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Abstract Background The pituitary directly regulates the reproductive process through follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Transcriptomic research on the pituitaries of ewes with different FecB (fecundity Booroola) genotypes has shown that some key genes and lncRNAs play an important role in pituitary function and sheep fecundity. Our previous study found that ewes with FecB + + genotypes (without FecB mutation) still had individuals with more than one offspring per birth. It is hoped to analyze this phenomenon from the perspective of the pituitary transcriptome. Results The 12 Small Tail Han Sheep were equally divided into polytocous sheep in the follicular phase (PF), polytocous sheep in the luteal phase (PL), monotocous sheep in the follicular phase (MF), and monotocous sheep in the luteal phase (ML). Pituitary tissues were collected after estrus synchronous treatment for transcriptomic analysis. A total of 384 differentially expressed genes (DEGs) (182 in PF vs. MF and 202 in PL vs. ML) and 844 differentially expressed lncRNAs (DELs) (427 in PF vs. MF and 417 in PL vs. ML) were obtained from the polytocous-monotocous comparison groups in the two phases. Functional enrichment analysis showed that the DEGs in the two phases were enriched in signaling pathways known to play an important role in sheep fecundity, such as calcium ion binding and cAMP signaling pathways. A total of 1322 target relationship pairs (551 pairs in PF vs. MF and 771 pairs in PL vs. ML) were obtained for the target genes prediction of DELs, of which 29 DEL-DEG target relationship pairs (nine pairs in PF vs. MF and twenty pairs in PL vs. ML). In addition, the competing endogenous RNA (ceRNA) networks were constructed to explore the regulatory relationships of DEGs, and some important regulatory relationship pairs were obtained. Conclusion According to the analysis results, we hypothesized that the pituitary first receives steroid hormone signals from the ovary and uterus and that VAV3 (Vav Guanine Nucleotide Exchange Factor 3), GABRG1 (Gamma-Aminobutyric Acid A Receptor, Gamma 1), and FNDC1 (Fibronectin Type III Domain Containing 1) played an important role in this process. Subsequently, the reproductive process was regulated by gonadotropins, and IGFBP1 (Insulin-like Growth Factor Binding Protein 1) was directly involved in this process, ultimately affecting litter size. In addition, TGIF1 (Transforming Growth Factor-Beta-Induced Factor 1) and TMEFF2 (Transmembrane Protein With EGF Like And Two Follistatin Like Domains 2) compensated for the effect of the FecB mutation and function by acting on TGF-β/SMAD signaling pathway, an important pathway for sheep reproduction. These results provided a reference for understanding the mechanism of multiple births in Small Tail Han Sheep without FecB mutation.

The Booroola fecundity mutation (FecB) in Small Tail Han sheep has been shown to enhance ovulation rates and litter sizes by affecting the hypothalamic–pituitary–gonadal (HPG) axis. Despite the pituitary’s role in reproductive regulation, its involvement in FecB-induced ovulation remains understudied. Our study aimed to fill this gap by analyzing pituitary tissues from FecB homozygous (BB) and wild-type (WW) ewes during luteal and follicular phases using tandem mass tag–based protein quantification and the DIABLO framework for proteomic and transcriptomic data integration. Significant differences in 277 proteins were observed across estrus periods, with network analysis highlighting the voltage-dependent calcium channel L-type alpha-1C as a key convergence point in oxytocin signaling and GnRH secretion pathways. The DIABLO method revealed a strong correlation (0.98) between proteomic and transcriptomic datasets, indicating a coordinated response in FecB ewes. Notably, higher expression levels of Follicle Stimulating Hormone Subunit Beta (FSHB) and Luteinizing Hormone Subunit Beta (LHB) were found in BB ewes during the follicular phase, potentially due to elevated E2 concentrations. Furthermore, our analysis identified genes related to the Gamma–aminobutyric acid type A receptor family (GABRA2, GABRG1, GABRB1) in the pituitary, with GABRB1 showing higher expression in BB ewes. This suggests a role for GABA in modulating GnRH and gonadotropin feedback loops, potentially contributing to the FecB mutation’s effect on ovulation. This study provides novel insights into the pituitary’s role in fertility among FecB sheep, identifying GABA as a potential regulatory factor within the HPG axis. The findings also open avenues for discovering new biomarkers in pituitary endocrinology for sheep breeding purposes.

Small Tail Han (STH) sheep, a unique Chinese breed, is recognized for its early maturity, year-round estrus, and prolificacy. However, the molecular mechanism of its high prolificacy has not been fully elucidated. The Proteomics approach is feasible and effective to reveal the proteins involved in the complex physiological processes of any organism. Given this, we performed the protein expression profiling of ovarian tissues during the luteal phase using polytocous STH sheep (litter size ≥2, three consecutive lambings) and monotocous STH sheep (litter size =1, three consecutive lambings) (PL vs. ML), and the follicular phase using polytocous STH sheep (litter size ≥2, three consecutive lambings) and monotocous STH sheep (litter size =1, three consecutive lambings) (PF vs. MF), respectively. Parallel Reaction Monitoring (PRM) was conducted to validate the differentially abundant proteins (DAPs). The tandem mass tag (TMT) quantitative proteomic results showed that a total of 5,237 proteins were identified, of which 49 and 44 showed differential abundance in the PL vs. ML and PF vs. MF groups, respectively. Enrichments analyses indicated that the DAPs including TIA1 cytotoxic granule-associated RNA-binding protein-like 1 (TIAL1), nicotinamide phosphoribosyltransferase (NAMPT), and cellular retinoic acid-binding protein 1 (CRABP1) were enriched at the luteal phase, while TIAL1, inhibin beta-a-subunit (A2ICA4), and W5PG55 were enriched at the follicular phase, potentially mediating reproductive processes in polytocous ewes. Furthermore, six DAPs were verified using PRM, confirming the accuracy of the TMT data acquired in this study. Together, our work expanded the database of indigenous sheep breeds and provided new ovarian candidate molecular targets, which will help in the study of the genetic mechanisms of ovine prolificacy.

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.

Circular RNAs (circRNAs) are a specific type of noncoding RNA, and some have defined roles in cellular and biological processes. However, little is known about the role of circRNAs in follicular development in sheep with FecB (fecundity Booroola) mutations. Here, the expression profiles of circRNAs were investigated using RNA sequencing (RNA-seq) in the follicular phase (F) and the luteal phase (L) of FecB mutant homozygous (BB) and wild-type (WW) Small Tail Han sheep. A total of 38,979 circRNAs were identified, and 314, 343, 336, and 296 of them were differentially expressed (DE) between BB_F and BB_L, WW_F and WW_L, BB_F and WW_F, and BB_L and WW_L, respectively. The length, type, and chromosome distribution of the circRNAs and the expression characteristic between the circRNAs and their host genes in the sheep hypothalamus were ascertained. Enrichment analysis showed that the host genes of DE circRNAs in the follicular and luteal phases were annotated to MAPK, gap junctions, progesterone-mediated oocyte maturation, oocyte meiosis, and other hormone-related signaling pathways, and the different FecB genotypes were annotated to the gap junctions, circadian entrainment, MAPK, and other hormone-related signaling pathways. The competing endogenous RNA network prediction revealed that the 129 target miRNAs might be bound to 336 DE circRNAs. oar_circ_0000523 and oar_circ_0028984, which were specifically expressed during the follicular phase in the BB genotype sheep, probably acted as miRNA sponges involved in the regulation of LH synthesis and secretion. This study reveals the expression profiles and characterization of circRNAs at two phases of follicular development considering different FecB genotypes, thereby providing an improved understanding of the roles of circRNAs in the sheep hypothalamus and their involvement in follicular development and ovulation.

The Booroola fecundity (FecB) mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene increases ovulation in sheep. However, its effect on follicular maturation is not fully understood. Therefore, we collected granulosa cells (GCs) at a critical stage of follicle maturation from nine wild-type (WW), nine heterozygous FecB mutant (WB), and nine homozygous FecB mutant (BB) Small Tail Han sheep. The GCs of three ewes were selected at random from each genotype and consolidated into a single group, yielding a total of nine groups (three groups per genotype) for proteomic analysis. The tandem mass tag technique was utilized to ascertain the specific proteins linked to multiple ovulation in the various FecB genotypes. Using a general linear model, we identified 199 proteins significantly affected by the FecB mutation with the LIMMA package (p < 0.05). The differential abundance of proteins was enriched in pathways related to cholesterol metabolism, carbohydrate metabolism, amino acid biosynthesis, and glutathione metabolism. These pathways are involved in important processes for GC-regulated ‘conservation’ of oocyte maturation. Further, the sparse partial least-squares discriminant analysis and the Fuzzy-C-mean clustering method were combined to estimate weights and cluster differential abundance proteins according to ovulation to screen important ovulation-related proteins. Among them, ZP2 and ZP3 were found to be enriched in the cellular component catalog term “egg coat”, as well as some apolipoproteins, such as APOA1, APOA2, and APOA4, enriched in several Gene Ontology terms related to cholesterol metabolism and lipoprotein transport. A higher abundance of these essential proteins for oocyte maturation was observed in BB and WB genotypes compared with WW ewes. These proteins had a high weight in the model for discriminating sheep with different FecB genotypes. These findings provide new insight that the FecB mutant in GCs improves nutrient metabolism, leading to better oocyte maturation by altering the abundance of important proteins (ZP2, ZP3, and APOA1) in favor of increased ovulation or better oocyte quality.

Litter size is an economically important trait in sheep, and it is a complex trait controlled by multiple genes in multiple organs. Among them, the regulation of lamb number trait by the thyroid gland is a very important part. However, the molecular mechanisms of the thyroid gland in sheep reproduction remain unclear. Here, RNA-seq was used to detect transcriptome expression patterns in the thyroid gland between follicular phase (FP) and luteal phase (LP) in FecB BB (MM) and FecB ++ (ww) STH sheep, respectively, and to identify differentially expressed circRNAs (DECs) associated with reproduction. Bioinformatic analysis of the source genes of these DECs revealed that they can be enriched in multiple signaling pathways involved in the reproductive process of animals. We found that the source genes of these DECs, such as GNAQ, VEGFC, MAPK1, STAT1, and HSD17B7, may play important roles in the reproductive process of animals. To better understand the function of these DECs, we constructed circRNA–miRNA co-expression networks. Dual luciferase reporter assays suggested that a ceRNA regulatory mechanism between circ_0003259-oar-miR-133-TXLNA and circ_0012128-oar-miR-370-3p-FGFR1 may hold. All of these DEC expression profiles in the thyroid gland provide a novel resource for elucidating the regulatory mechanisms underlying STH sheep prolificacy.

Abstract Background Hu sheep and Tibetan sheep in China are characterized by fat tails and thin tails, respectively. Several transcriptomes have been conducted in different sheep breeds to identify the differentially expressed genes (DEGs) underlying this trait. However, these studies identified different DEGs in different sheep breeds. Results Hence, RNA sequencing was performed on Hu sheep and Tibetan sheep. We obtained a total of 45.57 and 43.82 million sequencing reads, respectively. Two libraries mapped reads from 36.93 and 38.55 million reads after alignment to the reference sequences. 2108 DEGs were identified, including 1247 downregulated and 861 upregulated DEGs. GO and KEGG analyses of all DEGs demonstrated that pathways were enriched in the regulation of lipolysis in adipocytes and terms related to the chemokine signalling pathway, lysosomes, and glycosaminoglycan degradation. Eight genes were selected for validation by RT–qPCR. In addition, the transfection of BMP2 overexpression into preadipocytes resulted in increased PPAR-γ expression and expression. BMP2 potentially induces adipogenesis through LOX in preadipocytes. The number of lipid drops in BMP2 overexpression detected by oil red O staining was also greater than that in the negative control. Conclusion In summary, these results showed that significant genes (BMP2, HOXA11, PPP1CC and LPIN1) are involved in the regulation of adipogenesis metabolism and suggested novel insights into metabolic molecules in sheep fat tails.

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.

As a novel class of small RNAs, piRNAs are highly expressed in the animal gonads and their main known role is to inhibit transposon activity for ensuring the correctness and integrity of genome. In order to explore the characteristics of piRNAs in sheep testis and their possible regulatory roles on male reproduction, deep sequencing technology was used to sequence small RNAs and identify piRNAs in testes of sheep. The length of piRNAs in sheep testes showed a unimodal distribution between 26 and 31 nt, with a peak at 29 nt. These piRNAs exhibited obvious ping-pong signature and strand specificity. In the genome, they were mainly aligned to CDS, intron, repetitive sequence regions and unannotated regions. Furthermore, in transposon analysis, piRNAs were aligned predominantly to LINE, SINE, and LTR types of retrotransposon in sheep testes, and the piRNAs derived from each type showed obvious ping-pong signature. The piRNA clusters identified in sheep testes were mainly distributed on chromosomes 3, 7, 15, 17, 18 and 20. The results combining semen determination with pathway enrichment analysis implied that differentially expressed piRNAs between the testes of rams with different fertility might participate in spermatogenesis by regulating multiple pathways closely related to stabilization of blood-testis barrier and renewal and differentiation of spermatogonial stem cell. Taken together, the study provided new insights into the characteristics, origin and expression patterns of piRNAs in sheep testes tissue, which would help us better understand the role of piRNAs in sheep reproduction.

The mammalian oviduct is functionally highly diverse during the estrus cycle. It provides a suitable milieu for oocyte maturation, sperm capacitation, fertilization, early embryo development and transportation. While there have been many studies of molecular mechanisms on the kidding number of goats, a systematic analysis by which the underlying circular RNAs (circRNAs) changes in the oviduct related to prolificacy traits is lacking. Herein, we present a comprehensive circRNA atlas of the oviduct among high- and low-fecundity goats in the follicular phase (FH vs. FL), luteal phase (LH vs. LL), and estrus cycle (FH vs. LH; FL vs. LL) to unravel their potential regulatory mechanisms in improving kidding number. We generated RNA sequencing data, and identified 4,078 circRNAs from twenty sampled Yunshang black goats. Many of these circRNAs are exon-derived and differentially expressed between each comparison group. Subsequently, eight differentially expressed (DE) circRNAs were validated by RT‒qPCR, which was consistent with the RNA-seq data. GO and KEGG enrichment analyses suggested that numerous host genes of DE circRNAs were involved in the hormone secretion, gamete production, fertilization, and embryo development processes. The competing endogenous RNA (ceRNA) interaction network analysis revealed that 2,673 circRNA–miRNA–mRNA axes (including 15 DE circRNAs, 14 miRNAs, and 1,699 mRNAs) were formed, and several target genes derived from the ceRNA network were associated with oviduct functions and reproduction, including SMAD1, BMPR1B, IGF1, REV1, and BMP2K. Furthermore, miR-15a-5p, miR-181b-5p, miR-23b-5p, miR-204-3p, and miR-145-5p might play important roles in reproduction. Finally, a novel circRNA, circIQCG, was identified as potentially involved in embryo development. Overall, our study provides a resource of circRNAs to understand the oviductal function and its connection to prolificacy trait of goats in the differentiation estrus cycle.

PIWI-interacting RNAs (piRNAs), as a novel class of small non-coding RNAs that have been shown to be indispensable in germline integrity and stem cell development. However, the expressed characteristics and regulatory roles of piRNAs during different reproductive phases of animals remain unknown. In this study, we investigated the piRNAs expression profiles in ovaries of sheep during the luteal phase (LP) and follicular phase (FP) using the Solexa sequencing technique. A total of 85,219 and 1,27,156 piRNAs tags were identified in ovine ovaries across the two phases. Most expressed piRNAs start with uracil. piRNAs with a length of 24 nt or 27–29 nts accounted for the largest proportion. The obvious ping-pong signature appeared in the FP ovary. The piRNA clusters in the sheep ovary were unevenly distributed on the chromosomes, with high density on Chr 3 and 1. For genome distribution, piRNAs in sheep ovary were mainly derived from intron, CDS, and repeat sequence regions. Compared to the LP ovary, a greater number of expressed piRNA clusters were detected in the FP ovary. Simultaneously, we identified 271 differentially expressed (DE) piRNAs between LP and FP ovaries, with 96 piRNAs upregulated and 175 piRNAs downregulated, respectively. Functional enrichment analysis (GO and KEGG) indicated that their target genes were enriched in reproduction-related pathways including oocyte meiosis, PI3K-Akt, Wnt, and TGF-β signaling pathways. Together, our results highlighted the sequence and expression characteristics of the piRNAs in the sheep ovary, which will help us understand the roles of piRNAs in the ovine estrus cycle.

CircRNAs have been found to play key roles in many biological processes and have diverse biological functions. There have been studies on circRNAs in sheep pituitary, and some important circRNAs have been found. But there are still few studies on circRNAs in sheep pituitary with different fecundity. In this study, we obtained the circRNAs expression profiles in the pituitary of FecB ++ genotype Small Tail Han sheep with different fecundity and estrous phases. A total of 34,878 circRNAs were identified in 12 pituitary samples, 300 differentially expressed circRNAs (DE circRNAs) (down: 104; up: 196) were identified in polytocous sheep in the follicular phase (PF) and monotocous sheep in the follicular phase (MF) (PF vs. MF), and 347 DE circRNAs (down: 162; up: 185) were identified in polytocous sheep in the luteal phase (PL) and monotocous sheep in the luteal phase (ML) (PL vs. ML). Cortisol synthesis and secretion pathway (follicular phase) and estrogen signaling pathway (luteal phase) were obtained by functional enrichment analysis of circRNAs source genes. Competing endogenous RNA (ceRNA) network analysis of key DE circRNAs revealed that oar-circ-0022776 (source gene ITPR2, follicular phase) targeted oar-miR-432, oar-circ-0009003 (source gene ITPR1, luteal phase) and oar-circ-0003113 (source gene PLCB1, luteal phase) targeted oar-miR-370-3p. We also explored the coding ability of DE circRNAs. In conclusion, our study shows that changes in the pituitary circRNAs may be related to the response of the pituitary to steroid hormones and regulate the reproductive process of sheep by affecting the pituitary function. Results of this study provide some new information for understanding the functions of circRNAs and the fecundity of FecB ++ genotype sheep.

Circular RNAs (circRNAs) are non-coding RNAs newly identified and play important roles in RNA regulation. However, little is known regarding photoperiods induced circRNAs in the thyroid gland. In this study, we performed a comprehensive analysis of circRNA profiles in the thyroid gland of OVX+E2 ewes at different photoperiods by whole transcriptome sequencing. A total of 37,470 novel circRNAs were detected in different photoperiods (42 days of short photoperiod treatment, SP42; 42 days of long photoperiod treatment, LP42; SP42 transfer to LP42, SPLP42), with a total of 817 circRNAs for SP42-LP42 (down: 132; up: 114), LP42-SPLP42 (down: 136; up: 112) and SP42-SPLP42 (down: 182; up: 141) having differentially expressed. Functional enrichment annotation analysis of DE-circRNAs for GO and KEGG by R package, features that influence photoperiod response in Sunite ewes through the Inositol phosphate metabolism, cGMP-PKG signaling pathway, Calcium signaling pathway, MAPK signaling pathway, and Oocyte meiosis. In addition, competitive endogenous RNA (ceRNA) network analysis revealed target binding sites for identified miRNAs in DE-cirRNAs such as oar-miR-10b, oar-miR-200c, oar-miR-21, oar-miR-370-3p, oar-miR-377-3p, oar-miR-181a, oar-miR-432, and oar-miR-495-3p. These results of this study will provide some new information for understanding circRNA function as well as the changes in the sheep thyroid gland under different photoperiods.

In seasonal estrus, it is well known that melatonin-regulated biorhythm plays a key role. Some studies indicate that the adrenal gland plays an important role in reproduction in mammals, but the molecular mechanism is not clear. This study used an artificially controlled light photoperiod model, combined with RNA-seq technology and bioinformatics analysis, to analyze the messenger RNA (mRNA) and microRNA (miRNA) of ewe (Sunite) adrenal glands under different photoperiod treatments. After identification, the key candidate genes GRHL2, CENPF, FGF16 and SLC25A30 that photoperiod affects reproduction were confirmed. The miRNAs (oar-miR-544-3p, oar-miR-411b-5p, oar-miR-376e-3p, oar-miR-376d, oar-miR-376b-3p, oar-miR-376a-3p) were specifically expressed in the adrenal gland. The candidate mRNA-miRNA pairs (e.g., SLC25A30 coagulated by novel miRNA554, novel miRNA555 and novel miRNA559) may affect seasonal estrus. In summary, we constructed relation network of the mRNAs and miRNAs of sheep adrenal glands using RNA sequencing and bioinformatics analysis, thereby, providing a valuable genetic variation resource for sheep genome research, which will contribute to the study of complex traits in sheep.

Abstract Background Many recent studies have shown that miRNAs play important roles in the regulation of animal reproduction, including seasonal reproduction. The pineal gland is a crucial hub in the regulation of seasonal reproduction. However, little is known about the expression characteristics of pineal miRNAs in different reproductive seasons (anestrus and breeding season). Therefore, the expression profiles and regulatory roles of ovine pineal miRNAs were investigated during different reproductive stages using Solexa sequencing technology and dual luciferase reporter assays. Results A total of 427 miRNAs were identified in the sheep pineal gland. Significant differences in miRNA expression were demonstrated between anestrus and the breeding season in terms of the frequency distributions of miRNA lengths, number of expressed miRNAs, and specifically and highly expressed miRNAs in each reproductive stage. KEGG analysis of the differentially expressed (DE) miRNAs between anestrus and the breeding season indicated that they are significantly enriched in pathways related to protein synthesis, secretion and uptake. Furthermore, transcriptome analysis revealed that many target genes of DE miRNAs in the ribosome pathway showed relatively low expression in the breeding season. On the other hand, analyses combining miRNA-gene expression data with target relationship validation in vitro implied that miR-89 may participate in the negative regulation of aralkylamine N-acetyltransferase (AANAT) mRNA expression by targeting its 3’UTR at a unique binding site. Conclusions Our results provide new insights into the expression characteristics of sheep pineal miRNAs at different reproductive stages and into the negative regulatory effects of pineal miRNAs on AANAT mRNA expression.

The fat tail is a unique characteristic of sheep that represents energy reserves and is a complex adaptative mechanism of fat-tailed sheep to environmental stress. MicroRNA plays a significant role as regulators at the posttranscriptional level, but no studies have explained the molecular mechanisms of miRNA which regulate fat deposition in sheep tails. In this study, mRNA and miRNA analysis examined tail fat tissue from three Hu fat-tailed and three Tibetan thin-tailed sheep. After aligning to the reference sequences, 2,108 differentially expressed genes and 105 differential expression miRNAs were identified, including 1,247 up- and 861 downregulated genes and 43 up- and 62 downregulated miRNAs. Among these differentially expressed miRNAs, oar-miR-432 was one of the most downregulated miRNAs between Hu sheep and Tibetan sheep, and 712 genes were predicted to be targeted by oar-miR-432, 80 of which overlapped with DEGs. The Gene Ontology analysis on these genes showed that BMP2, LEP, GRK5, BMP7, and RORC were enriched in fat cell differentiation terms. The genes for BMP2 targeted by oar-miR-432 were examined using dual-luciferase assay. The oar-miR-432 mimic transfected into preadipocytes resulted in increased expression of BMP2. The marker gene PPAR-γ of fat differentiation had a lower expression than the negative control on days 0, 2, and 4 after induced differentiation. The decrease in the number of lipids in the oar-miR-432 mimic group detected by oil red O stain was also less than that in the negative control. This is the first study to reveal the fat mechanisms by which oar-miR-432 inhibits fat differentiation and promotes the expression of BMP2 in sheep tails.

Accurate timing of seasonal changes is an essential ability for an animal’s survival, and the change in the photoperiod is the key factor affecting reproductive seasonality in mammals. Emerging evidence has suggested that multiple hypothalamic genes participate in the photoperiod-induced regulation of reproductive activities in sheep, but the mechanism is still unclear. In this study, we initially examined the plasma level of two major reproductive hormones, namely, follicle-stimulating hormone (FSH) and prolactin (PRL), under different photoperiods in ovariectomized and estradiol-treated (OVX + E2) sheep using radioimmunoassay (RIA). Of the two hormones, the concentration of PRL significantly increased with the extension of the photoperiod, while FSH showed the opposite trend. Subsequently, an examination of the transcriptomic variation between the short photoperiod (SP) and long photoperiod (LP) was conducted. Differential expression analyses and functional annotation showed that several key genes in the insulin secretion (VAMP2, PRKACB, PRKCG, and PLCB1), GnRH (MAPK13, CGA, CDC42, ATF4, and LHB) pathways, and circadian entrainment (KCNJ5, PER1, GNB2, MTNR1A, and RASD1), as well as numerous lncRNAs, including XR_173257.3, XR_173415.3, XR_001435315.1, XR_001024596.2, and XR_001023464.2, were shown potentially vital for the hypothalamic photoperiodic response. Four of the differentially expressed mRNAs and lncRNAs were validated by qPCR. The constructed mRNA–mRNA interaction networks further revealed that transcripts potentially participated in hypothalamic thyroid hormone synthesis, endocrine resistance, and neuroactive ligand–receptor interactions. The interactome analysis of lncRNAs and their targets implied that XR_173257.3 and its target arylalkylamine N-acetyltransferase (AANAT) and XR_173415.3 and its target TH might participate in the regulation of seasonal reproduction. Together, the changes in reproductive hormones and transcriptome will help to determine the important photoperiod-induced lncRNAs and mRNAs and provide a valuable resource for further research on reproductive seasonality in sheep.

The hypothalamic–pituitary–adrenal (HPA) axis plays an important role in the growth and development of mammals. Recently, lncRNA transcripts have emerged as an area of importance in sheep photoperiod and seasonal estrus studies. This research aims to identify lncRNA and mRNA that are differentially expressed in the sheep adrenal gland in long (LP) or short (SP) photoperiods using transcriptome sequencing and bioinformatics analysis based on the OVX + E2 (Bilateral ovariectomy and estradiol-implanted) model. We found significant differences in the expression of lncRNAs in LP42 (where LP is for 42 days) vs. SP-LP42 (where SP is for 42 days followed by LP for 42 days) (n = 304), SP42 (where SP is for 42 days) vs. SP-LP42 (n = 1,110) and SP42 vs. LP42 (n = 928). Cluster analysis and enrichment analysis identified SP42 vs. LP42 as a comparable group of interest and found the following candidate genes related to reproductive phenotype: FGF16, PLGF, CDKN1A, SEMA7A, EDG1, CACNA1C and ADCY5. FGF16 (Up-regulated lncRNA MSTRG.242136 and MSTRG.236582) is the only up-regulated gene that is closely related to oocyte maturation. However, EDG1 (Down-regulated lncRNA MSTRG.43609) and CACNA1C may be related to precocious puberty in sheep. PLGF (Down-regulated lncRNA MSTRG.146618 and MSTRG.247208) and CDKN1A (Up-regulated lncRNA MSTRG.203610 and MSTRG.129663) are involved in the growth and differentiation of placental and retinal vessels, and SEMA7A (Up-regulated lncRNA MSTRG.250579) is essential for the development of gonadotropin-releasing hormone (GnRH) neurons. These results identify novel candidate genes that may regulate sheep seasonality and may lead to new methods for the management of sheep reproduction. This study provides a basis for further explanation of the basic molecular mechanism of the adrenal gland, but also provides a new idea for a comprehensive understanding of seasonal estrus characteristics in Sunite sheep.

Intense artificial selection has been imposed to Luzhong mutton sheep population in the past years. Improvements on growth and reproductive performance are two breeding goals in the present herd. Although some progresses were phenotypically observed possibly due to inbreeding induced by strong selection in terms of these traits, the genomic evaluation was poorly understood. Therefore, a high-density SNP array was used to characterize the pattern of runs of homozygosity (ROH), estimate inbreeding and inbreeding depressions on early growth performance and litter size based upon ROH, and scan positive selection signatures of recent population. Consequently, a low inbreeding level was observed which had negative effects on litter size, but not on early growth performance. And 160 genes were under selection, of which some were reported to be linked to several traits of sheep including body weight, litter size, carcass and meat quality, milk yield and composition, fiber quality and health, and the top genes were associated with growth (growth hormone [GH]- growth hormone receptor [GHR]- Insulin-like growth factor 1 [IGF1] axis) and litter size (bone morphogenic proteins [BMPs]-associated). The effectiveness of previous breeding measures was highlighted, but purging selection was proposed to alleviate the inbreeding depression on litter size, providing some genomic insights to breeding management of Luzhong mutton sheep.

The piRNA-PIWI protein complex plays crucial roles in safeguarding the genome against inordinate transposon mobilization and regulation of embryonic development. A previous study indicated the presence of piRNA in sheep reproductive organs. However, the tissue distribution and cellular localization of PIWI proteins in sheep remains unclear. Therefore the present study aimed to explore the expression profiles of mRNAs of mammalian PIWI proteins (PIWIL1, PIWIL2, PIWIL4 and AGO3) in 9 tissues derived from adult male and female sheep. Results showed the expression of PIWIL1, PIWIL2, and PIWIL4 was significantly higher in the testis and ovary than in the other tissues. Immunohistochemistry analysis of testes indicated that each of the 4 proteins had specific cellular localizations, and some of the localizations were different from those of other species. All the proteins were mainly localized in the primary spermatocytes, suggesting that they are crucial for silencing of transposon to guarantee the integrity of the gamete genome during early stage of spermatogenesis. In the ovaries, the PIWI proteins were mainly localized in oocytes from antral follicles and leukocytes in ovarian blood. Our results provide insights to better understand the functions of PIWI proteins during spermatogenesis, oogenesis and immune defence in sheep.

The pituitary is a remarkably dynamic organ with roles in hormone (FSH and LH) synthesis and secretion. In animals with the FecB (fecundity Booroola) mutation, the pituitary experiences hormone fluctuations during the follicular–luteal transition, which is implicated in the expression and regulation of many genes and regulators. Long non-coding RNAs (lncRNAs) are a novel type of regulatory factors for the reproductive process. Nevertheless, the expression patterns of lncRNAs and their roles in FecB-mediated follicular development and ovulation remain obscure. Thus, we profiled the pituitary transcriptome during the follicular (F, 45 h after evacuation vaginal sponges) and luteal (L, 216 h after evacuation vaginal sponges) phases in FecB-mutant homozygous (BB) and wild-type (WW) Small Tail Han sheep. We identified 78 differentially expressed genes (DEGs) and 41 differentially expressed lncRNAs (DELs) between BB_F and BB_L, 32 DEGs and 26 DELs between BB_F and WW_F, 16 DEGs and 29 DELs between BB_L and WW_L, and 50 DEGs and 18 DELs between WW_F and WW_L. The results of real-time quantitative PCR (RT-qPCR) correlated well with the transcriptome data. In both the follicular and luteal phases, DEGs (GRID2, glutamate ionotropic receptor delta type subunit 2; ST14, ST14 transmembrane serine protease matriptase) were enriched in hormone synthesis, secretion, and action. MSTRG.47470 and MSTRG.101530 were the trans-regulated elements of ID1 (inhibitor of DNA binding 3, HLH protein) and the DEG ID3 (inhibitor of DNA binding 3, HLH protein), and EEF2 (eukaryotic translation elongation factor 2), respectively; these factors might be involved in melatonin and peptide hormone secretion. In the FecB-mediated follicular phase, MSTRG.125392 targeted seizure-related 6 homolog like (SEZ6L), and MSTRG.125394 and MSTRG.83276 targeted the DEG KCNQ3 (potassium voltage-gated channel subfamily Q member 3) in cis, while MSTRG.55861 targeted FKBP4 (FKBP prolyl isomerase 4) in trans. In the FecB-mediated luteal phase, LOC105613905, MSTRG.81536, and MSTRG.150434 modulated TGFB1, SMAD3, OXT, respectively, in trans. We postulated that the FecB mutation in pituitary tissue elevated the expression of certain genes associated with pituitary development and hormone secretion. Furthermore, this study provides new insights into how the pituitary regulates follicular development and ovulation, illustrated by the effect of the FecB mutation.

MicroRNA (miRNA) is a kind of noncoding RNA whose function involved in various biological processes in neuronal maturation and adipocyte cells, such as differentiation, proliferation, development, apoptosis, and metabolism. Herein, miRNA-Seq was used to identify miRNAs in the tail fat tissue of Hu sheep (short-fat-tailed) and Tibetan sheep (short-thin-tailed). In this study, 155 differentially expression miRNAs (DE miRNAs) were identified, including 78 up-regulated and 77 down-regulated. Among these DE miRNAs, 17 miRNAs were reported and related with lipid metabolism. MiRanda and RNAhybrid software were used to predict the target genes of DE miRNAs, obtaining the number of targeting relationships is 38553. Target genes were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 742 terms and 302 single pathways are enriched, including lipid metabolic process, response to lipid, cellular lipid catabolic process, lipid catabolic process, cellular lipid metabolic process, inositol lipid-mediated signaling, calcium channel activity, PI3K-Akt signaling pathway, MAPK signaling pathway, ECM-receptor interaction, AMPK signaling pathway, Wnt signaling pathway and TGF-beta signaling pathway. Notably, miR-379-5p was associated with tail fat deposition of sheep. Dual-Luciferase reporter assays showed miR-379-5p and HOXC9 had targeted relationship. The result of RT-qPCR showed that the expression trend of miR-379-5p and HOXC9 was opposite. miR-379-5p was down-regulated and highly expressed in tail adipose tissue of Tibetan sheep. HOXC9 was highly expressed in adipose tissue of Hu sheep. These results could provide a meaningful theoretical basis for studying the molecular mechanisms of sheep tail adipogenesis.

Pituitary pars tuberalis (PT) plays an important role as the transmission center in the seasonal reproduction of animals. It helps convert external photoperiod signals into intrinsic seasonal reproduction signals. In sheep PT, specific expression patterns of several genes (including short photoperiod-induced gene CHGA and long photoperiod genes EYA3 and TSHβ) under different photoperiods are crucial characteristics during this signal transduction. Recent studies have revealed the role of epigenetics in regulating the expression of seasonal reproductive key genes. Therefore, we explored whether microRNAs and LncRNAs regulated the expressions of the above key genes. Firstly, the expression of miR-25 and CHGA showed a significant negative correlation in sheep PT. Results of the dual luciferase reporter assay and miR-25 overexpression indicated that miR-25 could inhibit the expression of CHGA by specifically binding to its 3′UTR region in pituitary cells. Then, expression negative correlation and dual luciferase reporter analyses were used to screen and identify the candidate LncRNA (Lnc107153) targeted by miR-25. Finally, the results of fluorescence in situ hybridization and Lnc107153 overexpression suggested that Lnc107153 and miR-25 were involved in the epigenetic regulation of CHGA expression. However, the expressions of EYA3 and TSHβ were not regulated by miRNAs. These results will provide new insights into the epigenetic regulatory network of key genes in sheep seasonal reproduction.

The litter size and ovulation rate are different among ewes of different FecB genotypes in Small Tail Han sheep. These variants in reproductive phenotypes may be regulated by hormones released by the hypothalamic–pituitary–ovarian axis. However, there have been few reports on the hypothalamus regarding regulating an increase in ovulation in sheep with FecB mutation at different estrous stages. Thus, we examined the abundance of hypothalamus tissue protein profiles of six FecB mutant homozygous (BB) and six wild-type (WW) ewes at the luteal and follicular phases. We determined this abundance by tandem mass tag-based quantitative analysis and parallel reaction monitoring methods. Furthermore, an integrated proteotranscriptomic analysis was performed by the Data Integration Analysis for Biomarker discovery using the latent variable approaches for Omics studies (DIABLO) framework to examine biological processes and pathway alterations by the FecB mutant. The abundance of 154 proteins was different between the two estrous stages. Growth hormone and prolactin were particularly enriched in the neuroactive ligand–receptor interactions, the prolactin signaling pathway, and the PI3K-Akt signaling pathway which are related to hypothalamic function and reproduction. We combined proteome and transcriptome data from different estrous stages and genotypes. There is a high correlation (Pearson correlation coefficient = 0.99) between the two datasets in the first two components. We applied the traditional single-omic multivariate approach to obtain differentially abundant proteins and differentially expressed genes. The major fertility related biomarkers were selected using the two approaches mentioned above. Several key pathways (GABAergic synapse, neuroactive ligand–receptor interaction, estrogen and MAPK signaling pathways) were enriched, which are central to gonadotrophin-releasing hormone (GnRH) secretion and reproduction. A higher level of gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and gamma-aminobutyric acid type A receptor subunit beta2 (GABRB2) expression was observed in BB ewes as compared to WW ewes. This finding suggested that a greater production of GnRH during follicular development in BB ewes may explain the higher mature follicle number in mutant ewes. FKBP prolyl isomerase 1A (FKBP1A), which was a major feature factor in the proteome selected by DIABLO, was an important switch for activating the transforming growth factor beta (TGFβ) pathway, and its expression was higher in the WW ewes than in the BB ewes. We suggest that BB sheep maintain TGFβ pathway activity by reducing FKBP1A protein abundance. This innovative data integration in the hypothalamus may provide fresh insight into the mechanisms by which the FecB mutation affects sheep fertility, while providing novel biomarkers related to reproductive endocrinology in sheep breeding.

The Booroola fecundity gene (FecB) has a mutation that was found to increase the ovulation rate and litter size in Booroola Merino sheep. This mutation is also associated with the fecundity of small-tail han (STH) sheep, an important maternal breed used to produce hybrid offspring for mutton production in China. Previous research showed that the FecB gene affects reproduction in STH sheep, based on litter size records. However, the effects of this gene on estrus, ovulation, and endocrine characteristics in these sheep remain unclear. Here, we analyzed the traits mentioned earlier and compared them among the three FecB genotypes of STH ewes using estrus synchronization. Overall, 53 pluriparous ewes were selected from among 890 STH ewes and subjected to FecB genotyping for experiments to characterize estrous and ovulation rates. FecB heterozygous (+B) ewes presented an earlier onset of estrus (42.9 ± 2.2 h) and a shorter estrous cycle (17.2 ± 0.2 days) (P ≤ 0.05). The ovulation rates increased with the increasing copy number of the B allele (P ≤ 0.01). Ovulation time showed no significant differences among the three FecB genotypes. The serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone, estrogen (E2), and progesterone (P4) were measured in 19 of the ewes. Serum concentrations of E2 and FSH dramatically varied around the time of behavioral estrus. In FecB mutant homozygous (BB) ewes, E2 concentration had two peaks, which were higher (P ≤ 0.05) than those of ++ genotypes. FSH concentration of BB ewes was higher (P ≤ 0.05) than that of the ++ ewes just after estrus. The expression of the estrogen receptor 1 (ESR1) gene in the +B genotype was higher than in the other genotypes. Based on the data for the reproductive performance of STH ewes with the three FecB genotypes, our study suggests that the development of follicles in ewes with the B allele is dependent on the response to FSH regulated by E2 in the early stage. +B ewes, exhibiting moderate ovulation and litter size and a shorter estrous cycle, can be highly recommended in sheep crossbreeding systems for commercial mutton production. Moreover, this study provides useful information to conserve better and use the genetic resources of STH sheep in China.

The oviduct is a dynamic reproductive organ for mammalian reproduction and is required for gamete storage, maturation, fertilization, and early embryonic development, and it directly affects fecundity. However, the molecular regulation of prolificacy occurring in estrous periods remain poorly understood. This study aims to gain a better understanding of the genes involved in regulating goat fecundity in the proteome and transcriptome levels of the oviducts. Twenty female Yunshang black goats (between 2 and 3 years old, weight 52.22 ± 0.43 kg) were divided into high- and low-fecundity groups in the follicular (FH and FL, five individuals per group) and luteal (LH and LL, five individuals per group) phases, respectively. The DIA-based high-resolution mass spectrometry (MS) method was used to quantify proteins in twenty oviducts. A total of 5409 proteins were quantified, and Weighted gene co-expression network analysis (WGCNA) determined that the tan module was highly associated with the high-fecundity trait in the luteal phase, and identified NUP107, ANXA11, COX2, AKP13, and ITF140 as hub proteins. Subsequently, 98 and 167 differentially abundant proteins (DAPs) were identified in the FH vs. FL and LH vs. LL comparison groups, respectively. Parallel reaction monitoring (PRM) was used to validate the results of the proteomics data, and the hub proteins were analyzed with Western blot (WB). In addition, biological adhesion and transporter activity processes were associated with oviductal function, and several proteins that play roles in oviductal communication with gametes or embryos were identified, including CAMSAP3, ITGAM, SYVN1, EMG1, ND5, RING1, CBS, PES1, ELP3, SEC24C, SPP1, and HSPA8. Correlation analysis of proteomics and transcriptomic revealed that the DAPs and differentially expressed genes (DEGs) are commonly involved in the metabolic processes at the follicular phase; they may prepare the oviductal microenvironment for gamete reception; and the MAP kinase activity, estrogen receptor binding, and angiotensin receptor binding terms were enriched in the luteal phase, which may be actively involved in reproductive processes. By generating the proteome data of the oviduct at two critical phases and integrating transcriptome analysis, we uncovered novel aspects of oviductal gene regulation of fecundity and provided a reference for other mammals.

The pituitary pars tuberalis (PT) is the regulating center of seasonal reproduction, which can sense the melatonin signal and eventually cause downstream changes of GnRH secretion through TSHβ. Recently, lncRNAs have been identified in animal reproductive-related tissues, and they play important roles in reproductive regulation. Therefore, in this study, we expect to identify photoperiod-induced lncRNAs and genes in pituitary PT of sheep by comparison of expression profiles between short photoperiod (SP) and long photoperiod (LP). Through RNA-Seq, a total of 55,472 lncRNAs were identified in pituitary PT of Sunite ewes. The number of differentially expressed (DE) genes and lncRNAs between SP and LP increased gradually with the extension of LP (from LP7 to LP42). The notable LP-induced candidate genes included EYA3, TSHB, SIX1, DCT, VMO1, AREG, SUV39H2, and EZH2, and SP-induced genes involved ENSOARG00000012585, CHGA, FOS, SOCS3, and TH. In enriched pathways for DE genes and lncRNA target genes between SP and LP, the reproduction- and circadian-related pathways were highlighted. In addition, the interactome analysis of lncRNAs and their targets implied that MSTRG.209166 and its trans-target TSHB, MSTRG.288068 and its cis-target SIX1, and ENSOARG00000026131 and its cis-target TH might participate in regulation of seasonal reproduction. Together, these results will help to determine important photoperiod-induced lncRNAs and genes and give us some new insights into the epigenetic regulation of seasonal reproduction in sheep.

To elucidate how expression characteristics of BMPR1B, BMP15, and GDF9 are associated with sheep reproduction, tissue-specific mRNA expression of these genes in Small Tail Han (STH) ewes (a polytocous breed) and Sunite (SNT) ewes (a monotocous breed) in the follicular phase were investigated using transcription profiling and quantitative real-time PCR (qPCR). Expression levels of the three genes were all highest in ovaries of the two sheep breeds, and BMPR1B and GDF9 expression in the ovarian tissue was significantly higher in STH sheep compared with SNT sheep (P < 0.01), whereas BMP15 expression in pituitary, ovarian, oviduct, and uterine tissues was significantly lower in STH sheep compared with SNT sheep (P < 0.01). This study revealed that BMPR1B, BMP15, and GDF9 may play important roles in the hypothalamic-pituitary-gonadal axis, especially in the ovary, which may affect sheep prolificacy. BMPR1B and GDF9 expression might increase litter size, whereas BMP15 expression may decrease litter size. These results provide preliminary information regarding molecular regulatory mechanisms underlying sheep prolificacy.

Abstract Background Sheep have developed the ability to store fat in their tails, which is a unique way of reserving energy to survive a harsh environment. However, the mechanism underlying this adaptive trait remains largely unsolved. Results In the present study, we provide evidence for the genetic determinants of fat tails, based on whole genome sequences of 89 individual sheep. A genome-wide scan of selective sweep identified several candidate loci including a region at chromosome 13, a haplotype of which underwent rapid evolution and spread through fat-tailed populations in China and the Middle East. Sequence analysis revealed an inter-genic origin of this locus, which later became a hotspot of ruminant-specific retro-transposon named BovB. Additionally, the candidate locus was validated based on a fat- and thin-tailed cross population. The expression of an upstream gene BMP2 was differentially regulated between fat-tailed and thin-tailed individuals in tail adipose and several other tissue types. Conclusions Our findings suggest the fixation of fat tails in domestic sheep is caused by a selective sweep near a retro-transposable hotspot at chromosome 13, the diversity of which specifically affects the expression of BMP2. The present study has shed light onto the understanding of fat metabolism.

Small-tailed Han sheep, with different FecB genotypes, manifest distinct ovulation rates and fecundities, which are due to differences in reproductive hormones secreted by the hypothalamic–pituitary–ovarian axis. Nevertheless, the function of the hypothalamus against a FecB mutant background on increasing ovulation rate is rarely reported. Therefore, we determined the expression profiles of hypothalamus tissue collected from six wild-type (WW) and six FecB mutant homozygous (BB) ewes at the follicular and luteal phases by whole-transcriptome sequencing. We identified 53 differentially expressed mRNAs (DEGs) and 40 differentially expressed long non-coding RNAs (DELs) between the two estrus states. Functional annotation analysis revealed that one of the DEGs, PRL, was particularly enriched in the hypothalamic function, hormone-related, and reproductive pathways. The lncRNA–target gene interaction networks and KEGG analysis in combination suggest that the lncRNAs LINC-676 and WNT3-AS cis-acting on DRD2 and WNT9B in different phases may induce gonadotropin-releasing hormone (GnRH) secretion. Furthermore, there were differences of regulatory elements and WNT gene family members involved in the follicular–luteal transition in the reproductive process between wild-type (WNT7A) and FecB mutant sheep (WNT9B). We combined the DEG and DEL data sets screened from different estrus states and genotypes. The overlap of these two sets was identified to select the mRNAs and lncRNAs that have major effects on ovulation. Among the overlapping molecules, seven DEGs and four DELs were involved in the follicular–luteal transition regulated by FecB mutation. Functional annotation analysis showed that two DEGs (FKBP5 and KITLG) were enriched in melanogenesis, oxytocin, and GnRH secretion. LINC-219386 and IGF2-AS were highly expressed in the BB ewes compared with WW ewes, modulating their target genes (DMXL2 and IGF2) to produce more GnRH during follicular development, which explains why mutated ewes produced more mature follicles. These results from expression profiling of the hypothalamus with the FecB mutation at different estrus states provide new insights into how the hypothalamus regulates ovulation under the effect of the FecB mutation.

The pituitary is a vital endocrine organ that regulates animal seasonal reproduction by controlling the synthesis and secretion of the hormone. The change of photoperiod is the key factor affecting the function of the pituitary in animals, but the mechanism is unclear. Here, we studied the transcriptomic variation in pars distalis (PD) of the pituitary between short photoperiod (SP) and long photoperiod (LP) using RNA sequencing based on the OVX+E2 sheep. 346 differentially expressed (DE) lncRNAs and 186 DE-mRNA were found in the PD. Moreover, function annotation analysis indicated that the reproductive hormones and photoperiod response-related pathways including aldosterone synthesis and secretion, insulin secretion, thyroid hormone synthesis, and circadian entrainment were enriched. The interaction analysis of mRNA-lncRNA suggested that MSTRG.240648, MSTRG.85500, MSTRG.32448, and MSTRG.304959 targeted CREB3L1 and DUSP6, which may be involved in the photoperiodic regulation of the PD. These findings provide resources for further study on the seasonal reproductive in ewes.

Early studies have provided a wealth of information on the functions of microRNAs (miRNAs). However, less is known regarding their functions in the hypothalamus involved in sheep reproduction. To explore the potential roles of hypothalamic messenger RNAs (mRNAs) and miRNAs in sheep without FecB mutation, in total, 172 and 235 differentially expressed genes (DEGs) and 42 and 79 differentially expressed miRNAs (DE miRNAs) were identified in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase versus monotocous sheep in the luteal phase (PL vs. ML), respectively, using RNA sequencing. We also identified several key mRNAs (e.g., POMC, GNRH1, PRL, GH, TRH, and TTR) and mRNA–miRNAs pairs (e.g., TRH co-regulated by oar-miR-379-5p, oar-miR-30b, oar-miR-152, oar-miR-495-3p, oar-miR-143, oar-miR-106b, oar-miR-218a, oar-miR-148a, and PRL regulated by oar-miR-432) through functional enrichment analysis, and the identified mRNAs and miRNAs may function, conceivably, by influencing gonadotropin-releasing hormone (GnRH) activities and nerve cell survival associated with reproductive hormone release via direct and indirect ways. This study represents an integral analysis between mRNAs and miRNAs in sheep hypothalamus and provides a valuable resource for elucidating sheep prolificacy.

Litter size is an important economic trait in the mutton sheep industry. BMP15 is one of the key candidate genes for litter size in sheep. In this study, the entire ORF region of BMP15 was sequenced in 154 Luzhong mutton ewes, and the novel variations were determined. The association between polymorphism in BMP15 and litter size was analyzed using a general linear model. Six out of a total of thirteen variations were identified to be novel. Association analysis indicated that four (SNPs ENSOART00000010201.1:c.352+342C>A, c.352+1232T>C, c.352+1165A>G and c.353-2036T>A) were significantly associated with litter size. The joint analysis among three major genes (BMP15, BMPR1B and GDF9) exhibited significant interaction effects in three combinations (FecB and c.352+1232T>C of BMP15; FecB and c.352+1165A>G of BMP15; c.352+342C>A of BMP15 and ENSOART00000014382.1:c.994G>A of GDF9). For the SNPs c.352+1232T>C and c.352+342C>A, the global distribution of allele frequencies showed that the highest variation frequency occurs in Western Europe. In conclusion, the results demonstrated that BMP15 is a major gene for litter size in Luzhong mutton sheep and candidate SNPs associated with litter size were identified.

Abstract Compared with the commercially available single nucleotide polymorphism (SNP) chip based on the Bead Chip technology, the solution hybrid selection (SHS)-based target enrichment SNP chip is not only design-flexible, but also cost-effective for genotype sequencing. In this study, we propose to design an animal SNP chip using the SHS-based target enrichment strategy for the first time. As an update to the international collaboration on goat research, a 66 K SNP chip for cashmere goat was created from the whole-genome sequencing data of 73 individuals. Verification of this 66 K SNP chip with the whole-genome sequencing data of 436 cashmere goats showed that the SNP call rates was between 95.3% and 99.8%. The average sequencing depth for target SNPs were 40X. The capture regions were shown to be 200 bp that flank target SNPs. This chip was further tested in a genome-wide association analysis of cashmere fineness (fiber diameter). Several top hit loci were found marginally associated with signaling pathways involved in hair growth. These results demonstrate that the 66 K SNP chip is a useful tool in the genomic analyses of cashmere goats. The successful chip design shows that the SHS-based target enrichment strategy could be applied to SNP chip design in other species.

China had the largest population of raising donkeys in the world, however the number of Chinese indigenous donkey decreased dramatically due to the increase of agriculture mechanization in the last century. The species has still been important in China because of its edible and medical value, therefore the survey on its genetic diversity in China is necessary for its conservation and utilization. In this study, 15 microsatellite markers were used to evaluate genetic diversity and population structure of five Chinese indigenous donkey breeds. The mean values of expected heterozygosity, allelic richness, and total number of alleles for all the tested Chinese donkeys were 0.70, 6.04, and 6.28 respectively, suggesting that the genetic diversity of Chinese indigenous donkeys is rich. The Bayesian analysis and principal component analysis plot yielded the same clustering result, which revealed that Guanzhong donkey was the most differentiated breed in all detected samples, and Jinnan (JN) and Guangling (GL) were genetically closed together. Additionally, our results indicated that the heterozygote deficit was severe in two Chinese indigenous donkey breeds (GL and JN), and it warned us that animal conservation activities on this species should be considered carefully in near future.

Abstract Background Alpha 1-antitrypsin (α1-AT) may affect the susceptibility of mastitis in dairy cattle for its possible role in the protection of lactoferrin from proteolytic degradation in the mammary. Milk somatic cell score (SCS) is a logarithmic transformation of the milk somatic cell count widely used as an index to evaluate mastitis. To study the relationships of α1-AT gene and SCS in Chinese Holstein cows, methods of PCR-SSCP, DNA sequencing, PCR-RFLP, and CRS-PCR technologies were used to detect single nucleotide polymorphisms sites in α1-AT gene. Results Two polymorphic sites at G5503A and G5746C of α1-AT gene were found. AA (0.3633), AB (0.4644) and BB (0.1723) genotypes were detected at G5503A site, CC (0.3483), CD (0.4906) and DD (0.1611) genotypes were found at G5746C in Chinese Holstein cows. Least squares mean of SCS for individuals with BB genotype was significantly lower than that with AA and AB genotype (p 0.05). Least squares mean of SCS for individuals with BBDD genotype combination were significantly lower than those with AACC and AACD (p

Background Long non-coding RNAs (lncRNAs) regulate endometrial secretion and uterine volume. However, there is little research on the role of lncRNAs in the uterus of Small Tail Han sheep (FecB++). Herein, RNA-seq was used to comparatively analyze gene expression profiles of uterine tissue between polytocous and monotocous sheep (FecB++) in follicular and luteal phases. Methods To identify lncRNA and mRNA expressed in the uterus, the expression of lncRNA and mRNA in the uterus of Small Tail Han sheep (FecB++) from the polytocous group (n = 6) and the monotocous group (n = 6) using RNA-sequencing and real-time polymerase chain reaction (RT-PCR). Identification of differentially expressed lncRNAs and mRNAs were performed between the two groups and two phases . Gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for the differentially expressed mRNAs. LncRNA-mRNA co-expression network was constructed to further analyses the function of related genes. Results In the follicular phase, 473 lncRNAs and 166 mRNAs were differentially expressed in polytocous and monotocous sheep; in the luteal phase, 967 lncRNAs and 505 mRNAs were differentially expressed in polytocous and monotocous sheep. GO and KEGG enrichment analysis showed that the differentially expressed lncRNAs and their target genes are mainly involved in ovarian steroidogenesis, retinol metabolism, the oxytocin signaling pathway, steroid hormone biosynthesis, and the Foxo signaling pathway. Key lncRNAs may regulate reproduction by regulating genes involved in these signaling pathways and biological processes. Specifically, UGT1A1, LHB, TGFB1, TAB1, and RHOA, which are targeted by MSTRG.134747, MSTRG.82376, MSTRG.134749, MSTRG.134751, and MSTRG.134746, may play key regulatory roles. These results offer insight into molecular mechanisms underlying sheep prolificacy.

Litter size is one of the most important economic traits in sheep. GDF9 and BMPR1B are major genes affecting the litter size of sheep. In this study, the whole coding region of GDF9 was sequenced and all the SNPs (single nucleotide polymorphisms) were determined in Luzhong mutton ewes. The FecB mutation was genotyped using the Sequenom MassARRAY®SNP assay technology. Then, the association analyses between polymorphic loci of GDF9 gene, FecB, and litter size were performed using a general linear model procedure. The results showed that eight SNPs were detected in GDF9 of Luzhong mutton sheep, including one novel mutation (g.41769606 T > G). The g.41768501A > G, g.41768485 G > A in GDF9 and FecB were significantly associated with litter size in Luzhong mutton ewes. The g.41768485 G > A is a missense mutation in the mature GDF9 protein region and is predicted to affect the tertiary structure of the protein. The results preliminarily demonstrated that GDF9 was a major gene affecting the fecundity of Luzhong mutton sheep and the two loci g.41768501A > G and g.41768485 G > A may be potential genetic markers for improving litter size.

Bamei mutton sheep is a Chinese domestic sheep breed developed by crossing German Mutton Merino sheep and indigenous Mongolian sheep for meat production. Here, we focused on detecting candidate genes associated with the increasing of the litter size in this breeds under recent artificial selection to improve the efficiency of mutton production. We selected five high- and five low-fecundity Bamei mutton sheep for whole-genome resequencing to identify candidate genes for sheep prolificacy. We used the FST and XP-EHH statistical approach to detect the selective sweeps between these two groups. Combining the two selective sweep methods, the reproduction-related genes JUN, ITPR3, PLCB2, HERC5, and KDM4B were detected. JUN, ITPR3, and PLCB2 play vital roles in GnRH (gonadotropin-releasing hormone), oxytocin, and estrogen signaling pathway. Moreover, KDM4B, which had the highest FST value, exhibits demethylase activity. It can affect reproduction by binding the promoters of estrogen-regulated genes, such as FOXA1 (forkhead box A1) and ESR1 (estrogen receptor 1). Notably, one nonsynonymous mutation (p.S936A) specific to the high-prolificacy group was identified at the TUDOR domain of KDM4B. These observations provide a new opportunity to research the genetic variation influencing fecundity traits within a population evolving under artificial selection. The identified genomic regions that are responsible for litter size can in turn be used for further selection.

Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction; however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small Tail Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as galectin 3 (LGALS3) in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction.

Inner Mongolia and Liaoning cashmere goats in China are well-known for their cashmere quality and yield. Thus, they are great models for identifying genomic regions associated with cashmere traits. Herein, 53 Inner Mongolia cashmere goats, Liaoning cashmere goats and Huanghuai goats were genotyped, and 53,347 single-nucleotide polymorphisms (SNPs) were produced using the Illumina Caprine 50K SNP chip. Additionally, we identified some positively selected SNPs by analyzing Fst and XP-EHH. The top 5% of SNPs had selection signatures. After gene annotation, 222 and 173 candidate genes were identified in Inner Mongolia and Liaoning cashmere goats, respectively. Several genes were related to hair follicle development, such as TRPS1, WDR74, LRRC14, SPTLC3, IGF1R, PADI2, FOXP1, WNT10A and CSN3. Gene enrichment analysis of these cashmere trait-associated genes related 67 enriched signaling pathways that mainly participate in hair follicle development and stem cell pluripotency regulation. Furthermore, we identified 20 overlapping genes that were selected in both cashmere goat breeds. Among these overlapping genes, WNT10A and CSN3, which are associated with hair follicle development, are potentially involved in cashmere production. These findings may improve molecular breeding of cashmere goats in the future.

Tibetan goat is an ancient breed, which inhabits the adverse conditions of the plateaus in China. To investigate the role of selection in shaping its genomes, we genotyped Tibetan goats (Nagqu Prefecture, above 4500 m) and three lowland populations (Xinjiang goats, Taihang goats and Huanghuai goats). The result of PCA, neighbor-joining (N-J) tree and model-based clustering showed that the genetic structure between the Tibetan goat and the three lowland populations has significant difference. As demonstrated by the di statistic, we found that some genes were related to the high-altitude adaptation of Tibetan goats. Functional analysis revealed that these genes were enriched in the VEGF (vascular endothelial growth factor) signaling pathway and melanoma, suggesting that nine genes (FGF2, EGFR, AKT1, PTEN, MITF, ENPEP, SIRT6, KDR, and CDC42) might have important roles in the high-altitude adaptation of Nagqu Tibetan goats. We also found that the LEPR gene was under the strongest selection (di value = 16.70), and it could induce upregulation of the hypoxic ventilatory response. In addition, five genes (LEPR, LDB1, EGFR, NOX4 and FGF2) with high di values were analyzed using q-PCR. Among them, we found that LEPR, LDB1 and FGF2 exhibited higher expression in the lungs of the Tibetan goats; LEPR, EGFR and LDB1 exhibited higher expression in the hearts of the Huanghuai goat. Our results suggest that LEPR, LDB1, EGFR and FGF2 genes may be related to the high-altitude adaptation of the goats. These findings improve our understanding of the selection of the high-altitude adaptability of the Nagqu Tibetan goats and provide new theoretical knowledge for the conservation and utilization of germplasm resources.

Previous studies showed that the NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 genes are two important reproductive genes; however, their effects on sheep litter size are unknown. Therefore, in this study, we first genotyped seven sheep breeds via the MassARRAY® SNP system at the loci g.60495375A > G, g.60495363G > A, and g.60499690C > A in NLRP5, and g.59030623T > C and g.59043397A > C in NLRP9. Our results revealed that each locus in most sheep breeds contained three genotypes. Then, we conducted population genetic analysis of single nucleotide polymorphisms in NLRP5 and NLRP9, and we found that the polymorphism information content value in all sheep breeds ranged from 0 to 0.36, and most sheep breeds were under Hardy–Weinberg equilibrium (p > 0.05). Furthermore, association analysis in Small Tail Han sheep indicated that two loci, g.60495363G > A in NLRP5 and g.59030623T > C in NLRP9, were highly associated with litter size. The mutation in g.60495363G > A may decrease interactions of NLRP5 with proteins, such as GDF9, whereas the mutation in g.59030623T > C may enhance the combining capacity of NLRP9 with these proteins; consequently, these mutations may influence the ovulation rate and even litter size. The findings of our study provide valuable genetic markers that can be used to improve the breeding of sheep and even other mammals.

Most ewes in China are seasonally polyestrous with normal ovulatory cycles, which is controlled by photoperiod (length of the daily light phase). These ewes are estrous in the short-day season and anestrus in the long-day season and cannot mate during anestrus. Thus seasonal breeding limits both diversification and intensification of production. If sheep can estrus all round year, it can be mated twice per year, which can greatly improve the economic benefits. To change seasonal estrus at the genetic level and cultivating new sheep breeds, it is important to understand the molecular mechanisms of seasonal breeding trait in sheep. The BCL2-associated agonist of cell death (BAD) gene being a regulator of cellular apoptosis was identified by our previous RNA-Seq, which is associated with follicular development in mammalian ovaries. However, the mechanism how BAD can regulate estrus in sheep was poorly understood. In this study, we characterized ovine BAD, including full-length mRNA cloning and protein sequence prediction, as well as BAD expression profile in Small-tailed Han (STH) sheep. The highest expression levels of BAD were observed in sheep hypothalamus, lung, and pituitary, while the lowest expression was in liver. Functional analysis of BAD was performed in primary granulosa cells of sheep. The concentration of P4 was significantly increased after RNAi interference of BAD, while P4 level was shown to be opposite after BAD overexpression in vitro. It has been found that BAD can reduce progesterone levels by promoting ovarian GC apoptosis, which might be involved in regulating the estrus cycle in sheep.

CircRNA plays important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports on circRNAs related to livestock reproduction. In this study, we identified circRNAs by deep sequencing and analyzed their expression in the uteri of polytocous and monotocous sheep (FecB++) during follicular and luteal phases. There were 147 and 364 circRNAs with differential expression in the follicular and luteal phases, respectively. GO and KEGG enrichment analysis was performed for the host genes of the circRNAs to predict the functions of differentially expressed circRNAs. These source genes were mainly involved in the estrogen signaling pathway, TGFβ signaling pathway, GnRH signaling pathway, oxytocin signaling pathway, pentose phosphate pathway, and starch and sucrose metabolism related to reproduction and energy metabolism. CircRNA expression patterns were validated by RT-qPCR. Our findings provide a solid foundation for the identification and characterization of key important circRNAs involved in reproduction.

The diverse functions of long noncoding RNAs (lncRNAs), which execute their functions mainly through modulating the activities of their target genes, have been have been widely studied for many years (including a number of studies involving lncRNAs in the ovary and uterus). Herein, for the first time, we detect lncRNAs in sheep hypothalami with FecB++ through RNA Sequencing (RNA-Seq) and identify a number of known and novel lncRNAs, with 622 and 809 found to be differentially expressed in polytocous sheep in the follicular phase (PF) vs. monotocous sheep in the follicular phase (MF) and polytocous sheep in the luteal phase (PL) vs. monotocous sheep in the luteal phase (ML), respectively. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed based on the predicted target genes. The most highly enriched GO terms (at the molecular function level) included carbonyl reductase (NADPH), 15-hydroxyprostaglandin dehydrogenase (NADP+), and prostaglandin-E2 9-reductase activity in PF vs. MF, and phosphatidylinositol-3,5-bisphosphate binding in PL vs. ML was associated with sheep fecundity. Interestingly, the phenomena of valine, leucine, and isoleucine degradation in PL vs. ML, and valine, leucine, and isoleucine biosynthesis in PF vs. MF, were present. In addition, the interactome of lncRNA and its targets showed that MSTRG.26777 and its cis-targets ENSOARG00000013744, ENSOARG00000013700, and ENSOARG00000013777, and MSTRG.105228 and its target WNT7A may participate in the sheep reproductive process at the hypothalamus level. Significantly, MSTRG.95128 and its cis-target Forkhead box L1 (FOXG1) were shown to be upregulated in PF vs. MF but downregulated in PL vs. ML. All of these results may be attributed to discoveries of new candidate genes and pathways related to sheep reproduction, and they may provide new views for understanding sheep reproduction without the effects of the FecB mutation.

Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.

The expression characteristics of the prolific candidate genes, BMPR1B, BMP15, and GDF9, in the major visceral organs and hypothalamic–pituitary–gonadal (HPG) axis tissues of three FecB genotypes (FecB BB, FecB B+, and FecB ++) were explored in STH ewes using RT-PCR and qPCR. The results were as follows, BMPR1B was expressed in all FecB BB genotype (Han BB) tissues, and GDF9 was expressed in all selected tissues, but BMP15 was specifically expressed in the ovaries. Further study of ovarian expression indicated that there was no difference in BMPR1B expression between genotypes, but the FecB B+ genotype (Han B+) had greater expression of GDF9 and BMP15 than Han BB and FecB ++ genotype (Han ++) (p < 0.05, p < 0.01). BMP15 expression was lower in the ovaries of Han BB than in Han ++ sheep, but the reverse was shown for GDF9. The gene expression in non-ovarian tissues was also different between genotypes. Therefore, we consider that the three genes have an important function in ovine follicular development and maturation. This is the first systematic analysis of the tissue expression pattern of BMPR1B, BMP15, and GDF9 genes in STH sheep of the three FecB genotypes. These results contribute to the understanding of the molecular regulatory mechanism for ovine reproduction.