Previous studies have screened key candidate genes for litter size in sheep, including fibrillin-1 (FBN1), family with sequence similarity 184 member B (FAM184B) and zinc finger and AT-hook domain containing (ZFAT). Therefore, it is necessary to verify these genes in the Xinggao mutton sheep population and determine the associated loci for litter size. In this study, three loci (FBN1 g.160338382 T > C, FAM184B g.398531673 C > T and ZFAT g.20150315 C > T) were firstly screened based on the population differentiation coefficient between the polytocous and monotocous sheep groups. Then, population genetic analysis and association analysis were performed on these loci. The results revealed that the g.160338382 T > C in FBN1 was significantly associated with the litter size of sheep. Moreover, there was no significant interaction effect between the g.160338382 T > C locus and FecB on litter size. Notably, g.160338382 T > C is adjacent to the anterior border of exon 58 and belongs to a splice polypyrimidine tract variant, which may lead to alternative splicing and ultimately cause changes in the structure and function of the protein. In summary, our results provided a potentially effective genetic marker for improving the litter size of sheep.

Xinggao sheep are a breed of Chinese domestic sheep that are adapted to the extremely cold climatic features of the Hinggan League in China. The economically vital reproductive trait of ewes (litter size, LS) and productive traits of lambs (birth weight, BWT; weaning weight, WWT; and average daily gain, ADG) are expressed in females and later in life after most of the selection decisions have been made. This study estimated the genetic parameters for four traits to explore the genetic mechanisms underlying the variation, and we performed genome-wide association study (GWAS) tests on a small sample size to identify novel marker trait associations (MTAs) associated with prolificacy and growth. We detected two suggestive significant single-nucleotide polymorphisms (SNPs) associated with LS and eight significant SNPs for BWT, WWT, and ADG. These candidate loci and genes also provide valuable information for further fine-mapping of QTLs and improvement of reproductive and productive traits in sheep.

In previous studies, NOX4, PDE11A and GHR genes have been screened as important candidate genes for litter size in sheep by using the GWAS method; however, neither their effects on litter size nor the loci associated with litter size have been identified. In this study, three candidate loci (c.1057-4C > T in NOX4, c.1983C > T in PDE11A and c.1618C > T in GHR) were first screened based on our previous resequencing data of 10 sheep breeds. After the three loci were genotyped using Sequenom MassARRAY technology, we carried out population genetics analysis on the three loci and performed association analysis between the polymorphism of the three loci and the litter size of sheep. The results of population genetics analysis suggested that c.1057-4C > T in NOX4 and c.1983C > T in PDE11A may be subject to natural or artificial selection. The results of association analysis indicated that litter size was significantly associated with c.1057-4C > T in NOX4 and c.1983C > T in PDE11A (p < 0.05) in Small Tail Han sheep, and there was no significant interaction effect between the two loci on the litter size. In summary, c.1057-4C > T in NOX4 and c.1983 C > T in PDE11A can be considered candidate molecular markers for improving litter size in sheep.

In vitro maturation (IVM) of sheep oocytes and early embryonic development are of great scientific importance for the study of reproductive development in sheep. Ghrelin is an important hormone that regulates the secretion of the growth hormone (GH). In this study, different gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their cell morphology, and Hosesth 33342 staining was used to determine the time taken for oocytes to reach different developmental stages. We found 200 ng/mL ghrelin to be the optimal concentration. The RNA-seq analysis showed that many signaling pathways were significantly altered by ghrelin. Cell cycle, Wnt, and oxidative phosphorylation were activated; the P53 was inhibited. These pathways together regulate the maturation of oocytes and early embryonic development in vitro. The effects of the addition of ghrelin were verified by the expression of GLUT1 in early embryonic development. The results suggest that adding ghrelin shortens the duration of the IVM of sheep oocytes and hinders early embryonic development. This study provides new insights into the effects of exogenous ghrelin on sheep oocyte maturation and early embryonic development in vitro.

Wool is one of the most important animal fibers in the textile industry and the diameter directly affects its economic value. However, the molecular mechanisms underlying the wool diameter have not been fully elucidated. In the present study, high-throughput RNA-Seq technology was employed to explore the skin transcriptome using 3 sheep with fine wool (fiber diameter, FD27.0μm). In total, 28,607,228 bp clean reads were obtained, and 78.88%+/-3.84% was uniquely aligned to the reference genome across the six samples. In total, 19,914 mRNA transcripts were expressed (FPKM>0) in the six skin samples, among which there were certain well-known genes affecting the skin hair cycle, such as KRTAP7-1, KRT14, Wnt10b, Wnt2b, β-catenin, and FGF5. Furthermore, 467 expressed genes were significantly differentially expressed between the two groups, including 21 genes up-regulated and 446 genes down-regulated in the sheep with the smaller fiber diameter. To verify the results, 13 differentially expressed genes were randomly selected to validate the expression patterns using qRT-PCR, and the correlation between the mRNA expression level from qRT-PCR and RNA-Seq data was 0.999 ( P<0.05). These differentially expressed genes were particularly enriched in GO processes related to lipid metabolism, skin development, differentiation, and immune function (P<0.05). The biological processes were involved in collagen catabolism, negative regulation of macromolecule metabolism, steroid hormone stimulation and lipid metabolism. A significant KEGG pathway involving the “metabolism of lipids and lipoproteins” was also enriched. This study revealed that the lipid metabolism might constitute one of the major factors related to wool diameter.

The composition and function of the intestinal microbiota of Xinggao mutton sheep were explored before and post-weaning using non-target metabolomics. Both 16S rDNA amplicon sequencing technology and serum non-target metabolomics were used to determine changes in sheep metabolites and intestinal flora. A random sampling method was used to collect fecal samples from 20 pre-weaning lambs and 20 lambs post-weaning. Analysis of the intestinal flora identified Firmicutes as the predominant phylum pre-weaning and Bacteroidota as the dominant phylum post-weaning. Joint multi-omics analysis revealed that Oscillospiraceae UCG-005 and Prevotella_9 were significantly correlated with serum L-tyrosine, Indole-3-acrylic acid, and taurine-related metabolites, suggesting their potential roles in modulating digestive health in Xinggao mutton sheep.