Transcriptome analysis of fasting caecotrophy on hepatic lipid metabolism in New Zealand rabbits

Background Rabbit produce two kinds of feces: hard and soft feces, and they have a preference for consuming the latter. Although this habit of rabbits has been reported for many years, little is known on whether this behavior will impact growth performance and metabolism. The RNA-Seq technology is an effective means of analyzing transcript groups to clarify molecular mechanisms. The aim of the present study was to investigate the effects of fasting caecotrophy on growth performance and lipid metabolism in rabbits. Results Our results indicated that, compared with the control group, the final body weight, weight gain, liver weight, specific growth rate and feed conversion ratio were all decreased in the experimental group (P<0.05). Oil red staining of the liver tissue indicated that fasting caecotrophy resulted in decrease of lipid droplet accumulation. RNA sequencing (RNA-seq) analysis revealed a total of 301.2 million raw reads approximately 45.06 Gb of high-quality clean data. The data were mapped to the rabbit genome (http://www.ensembl.org/Oryctolagus_cuniculus). After a five step filtering process, 14964 genes were identified, including 444 differentially expressed genes (P<0.05, foldchange≥1). Especially for remarkable changes of genes related to lipid metabolism, RT-PCR further validated the remarkable decrease of these genes in fasting caecotrophy group, including CYP7A1, PPARG, ABCA1, ABCB1, ABCG1, GPAM, SREBP, etc. KEGG annotation of the differentially expressed genes indicated that the main pathways affected were retinol metabolism, pentose and glucuronide interactions, starch and sucrose metabolism, fatty acid degradation, steroid hormone biosynthesis. Conclusion In conclusion, the present study revealed that banning caecotrophy reduced growth rate and altered lipid metabolism, our results laid instructive basis for rabbit feeding and production. These data also provides a reference for studying the effects of soft feces on other small herbivores.


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Introduction 2 Many small herbivores have a natural instinct of caecotrophy [1]. Because of the 3 small body shape and their digestive tract volume is limited, the average residence 4 time of food in the digestive tract is relatively short [2,3]. In order to meet their 5 nutrition needs, small herbivores need to obtain adequate high-quality food [4]. Small 6 herbivores mainly rely on low-quality, high-fiber plant stems and leaves as food 7 sources, with cellulose from symbiotic microorganisms in the hind-gut aiding in 8 digestion [5,6]. Because microbial fermentation takes longer time than the average 9 residence time of food in the digestive tract, increasing digestibility by ingesting 10 incompletely-digested nutrients is an important nutritional strategy for small 11 herbivores [7][8][9]. 12 There are two types of feces excreted by rabbits: hard feces (nutrient-poor) and 13 soft feces (consist of protein, vitamins, and inorganic salts) [10][11][12]. Soft feces also 14 contain a large number of microorganisms, which are important for microbial 15 fermentation in herbivores. Thus, we speculate that rabbits might build their intestinal 16 microbial flora through their caecotrophy behavior [13][14][15]. Although modern feeding 17 and management techniques are designed to fully meet the nutritional needs for 18 growth, rabbits still maintain the habit of eating soft feces [16,17]. One study 19 comparing high and low body weight Rex rabbits, the high-weight group had more 20 abundant bacterial groups than the low-weight group, with bacterial groups in soft 21 feces being more abundant than hard feces [18][19][20]. Intestinal flora and their host have 22 a mutually beneficial symbiosis, with the host nutrition and metabolism, immune 23 system development, disease resistance and other physiological functions playing an 24 important role [21,22]. 25 Liver plays an important role in overall metabolism of lipids, including its 26 function in supporting digestion, absorption, decomposition, synthesis and 27 transportation of lipids [23,24]. Bile acids produced by the liver are conversion 6 28 products of cholesterol metabolism. These compounds are important for emulsifying 29 dietary lipids and promote the digestion and absorption of lipids [25]. By generating 30 ATP for its own utilization, the liver is also the main site for oxidative decomposition 31 of fatty acids and production of ketone bodies [26][27][28]. In monogastrics, liver is the 32 primary site for fatty acids oxidation and ketone bodies production [29,30] temperature was controlled at around 23±1℃ and rabbits had free access to food and 65 water. Specifically, they were fed with the same pelleted diet at the breeding farm, 66 and the diet was replaced with the experimental diet gradually.  Bioengineering Institute, Nanjing, China). Liver, spleen, kidney, lung, heart, muscle 77 and adipose were collected and weighted, and were put into liquid nitrogen 78 immediately until analysis. Body weight and feed intake was recorded every week.   The high-quality total RNA of the rabbit livers (n = 3 per group) were send for 104 the preparation of RNA-seq libraries. All the libraries were sequenced by the Illumina 105 HiSeq TM 2500 platform (Biomarker technologies Corporation, Beijing, China).  were estimated by fragments per kilobase of million fragments mapped. Differential 123 expression analysis of two groups was performed using the DESeq R package. DESeq 124 provide statistical routines for determining differential expression in digital gene 125 expression data using a model based on the negative binomial distribution. The 126 resulting P value was using the Benjamini and Hochberg's approach for controlling 127 the false discovery rate. Here, only unique reads with an absolute value of log 2 ratio ≥ 128 1 and FDR significance score < 0.01 were used for the subsequent analysis.    Table 1, compared with the control group, the bodyweight, food 163 intake, specific growth rate (SGR) and feed conversion rate (FCR) of the experimental 164 group rabbits were significantly decreased (P<0.05), while the hepatosomatic index 165 (HSI) did not show significant differences between these two groups (P>0.05).

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During the experimental period, food intake of these two group did not show 167 significant change (Fig. 2). The liver weight and the perirenal fat in the experimental 168 group were significant decreased compared to the control group (P<0.05) (Fig. 3). 169 We also measured lipid droplet accumulation in liver tissue by using Oil red O 170 staining, results showed that lipid droplet accumulation in the liver tissue of the 171 experimental group is significantly low than the control group (P<0.05) (Fig. 4).
172 Table 1 Growth index changes of the experimental group and control group 173 In the same row, values with no letter or the same latter superscripts mean no significant   Table). The DEGs had 63 GO terms (S3 Table) and were mapped 205 onto 217 KEGG pathways (S4 Table). Among them, the most significant GO term represents the pathway name, and the abscissa is the enrichment factor, which represents the ratio 233 of the proportion of genes in the differential gene that are annotated to the pathway to the 234 proportion of genes that all genes are annotated to. The larger the enrichment factor, the more 235 significant the level of enrichment of the differentially expressed genes in this pathway. The color 236 of the circle represents q-value, q-value is the p value after correction by multiple hypothesis test.

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The smaller the q-value, the more reliable the significance of the differential expression of the (Pearson'r=0.929). All the genes were related to the lipid metabolism (Fig.8).

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In order to analyze the correlation between differentially expressed genes and

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In the present study, growth performance results showed that banned the rabbit 263 from eating their soft feces had great effects on body weight, weight gain, and SGR.

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After slaughter, weight of liver weight, tissue and perirenal fat weight of the 265 experimental group was lower than the control group. Further analysis was performed 266 to measure the lipid droplet accumulation in liver tissue between these two groups,

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Oil red O staining results of the liver tissue indicate that lipid droplet accumulation 268 was decreased in the experimental group compared with the control group, so as the 269 quantified data, Results in our present study is consistent with the previous study [35].

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Some research showed that, under a normal diet condition, prevent the rabbits from 271 eating soft feces leads them to develop malnutrition [36,37]. It is speculated that there 272 may be two main reasons for this phenomenon, one is that soft feces is rich in 273 vitamins and microbial proteins, with the same feed intake, rabbits that were fasted to

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Liver is one of the most active organ for lipid metabolism, it is also a storage 318 organ of retinol, more than 90% of the retinol is stored in the liver [48]. Retinol in the 319 liver undergoes a biological role in the transport of plasma to peripheral target organs, 320 and this process required the involvement of the specific vector retinol-binding white rabbits, as well as to study the fecal behavior of other small mammals. Research System (G2013-08)".

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The authors declare no competing financial interest.