| Small Tail Han(STH)Sheep is famous for its high litter size and tall in body size,it is an important female parent in the production of mutton sheep.Exploring the molecular mechanism of FecB gene in the reproductive traits of STH sheep will be benefit for the discovery of new molecular markers for reproduction.In this study,STH sheep from the core group in Shandong province were selected as our research object.The distribution of FecB gene in the core group of STH sheep was genotyped using TaqMan probe.The frequencies of ++,+B and BB were 0.160(142),0.464(413)and 0.376(335),respectively.And the allele of B frequency was 0.608.Statistical analysis of the litter size in multiple paritis,we verified the result that the litter size of STH sheep was significantly increased accompanied with the copies of FecB gene increasing(P≤0.01).Estrus synchronization of three groups of different FecB genotypes STH sheep were all performed.Teasers were used to determine the phenotype of estrus,and blood samples were collected to determine the level of reproductive hormones in serum.During the luteal phase,the ovulation rate of ewes were observed through laparoscope.After CIDR withdrawal,statistical data showed that the estrus of +B sheep was significantly earlier than that of BB and ++ individuals(P≤0.05),and the estrus cycle of +B STH sheep was also shorter than others(P≤0.05).There was no significant difference in level of reproductive hormones among the three groups of sheep.The ovulation rate increased significantly accompanied with the copies of FecB gene increasing(P≤0.01).This study showed that in the breeding of STH sheep,FecB heterozygotes(+B)with moderate ovulation rate,higher litter size,and shorter estrus cycle should be valued and promoted by livestock workers.As a unique microenvironment,follicular fluid can provide energy,nutrition and regulatory factors for oocyte development and ovulation.In this study,the metabolome differences of follicular fluid(FF)and ovarian vein serum(OVS)among three FecB genotypes of STH sheep were determined by LC-MS and GC-MS combined techniques.A total of 236 and 310 known biochemical compounds have been identified in ovine FF and OVS samples,respectively.Compared with OVS,the difference in metabolome of FecB gene is mainly reflected in FF.With the increasing copies of FecB gene,the concentration of oxidized glutathione in FF increased significantly(P≤0.01).Combined with the high levels of gamma-glutamyl amino acids detected in individuals which carrying the FecB gene,we suggested that antioxidant defenses are essential for high ovulation rates in ewes.Oocytes and cumulus granulosa cells were collected through mouth pipette,then single cell RNA-Seq was performed separately.In the three FecB genotypes of STH sheep,three replicates of two cell types were selected to construct 18 libraries for sequencing.An average total of 104397835 clean reads were obtained from each library.There is a clear heterogeneity of gene expression between the two cell typs.In oocytes and cumulus granulosa cells,the differentially expressed genes between ++ and BB genotype were 683 and 786,respectively(FDR ≤ 0.05).The FecB gene was mainly expressed in cumulus granulosa cells,when the FecB gene mutated,the expression of its downstream gene SMAD4 and related reproductive hormone receptor genes(FSHR,LHCGR)were significantly changed(P ≤ 0.05).No significant difference had been observed for reproductive hormones among the three FecB genotypes in STH sheep,while the ability to receive hormonal signals may be regulated by differential expression level of hormone receptor genes.In oocytes and cumulus granulosa cells,differentially expressed genes were significantly enriched in oocyte meiosis pathway and TGF-beta signaling pathway(qvalue ≤ 0.05),respectively.This study indicated that FecB mutation may alter the development of oocytes and the proliferation of cumulus granulosa cells. |
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