of Firmicutes/ Bacteroides, the more likely for the host to become obese. Obesity was associated with the composition of the host gut flora by comparing the intestinal flora of hereditary obese mice with lean littermates (Backhed et al., 2004; Ley, Turnbaugh, Klein, & Gordon, 2006; Turnbaugh et al., 2006). An investigation of obese and thin human volunteers found that obesity was associated with changes in the relative abundance of Bacteroidetes and Firmicutes. Dicksved found that the relative proportion of Bacteroidetes in the intestines of obese people was lower than in lean people, which is consistent with the results of this experiment. He also transplanted the intestinal flora of normal mice into sterile mice and found that the body fat of the sterile mice increased, indicating some bacteria in the intestine have a strong ability to release calories. Obese people have a lower proportion of Bacteroides in the intestine (Dicksved et al., 2008). Moreover, 75% of intestinal microbial genes of obese volunteers derived from Actinobacteria, while 42% of intestinal microbial genes of lean volunteers derived from Bacteroidetes. In this study, after soft faeces fasting, the proportion of Bacteroides in the caeca of NZW rabbits was reduced, and the result was consistent. Actinobacteria is Gram-positive bacteria, the GC content of Actinobacteria is higher, and it has no cell nucleus. There are many Actinobacteria in the guts of animals that play important roles in the health and immunity of hosts (Miao & Davies, 2010). The abundance of Actinobacteria was lower in the soft faeces fasting group, which may be related to the health of NZW rabbits after fasting from soft faeces. Therefore, cecotrophy in rabbits changes caecal microflora, in turn affecting healthy and normal growth and development.
Nucleotide excision repair and Homologous recombination pathways were related to the regulation of microbial expression, indicating that the expression and regulation of caecal microbes were depressed after soft faeces fasting. Protein export is mainly the ability of microbes to secrete proteins; therefore, it may be concluded that the ability of caecal microbes to secrete proteins was reduced after soft faeces fasting. In the soft faeces fasting group, the microbial genes enriched in Carbon fixation pathways in prokaryotes and in Fructose and mannose metabolism were significantly higher than in the control group (p < .05), indicating that after soft faeces fasting, the ability of microorganisms to degrade cellulose in the caeca of NZW rabbits was enhanced. ascorbate and aldarate metabolism, riboflavin metabolism and bile secretion exhibited significant differences (p < .05) between the soft faeces fasting group and the control group and can participate in the metabolic processes of NZW rabbits. In our previous study, riboflavin metabolism genes enriched in the intestinal microbes of NZW rabbits were significantly higher in the soft faeces fasting group than in the control group (Wang et al., 2019). A high level of riboflavin can reduce the concentration of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in the liver (Cogburn et al., 2018). HMGR is the rate-limiting enzyme in cholesterol synthesis, and a decrease in HMGR is beneficial through the reduction of cholesterol. The previous study reported that the lack of riboflavin promotes the synthesis of triglycerides as well as endoplasmic reticulum stress, which leads to high expression of SREBP-1c to increase the synthesis of triglycerides (Liu, Li, Ning, & Yang, 2015). Caecal flora can affect the lipid metabolism of NZW rabbits through riboflavin metabolism and bile secretion. In this study, the genes of riboflavin metabolism were significantly more enriched in the intestinal microbes of NZW rabbits in the soft faeces fasting group than in those of the control group. The TC content was lower in the soft faeces fasting group than in the control group; the results are consistent (Wang et al., 2019).
Correlation analysis among the production index, serum index and the KEGG function of the microbial annotation revealed that Oscillibacter abundance was positively correlated with body weight and lipid metabolism, while Akkermansia had a negative effect. In the different pathological stages of liver disease, the Akkermansia genus was significantly negatively correlated with liver weight and blood lipids (Dicksved et al., 2008). There was a strain under the genus Akkermansia—Akkermansia muciniphila was isolated and named in 2004 (Derrien, Vaughan, Plugge, & Vos, 2004). The epithelial cells of the human intestine are covered with a layer of mucous membrane, which contains much mucin. A mucous membrane can act as a binder for much intestinal flora, promoting interaction between humans and microorganisms, while Akkermansia muciniphila can use this mucin as its energy source to protect the intestine from pathogens through competition (Clara Belzer & Vos, 2012). Unlike other bacteria, Akkermansia muciniphila can store and secrete mucin, even if there are no nutrients in the intestine, especially during fasting (Muriel Derrien, Belzer, & Vos, 2017). In addition, Akkermansia muciniphila can release various kinds of by-products, for example acetic acid, which can play a role in weight control by its anorexic effect. Akkermansia muciniphila shows a strong correlation between gut microbiome richness and serum acetate (Dao et al., 2016). Moreover, Akkermansia muciniphila can induce the expression of fasting-induced adipose factor (FIAF), which can reduce fat expression of storage capacity. Feeding mice with live Akkermansia muciniphila can prevent diet-induced obesity without affecting appetite or eating habits (Everard et al., 2013). Nehra discovered that the abundance of Akkermansia muciniphila in the gut of humans and animals was inversely related to host body weight, fat content and insulin resistance, which can be used as a potential biomarker for assessing nutritional status and metabolic diseases (Derrien et al., 2004; Nehra, Allen, Mailing, Kashyap, & Woods, 2016). Obese mice with type 2 diabetes have a reduced abundance of Akkermansia muciniphila in a high-fat diet, and its abundance was increased after intervention with a drug, with increased body fat, fatty inflammation and insulin resistance having also been reversed (Plovier et al., 2016). Subsequently, researchers also found that Akkermansia muciniphila inhibits the negative effects of IFN-γ in glucose metabolism and improves the body's tolerance to glucose (Xie et al., 2016). All studies have shown that Akkermansia muciniphila is negatively correlated with lipid metabolism. Oscillibacter is a member of the thick-walled bacteria. Under the same feeding conditions, the ratio of Firmicutes to Bacteroides is higher in the intestines of obese mice (Frank A Duca et al., 2014). Three obesity-related bacteria were identified, including Oscillibacter, Clostridium 4 clusters and 14a clusters. If the intestinal flora of obese mice is transplanted into sterile mice, the sterile mice will gradually become obese, indicating Oscillibacter has a significant relationship with host lipid metabolism.
5 CONCLUSIONS
Through metagenomic sequencing technology, we discovered that cecotrophy can affect the diversity of caecal microbes, especially Bacteroides; its abundance was elevated after soft faeces fasting. Moreover, riboflavin metabolism, which can participate in the metabolic processes of NZW rabbits, differed significantly (p < .05) between the soft faeces fasting group and the control group. Bacteroides affects body weight and TC by participating in the riboflavin metabolic pathway. This study laid the foundation for finding the key microorganisms that affect rapid growth and development of rabbits and provided a useful reference for the future research and development of microecological preparations for NZW rabbits. However, due to the limited sample size, more studies within large-scale populations are required to support the results of present study.
ACKNOWLEDGEMENTS
This work was supported by the National Key R&D Program of China (2018YFD0502203) and the Special Fund for the Henan Agriculture Research System (S2013-08-G01).
CONFLICT OF INTEREST
The authors declare that they have no competing interests.
ANIMAL WELFARE STATEMENT
The study was designed and performed according to the guidelines of the Institutional Animal Care and Use Committee, College of Animal Husbandry and Veterinary Medicine of Henan Agricultural University (Permit Number: 17-0118).
