4D label-free quantitative proteomic analysis identifies CRABP1 as a novel candidate gene for litter size in rabbits.

In commercial rabbit breeding, litter size is a crucial reproductive trait. This trait directly determines the reproductive ability of female rabbits and is crucial for evaluating the production efficiency. We here compared differentially expressed proteins of in the ovary tissue from New Zealand female rabbits with high (H) and low (L) litter sizes by using 4D label-free quantitative proteomic technology and identified 92 differential proteins. The biological functions of these proteins were revealed through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Most distributions of GO and KEGG were related to reproduction, growth development, and metabolism. Furthermore, a novel candidate gene Cellular Retinoic Acid Binding Protein-1 (CRABP1), which was highly expressed in the L group, was selected for further biological function verification. The Cell Counting Kit-8 assay and flow cytometry analysis revealed that CRABP1 can promote granulosa cell (GC) apoptosis and inhibit GC proliferation. Furthermore, qRT-PCR and western blotting analysis revealed that CRABP1 regulates the genes (HSD17B1, Wnt-10b, FSHR, TAF4B, BMP15, and BMP6) and protein (Wnt-10b) associated with steroid hormone synthesis and follicle development. The PCR product direct sequencing method revealed single nucleotide polymorphisms in the core promoter region of CRABP1. Luciferase activity assays revealed that the transcriptional activity of the GG genotype was significantly higher than that of the TT or TG genotype. Different genotypes are accompanied by changes in transcription factors, which indicates that T-359G polymorphism can regulate CRABP1 expression. In general, we identified litter size-related genes and revealed the mechanism underlying the effect of CRABP1 on litter size. CRABP1 serves as a key factor in the reproductive capacity of rabbits and can act as a molecular biomarker for the breeding of New Zealand rabbits.

Effects of organic fertilizers on yield, soil physico-chemical property, soil microbial community diversity and structure of Brassica rapa var. Chinensis.

The amount of chemical fertilizer for vegetables is on the high level in China. The use of organic fertilizers to meet the nutrient requirement of crops will be an inevitable practice in sustainable agriculture. In this study, we compared the effects of pig manure fertilizer, rabbit manure fertilizer and chemical fertilizer on yield, quality of Brassica rapa var. Chinensis, soil physico-chemical properties and microbial community by using two consecutive seasons of three fertilizers in a pot experiment. The results were as follows: (1) In the first season, the fresh yield of Brassica rapa var. Chinensis applying chemical fertilizer was significantly (p ≤ 5%) higher than those of applying the pig manure and rabbit manure fertilizer, and the results were the opposite in the second season. The total soluble sugar concentration of fresh Brassica rapa var. Chinensis applying rabbit manure fertilizer was significantly (p ≤ 5%) higher than those of applying pig manure fertilizer and chemical fertilizer in the first season, and the NO3-N content of fresh Brassica rapa var. Chinensis on the contrary. (2) The organic fertilizer increased the concentration of total nitrogen, total phosphorus and organic carbon in soil in both two seasons. Rabbit manure fertilizer increased the soil pH and EC and significantly (p ≤ 5%) reduced the soil NO3-N content. (3) The pig manure and rabbit manure fertilizer significantly (p ≤ 5%) increased the diversity and abundance of soil bacterial of Brassica rapa var. Chinensis, but had no significant effect on soil fungi. Pearson correlation analysis showed that soil TN, TP, organic carbon content and EC were significantly correlated with soil bacterial α - diversity. There were significant differences (p ≤ 5%) in the bacterial community structures between three treatments in two seasons, and significant differences (p ≤ 5%) in the fungal community structures between fertilizer treatments while not between two seasons. Pig manure and rabbit manure fertilizer decreased the relative abundance of soil Acidobacteria and Crenarchaeota, rabbit manure fertilizer significantly increased the abundance of Actinobacteria in the second season. Distance-based redundancy analysis (dbRDA) showed that soil EC, TN, and organic carbon content were key physico-chemical factors in determining bacterial community structure in Brassica rapa var. Chinensis soil, and soil NO3-N, EC, SOC concentration and soil pH in the fungal community structure.

NRAS promotes the proliferation of melanocytes to increase melanin deposition in Rex rabbits.

Melanocytes play a major role in the formation of mammalian fur color and are regulated by several genes. Despite playing the pivotal role in the study of melanoma, the mechanistic role of NRAS (neuroblastoma RAS viral oncogene homolog) in the formation of mammalian epidermal color is still elusive. First of all, the expression levels of NRAS mRNA and protein in the dorsal skin of different colored Rex rabbits were detected by qRT-PCR and Western blot. Then, the subcellular localization of NRAS was identified in melanocytes by indirect immunofluorescence. Next, the expression of NRAS was overexpressed and knocked down in melanocytes, and its efficiency was verified by qRT-PCR and Western blot. Subsequently, NaOH, CCK-8, and Annexin V-FITC were used to verify the changes in melanin content, proliferation, and apoptosis in melanocytes. Finally, we analyzed the regulation of NRAS on other genes (MITF, TYR, DCT, PMEL, and CREB) that affect melanin production. In silico studies showed NRAS as a stable and hydrophilic protein, and it is localized in the cytoplasm and nucleus of melanocytes. The mRNA and protein expression levels of NRAS were significantly different in skin of different colored Rex rabbits, and the highest level was found in black skin (P < 0.01). Moreover, the NRAS demonstrated impact on the proliferation, apoptosis, and melanin production of melanocytes (P < 0.05), and the strong correlation of NRAS with melanin-related genes was evidently observed (P < 0.05). Our results suggested that NRAS can be used as a gene that regulates melanin production and controls melanocyte proliferation and apoptosis, providing a new theoretical basis for studying the mechanism of mammalian fur color formation.

Characterization and functional analysis of lncRNA2690 in regulating the growth cycle of the hair follicle in rabbits.

Hair follicles (HFs) achieve hair growth and renewal by periodic regeneration. Therefore, exploring the key factors affecting hair growth in rabbits is of great significance for precisely breeding Angora rabbits and improving the competitiveness of the rabbit industry. Based on the results of our previous studies, lncRNA2690 was differentially expressed in the HF cycle using lncRNA-Seq, and the full-length sequence was annotated by bioinformatics analysis. The lncRNA2690 is 363 nt long and is found on chromosome 14 from 163 321 514 to 163 321 872. The lncRNA2690 was predicted to not have the coding ability through open reading frame and CPC2, and the nuclear-cytoplasmic separation experiment showed the lncRNA2690 to be highly expressed in the nucleus (p < 0.01). The expression pattern of lncRNA2690 was further analyzed in the different HF development stages of Angora rabbits using quantitative real-time PCR. The results showed that lncRNA2690 was periodically expressed in HF development, and the expression level was found to be high in the HF resting phases. The overexpression and knockdown of lncRNA2690 were found to significantly upregulate and downregulate the expression of the genes WNT2, CCND1, BMP2, LEF1, and SIAH1 in the rabbit dermal papilla cells (p < 0.01), promoting cell apoptosis and inhibiting cell proliferation (p < 0.01). This indicated that lncRNA2690 negatively regulates the periodic regeneration of the HFs in rabbits. These results provide a basis for the further study of lncRNA2690 in the HF growth cycle of Angora rabbits.

CDK1 promotes the proliferation of melanocytes in Rex rabbits.

BACKGROUND: The fur color constitutes one of the most important economic characteristics of fur animals and is determined by the content of melanin. A previous study has shown that the cyclin-dependent kinase 1 (CDK1) is a member of the protein kinase family, involved in forming the color of the fur in Rex rabbits. However, its effect on the melanocytes remains unclear. OBJECTIVE: This study aimed to provide evidence for the role of CDK1 in melanogenesis. METHODS: This study measured the expression of CDK1 in Rex rabbit skins of six coat colors using qRT-PCR. The CDK1-mediated regulation of the pigmentation-related genes and cyclin-dependent kinases were analyzed. The melanin content, proliferation, and apoptosis of the melanocytes were analyzed using the NaOH, CCK8, and Annexin V-FITC methods. RESULTS: The CDK1 expression in the skin of the rex rabbits with different coat colors was found to be regular, and the expression level was found to be the highest in the skin of the black rex rabbits (P < 0.05). The overexpression/knockdown of CDK1 was found to significantly increase/decrease the melanin content in the melanocytes (P < 0.01). Besides, CDK1 was found to significantly promote the proliferation of the melanocyte and inhibit apoptosis (P < 0.01). Furthermore, the overexpression of CDK1 was found to significantly affect the expression of the other melanin-related genes like TYR, PMEL, DCT, as well as the mRNA expression of the cyclin-dependent kinases CDK4, CDK6, CDK8, CCNB1. CONCLUSIONS: The results indicated that CDK1 can serve as a key gene regulating melanogenesis, melanocyte proliferation, and apoptosis, providing a new theoretical basis for studying the mechanism by which the different colors of the fur evolve in mammals.

A Sperm Quality Detection System Based on Microfluidic Chip and Micro-Imaging System.

Sperm quality assessment is the main method to predict the reproductive ability of livestock. The detection of sperm quality of livestock is of great significance to the application of artificial insemination and in vitro fertilization. In order to comprehensively evaluate sperm quality and improve the real-time and portability of sperm quality detection, a portable microscopic imaging system based on microfluidic chip is developed in this paper. The system can realize the comprehensive evaluation of sperm quality by detecting sperm vitality and survival rate. On the hardware side, a microfluidic chip is designed, which can automatically mix samples. A set of optical system with a magnification of 400 times was developed for microscopic observation of sperm. In the aspect of software, aiming at the comprehensive evaluation of sperm quality based on OpenCV, a set of algorithms for identifying sperm motility and survival rate is proposed. The accuracy of the system in detecting sperm survival rate is 94.0%, and the error rate is 0.6%. The evaluation results of sperm motility are consistent with those of computer-aided sperm analysis (CASA). The system's identification time is 9 s. Therefore, the system is absolutely suitable for sperm quality detection.

The effects of light colour on female rabbit reproductive performance and the expression of key genes in follicular development.

The purpose of this study was to analyse the effects of light colour on rabbit reproductive performance and the expression of key follicular development genes. Rabbits (n = 1,068, 5 months old, 3.6-4.4 kg live body weight) were divided randomly into four groups, housed individually in wire mesh cages and exposed to red, green, blue, and white light-emitting diode (LED) light (control). The lighting schedule was 16 L : 8 D-15 d / 150 lx / 6:00 am-22:00 pm (3 d preartificial insemination to 12 d postartificial insemination). Red light and white light affected the conception rate and kindling rate and increased the total litter size at birth (p < 0.05). The effects of red light on litter size at weaning, litter weight at weaning, and individual weight at weaning increased compared with the green and blue groups. The effects of red light on live litter size at birth were increased compared with those in the blue group (p < 0.05). Compared to white light, green and blue light reduced the number of secondary follicles (p < 0.05). Compared to red light, green and blue light reduced the number of tertiary follicles (p < 0.05). Compared with white light, red LED light resulted in greater ovarian follicle stimulating hormone receptor and luteinizing hormone receptor mRNA expression (p < 0.05). Compared with green and blue LED light, red LED light resulted in greater B-cell lymphom-2 mRNA expression (p < 0.05). Compared with green LED light, red LED light inhibited FOXO1 mRNA expression in rabbit ovaries (p < 0.05). Red light can affect the reproductive performance of female rabbits and the expression of key genes for follicular development.

Promoter Methylation Changes in KRT17: A Novel Epigenetic Marker for Wool Production in Angora Rabbit.

Wool production is an important economic trait of Angora rabbits. Exploring molecular markers related to wool production is one of the essentials of Angora rabbits' breeding. KRT17 (Keratin 17) is an important gene of hair follicle development, which must be explored for genetic/epigenetic variation to assess its effect on wool production. Based on the effective wool production data of 217 Angora rabbits, the high and low yield groups were screened with 1.5 standard deviations of the population mean. The full-length sequence of KRT17 was obtained by rapid amplification of cDNA ends technology, and the polymorphism was analyzed in the promoter, exon, and intron regions by direct sequencing. KRT17, SP1 over-expression plasmids, and siRNA were constructed and transfected into dermal papilla cells. The mRNA expressions of relevant genes were analyzed by RT-qPCR. The methylation level of the KRT17 promoter was determined by Bisulfite Sequencing PCR. Dual-luciferase system, site-directed mutagenesis, and electrophoretic mobility shift assays were used to analyze the binding relationship between SP1 and the promoter of KRT17. The structure map of KRT17 was drawn, and no SNPs were found in the promoter, exon, and intron, indicating a relatively conserved structure of KRT17. Expression of KRT17 was significantly higher in cutaneous tissues than in other tissues and was significantly upregulated in the high-yield group compared to the low-yield group (p < 0.05). Furthermore, the overall high methylation levels of KRT17 CpG I and CpG III showed significant association with low wool yield; the methylation levels of 5 CpG locus (CpG I site 4 and CpG III site 2−5) were significantly different between the high and low yield groups (p < 0.05). The methylation levels of 3 CpG locus (CpG I site 4 and CpG III site 4, 14) showed a significant correlation with KRT17 expression (p < 0.05). Overall, CpG III site 4 significantly affects wool production and KRT17 expressions (p < 0.05). This site promotes SP1 binding to the KRT17 promoter region (CGCTACGCCC) to positively regulate the KRT17 expression. KRT17 CpG III site 4 can be used as candidate epigenetic markers for the breeding of high wool-producing Angora rabbits.

Correction to: Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

The original article has been published with an incorrect grant number in the Funding section.

Morphological Characterization and Gene Expression Patterns for Melanin Pigmentation in Rex Rabbit.

Animal melanin has an important role in the formation of animal fur and skin, which is determined by its quantities, character, and distribution. To identify the effect of melanin on the formation of multi-colored Rex rabbits (Black, Chinchilla, Beaver, Protein cyan, Protein yellow, White), the structure of hair follicles and melanin content in multi-colored Rex rabbit skins were observed by Hematoxylin and Eosin (H&E) staining and melanin staining, respectively. The melanin granules were primarily found in the epidermis and hair follicle roots. The melanin content of skin was measured by extracting melanin from skin tissue. The results demonstrated that the melanin content was the highest in the skin of black Rex rabbit. Additionally, we measured the mRNA and protein expression levels of melanin-related key genes (MITF and TYR) in the skin of different hair color by quantitative real-time PCR and Wes assay, respectively. The results revealed that the mRNA expression levels in the skin of black Rex rabbit was highly expressed when as compared with other Rex rabbit skin (P < 0.01), and they were the lowest in the skin of white Rex rabbit. Finally, correlation analysis was conducted between melanin content and the expression levels of mRNA and protein. The results indicated a significant correlation between melanin content and the mRNA expression of MITF (P < 0.05), but it was not correlated with the mRNA expression of TYR (P > 0.05). In summary, melanin deposition has important economic value, and the coat color of fur-bearing animals is partly determined by the melanin-related genes.