Identification of N6-Methyladenosine-Related Factors and the Prediction of the Regulatory Mechanism of Hair Follicle Development in Rex and Hycole Rabbits.

Hair follicle development directly affects the development of the rabbit fur industry. The growth and development of a hair follicle is modified and regulated by many genes and mechanisms. M6A is an important RNA modification. However, there are few studies on the effects of the regulation of m6A on hair follicle growth and development. In this study, hematoxylin-eosin (HE) staining was used to explore the difference in hair follicle development between Rex rabbits and Hycole rabbits, and we performed m6A sequencing to identify the key genes with m6A modification in hair follicle growth. The results showed that the hair length, coarse hair percentage, primary hair follicle ratio, and skin thickness of Hycole rabbits were significantly higher than those of Rex rabbits. However, the proportion of secondary hair follicles in Hycole rabbits was significantly lower than that in Rex rabbits. In addition, we found five differential methylases, 20 differential genes, and 24 differential signaling pathways related to hair growth and development. The results of the Sankey diagram showed that 12 genes were related to 13 signal pathways. Finally, we found that five methylases regulated the development of hair follicles through differential genes/signal pathways. These findings laid a molecular foundation for the function of m6A modification in hair development.

Exploring the growing forest musk deer (Moschus berezovskii) dietary protein requirement based on gut microbiome.

It is necessary to assess the appropriate dietary protein level of the forest musk deer (FMD), as nutritional needs are unclear. The microbiome in gastrointestinal tracts plays an important role in regulating nutrient utilization, absorption and host growth or development. Thus, we aimed to evaluate growth performance, nutrient digestibility and fecal microbiome of growing FMD supplied with different protein levels of diets. Eighteen 6-month-old male FMD with an initial weight 5.0 ± 0.2 kg were used in a 62-day trial. The animals were randomly distributed to three groups, the dietary crude protein (CP) level was 11.51% (L), 13.37% (M), and 15.48% (H). The results showed that the CP digestibility decreased as dietary CP level increased (p < 0.01). Compared with group L and H, FMD in M group has higher average daily gain, feed efficiency and neutral detergent fiber digestibility. For the fecal bacterial community, the percentage of Firmicutes was increased, Bacteroidetes was decreased and the diversity of microbiota significantly reduced (p < 0.05) with the increasing of dietary protein. The proportion of Ruminococcaceae_005, Ruminococcaceae_UCG-014 and uncultured_bacterium_f_Lachnospiraceae were significantly increased wtih rising CP, the proportions of Bacteroides and Rikenellaceae_RC9_gut_group were significantly decrease nevertheless at the genus level. The higher abundance of f_Prevotellaceae and g_Prevotellaceae_UCG_004 were found at M group by LEfSe analysis. The relative abundance of uncultured_bacterium_f_Ruminococcaceae was positively correlated with the average daily gain and feed conversion ratio (p < 0.05), whereas Family_XIII_AD3011_group was negatively correlated with feed conversion ratio (p < 0.05). The UPGMA tree showed L and M groups were closer in clustering relationship, while H group was clustered separately into a branch, which indicated that the bacterial structure had changed greatly with protein level increased from 13.37 to 15.48%. Overall, our results indicated that the optimum dietary CP for the growing FMD was 13.37%.

The Characterization and Differential Analysis of m6A Methylation in Hycole Rabbit Muscle and Adipose Tissue and Prediction of Regulatory Mechanism about Intramuscular Fat.

N6-methyladenosine (m6A) widely participates in various life processes of animals, including disease, memory, growth and development, etc. However, there is no report on m6A regulating intramuscular fat deposition in rabbits. In this study, m6A modification of Hycole rabbit muscle and adipose tissues were detected by MeRIP-Seq. In this case, 3 methylases and 12 genes modified by m6A were found to be significantly different between muscle and adipose tissues. At the same time, we found 3 methylases can regulate the expression of 12 genes in different ways and the function of 12 genes is related to fat deposition base on existing studies. 12 genes were modified by m6A methylase in rabbit muscle and adipose tissues. These results suggest that 3 methylases may regulate the expression of 12 genes through different pathways. In addition, the analysis of results showed that 6 of the 12 genes regulated eight signaling pathways, which regulated intramuscular fat deposition. RT-qPCR was used to validate the sequencing results and found the expression results of RT-qPCR and sequencing results are consistent. In summary, METTL4, ZC3H13 and IGF2BP2 regulated intramuscular fat by m6A modified gene/signaling pathways. Our work provided a new molecular basis and a new way to produce rabbit meat with good taste.

FTO promoted adipocyte differentiation by regulating ADRB1 gene through m6A modification in Hycole rabbits.

N6-methyladenosine (m6A), the most abundant internal mRNA modification in eukaryotes, plays a vital role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Our previous study found that ADRB1 gene has m6A modification in both muscle and fat tissue. In this study, we interfered with FTO and ADRB1 genes After we cultured rabbit preadipocytes respectively. Oil red O staining and triglyceride assay were used to detect adipocyte differentiation. RT-qPCR was used to detect gene expression level and MeRIP-qPCR was used to detect the m6A modification level of gene. The results showed that FTO promoted the differentiation of adipocytes. At the same time, FTO up regulated the expression of ADRB1 gene and down regulated the m6A modification level of ADRB1 gene. Finally, we found that ADRB1 inhibited adipocyte differentiation. Together, we showed that FTO promoted adipocyte differentiation by regulating ADRB1 gene through m6A modification.

N6-Methyladenosine Methylome Profiling of Muscle and Adipose Tissues Reveals Methylase-mRNA Metabolic Regulatory Networks in Fat Deposition of Rex Rabbits.

N6-methyladenosine (m6A) is the most prevalent internal form of modification in messenger RNA in higher eukaryotes and plays an important role in cancer, immunity, reproduction, development, and fat deposition. Intramuscular fat is the main factor used to measure the meat quality of an animal. The deposition of intramuscular fat and perirenal fat increases with age. However, there is no data on m6A modification of Rex rabbits and its potential biological roles in adipose deposition and muscle growth. Here, we performed two high-throughput sequencing methods, m6A-modified RNA immunoprecipitation sequence (MeRIP-seq) and RNA sequence (RNA-seq), to identify key genes with m6A modification on fat deposition in the muscle and adipose tissues of Rex rabbits. Then, qRT-PCR was used to identify the differently methylated genes related to fat deposition. Our findings showed that there were 12,876 and 10,973 m6A peaks in the rabbit muscle and adipose tissue transcriptomes, respectively. Stop codons, 3'-untranslated regions, and coding regions were found to be mainly enriched for m6A peaks. In addition, we found 5 differential methylases and 12 key genes of methylation modification related to fat deposition between muscle and adipose tissues samples. The expression levels of six random key genes were significantly higher in the fat than that in the muscle of Rex rabbits at different stages (p < 0.01). Finally, five differential methylases were found to regulate adipogenesis by affecting the expression of screened genes in different ways. These findings provided a theoretical basis for our future research on the function of m6A modification during the growth of fat deposits.