Association of litter size with the ruminal microbiome structure and metabolomic profile in goats.

The Yunshang black goat is a renowned mutton specialist breed mainly originating from China that has excellent breeding ability with varying litter sizes. Litter size is an important factor in the economics of goat farming. However, ruminal microbiome structure might be directly or indirectly regulated by pregnancy-associated factors, including litter sizes. Therefore, the current experiment aimed to evaluate the association of different litter sizes (low versus high) with ruminal microbiome structure by 16S rRNA gene sequencing and metabolomic profiling of Yunshang black does. A total of twenty does of the Yunshang Black breed, approximately aged between 3 and 4 years, were grouped (n = 10 goats/group) into low (D-l) and high (D-h) litter groups according to their litter size (the lower group has ≤ 2 kids/litter and the high group has ≧ 3 kids/litter, respectively). All goats were sacrificed, and collected ruminal fluid samples were subjected to 16S rRNA sequencing and LC-MS/MC Analysis for ruminal microbiome and metabolomic profiling respectively. According to PCoA analysis, the ruminal microbiota was not significantly changed by the litter sizes among the groups. The Firmicutes and Bacteroidetes were the most dominant phyla, with an abundance of 55.34% and 39.62%, respectively. However, Ruminococcaceae_UCG-009, Sediminispirochaeta, and Paraprevotella were significantly increased in the D-h group, whereas Ruminococcaceae_UCG-010 and Howardella were found to be significantly decreased in the D-l group. The metabolic profiling analysis revealed that litter size impacts metabolites as 29 and 50 metabolites in positive and negative ionic modes respectively had significant differences in their regulation. From them, 16 and 24 metabolites of the D-h group were significantly down-regulated in the positive ionic mode, while 26 metabolites were up-regulated in the negative ionic mode for the same group. The most vibrant identified metabolites, including methyl linoleate, acetylursolic acid, O-desmethyl venlafaxine glucuronide, melanostatin, and arginyl-hydroxyproline, are involved in multiple biochemical processes relevant to rumen roles. The identified differential metabolites were significantly enriched in 12 different pathways including protein digestion and absorption, glycerophospholipid metabolism, regulation of lipolysis in adipocytes, and the mTOR signaling pathway. Spearman's correlation coefficient analysis indicated that metabolites and microbial communities were tightly correlated and had significant differences between the D-l and D-h groups. Based on the results, the present study provides novel insights into the regulation mechanisms of the rumen microbiota and metabolomic profiles leading to different fertility in goats, which can give breeders some enlightenments to further improve the fertility of Yunshang Black goats.

Oviduct Transcriptomic Reveals the Regulation of mRNAs and lncRNAs Related to Goat Prolificacy in the Luteal Phase.

The oviduct is associated with embryo development and transportation and regulates the pregnancy success of mammals. Previous studies have indicated a molecular mechanism of lncRNAs in gene regulation and reproduction. However, little is known about the function of lncRNAs in the oviduct in modulating goat kidding numbers. Therefore, we combined RNA sequencing (RNA-seq) to map the expression profiles of the oviduct at the luteal phase from high- and low-fecundity goats. The results showed that 2023 differentially expressed mRNAs (DEGs) and 377 differentially expressed lncRNAs (DELs) transcripts were screened, and 2109 regulated lncRNA-mRNA pairs were identified. Subsequently, the genes related to reproduction (IGF1, FGFRL1, and CREB1) and those associated with embryonic development and maturation (DHX34, LHX6) were identified. KEGG analysis of the DEGs revealed that the GnRH- and prolactin-signaling pathways, progesterone-mediated oocyte maturation, and oocyte meiosis were related to reproduction. GSEA and KEGG analyses of the target genes of DELs demonstrated that several biological processes and pathways might interact with oviduct functions and the prolificacy of goats. Furthermore, the co-expression network analysis showed that XLOC_029185, XLOC_040647, and XLOC_090025 were the cis-regulatory elements of the DEGs MUC1, PPP1R9A, and ALDOB, respectively; these factors might be associated with the success of pregnancy and glucolipid metabolism. In addition, the GATA4, LAMA2, SLC39A5, and S100G were trans-regulated by lncRNAs, predominantly mediating oviductal transport to the embryo and energy metabolism. Our findings could pave the way for a better understanding of the roles of mRNAs and lncRNAs in fecundity-related oviduct function in goats.

Genome-wide identification of long noncoding RNA genes and their potential association with mammary gland development in water buffalo.

OBJECTIVE: Water buffalo, an important domestic animal in tropical and subtropical regions, play an important role in agricultural economy. It is an important source for milk, meat, horns, skin, and draft power, especially its rich milk that is the great source of cream, butter, yogurt, and many cheeses. In recent years, long noncoding RNAs (lncRNAs) have been reported to play pivotal roles in many biological processes. Previous studies for the mammary gland development of water buffalo mainly focus on protein coding genes. However, lncRNAs of water buffalo remain poorly understood, and the regulation relationship between mammary gland development/milk production traits and lncRNA expression is also unclear. METHODS: Here, we sequenced 22 samples of the milk somatic cells from three lactation stages and integrated the current annotation and identified 7,962 lncRNA genes. RESULTS: By comparing the lncRNA genes of the water buffalo in the early, peak, and late different lactation stages, we found that lncRNA gene lnc-bbug14207 displayed significantly different expression between early and late lactation stages. And lnc-bbug14207 may regulate neighboring milk fat globule-EGF factor 8 (MFG-E8) and hyaluronan and proteoglycan link protein 3 (HAPLN3) protein coding genes, which are vital for mammary gland development. CONCLUSION: This study provides the first genome-wide identification of water buffalo lncRNAs and unveils the potential lncRNAs that impact mammary gland development.

Integrated Analysis of mRNAs and Long Non-Coding RNAs Expression of Oviduct That Provides Novel Insights into the Prolificacy Mechanism of Goat (Capra hircus).

Artificial directional selection has replaced natural selection and resulted in trait differences across breeds in domestic animal breeding. However, the molecular mechanism by which the oviduct regulates litter size remains largely elusive in goats during the follicular phase. Accumulating data have linked lncRNAs to reproductive activities; however, little is known about the modulation mechanism in the oviduct. Herein, RNA-seq was used to measure mRNA and lncRNA expression levels in low- and high-fecundity goats. We observed distinctive differences in mRNA and lncRNA in terms of different kidding numbers and detected the differential expression of 1640 mRNA transcripts and 271 lncRNA transcripts. Enrichment analysis of differentially expressed mRNAs (DEGs) suggested that multiple pathways, such as the AMPK, PI3K-Akt, calcium signaling pathway, oocyte meiosis, ABC transporter, and ECM-receptor interaction pathways, directly or indirectly affected goat reproduction. Additionally, coexpression of differentially expressed lncRNAs (DEL)-genes analysis showed that XLOC_021615, XLOC_119780, and XLOC_076450 were trans-acting as the DEGs ATAD2, DEPDC5, and TRPM6, respectively, and could regulate embryo development. Moreover, XLOC_020079, XLOC_107361, XLOC_169844, XLOC_252348 were the trans-regulated elements of the DEGs ARHGEF2 and RAPGEF6, and the target DEGs CPEB3 of XLOC_089239, XLOC_090063, XLOC_107409, XLOC_153574, XLOC_211271, XLOC_251687 were associated with prolificacy. Collectively, our study has offered a thorough dissection of the oviduct lncRNA and mRNA landscapes in goats. These results could serve as potential targets of the oviduct affecting fertility in goats.

Differentially Expressed Circular RNA Profile Signatures Identified in Prolificacy Trait of Yunshang Black Goat Ovary at Estrus Cycle.

CircRNAs acting as miRNA sponges play important roles in the growth process of animal individuals. The prolificacy trait of goats is involved in many pathways, however, the variation of circRNA expression profiles in the different phases of the estrus cycle at high and low fecundity groups is still unknown. Here, we analyzed the circRNA profiles of ovarian tissues among high and low fecundity groups in the follicular phase (HF vs LF), high and low fecundity groups in the luteal phase (HL vs LL), and high and low fecundity in the whole estrus cycle (HF vs HL and LF vs LL) using RNA-seq. A total of 283 (114 upregulated and 169 downregulated), 559 (299 upregulated and 260 downregulated), 449 (254 upregulated and 195 downregulated), and 314 (210 upregulated and 104 downregulated) differentially expressed (DE) circRNAs were screened in HF vs LF, HF vs HL, HL vs LL, and LF vs LL groups, respectively. Enrichment analysis suggested that the targeting of DE circRNAs was mainly enriched in oocyte meiosis, the GnRH signaling pathway, and estrogen signaling pathway. After integrating our previous study of miRNA-seq, there were 56 miRNAs that could target to 192 DE circRNAs, including the miR-133 family (including miR-133a-3p and miR-133b), miR-129-3p, and miR-21, which also had important influence on the prolificacy trait of goats. Then, 18 circRNAs with coding potential were obtained by four software predictions, and 9 circRNAs were validated by RT-qPCR. Together, circRNAs play a key role in the prolificacy trait and the transformation of the follicular phase to the luteal phase in the estrus cycle of goats.

Hypothalamic Transcriptome Analysis Reveals the Crucial MicroRNAs and mRNAs Affecting Litter Size in Goats.

The hypothalamus was the coordination center of the endocrine system, which played an important role in goat reproduction. However, the molecular mechanism of hypothalamus regulating litter size in goats was still poorly understood. This study aims to investigate the key functional genes associated with prolificacy by hypothalamus transcriptome analysis of goats. In this research, an integrated analysis of microRNAs (miRNAs)-mRNA was conducted using the hypothalamic tissue of Yunshang black goats in the follicular stage. A total of 72,220 transcripts were detected in RNA-seq. Besides, 1,836 differentially expressed genes (DEGs) were identified between high fecundity goats at the follicular phase (FP-HY) and low fecundity goats at the follicular phase (FP-LY). DEGs were significantly enriched in 71 Gene Ontology (GO) terms and 8 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The transcriptome data suggested that DEGs such as BMPR1B, FGFR1, IGF1 and CREB1 are directly or indirectly involved in many processes like hypothalamic gonadal hormone secretion. The miRNA-seq identified 1,837 miRNAs, of which 28 differentially expressed miRNAs (DEMs). These DEMs may affect the nerve cells survival of goat hypothalamic regulating the function of target genes and further affect the hormone secretion activities related to reproduction. They were enriched in prolactin signaling pathway, Jak-STAT signaling pathway and GnRH signaling pathway, as well as various metabolic pathways. Integrated analysis of DEMs and DEGs showed that 87 DEGs were potential target genes of 28 DEMs. After constructing a miRNA-mRNA pathway network, we identified several mRNA-miRNAs pairs by functional enrichment analysis, which was involved in hypothalamic nerve apoptosis. For example, NTRK3 was co-regulated by Novel-1187 and Novel-566, as well as another target PPP1R13L regulated by Novel-566. These results indicated that these key genes and miRNAs may play an important role in the development of goat hypothalamus and represent candidate targets for further research. This study provides a basis for further explanation of the basic molecular mechanism of hypothalamus, but also provides a new idea for a comprehensive understanding of prolificacy characteristics in Yunshang black goats.

Genome-Wide Analyses Reveal Genetic Convergence of Prolificacy between Goats and Sheep.

The litter size of domestic goats and sheep is an economically important trait that shows variation within breeds. Strenuous efforts have been made to understand the genetic mechanisms underlying prolificacy in goats and sheep. However, there has been a paucity of research on the genetic convergence of prolificacy between goats and sheep, which likely arose because of similar natural and artificial selection forces. Here, we performed comparative genomic and transcriptomic analyses to identify the genetic convergence of prolificacy between goats and sheep. By combining genomic and transcriptomic data for the first time, we identified this genetic convergence in (1) positively selected genes (CHST11 and SDCCAG8), (2) differentially expressed genes (SERPINA14, RSAD2, and PPIG at follicular phase, and IGF1, GPRIN3, LIPG, SLC7A11, and CHST15 at luteal phase), and (3) biological pathways (genomic level: osteoclast differentiation, ErbB signaling pathway, and relaxin signaling pathway; transcriptomic level: the regulation of viral genome replication at follicular phase, and protein kinase B signaling and antigen processing and presentation at luteal phase). These results indicated the potential physiological convergence and enhanced our understanding of the overlapping genetic makeup underlying litter size in goats and sheep.

Peroxisome proliferator-activated receptor gamma regulates genes involved in milk fat synthesis in mammary epithelial cells of water buffalo.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical transcription factor regulating lipid and glucose metabolism. However, the regulatory effect of PPARγ on milk fat synthesis in buffalo mammary gland is not clear. In order to explore the role of buffalo PPARG gene in milk fat synthesis, lentivirus-mediated interference was used to knock it down and then the recombinant fusion expression vector was transfected into buffalo mammary epithelial cell (BMEC) to overexpress it. PPARG gene knockdown significantly decreased the expression of CD36, FABP3, FABP4, ACSS2, ELOVL6, DGAT2, BTN1A1, AGPAT6, LPIN1, ABCG2, PPARGC1A, INSIG1, FASN, and SREBF2 genes and significantly upregulated the expression of INSIG2 gene but had no significant effect on the expression of ACSL1, GPAM, and SREBF1 genes. PPARG overexpression significantly increased the relative mRNA abundance of CD36, FABP3, FABP4, ACSS2, ELOVL6, DGAT2, BTN1A1, AGPAT6, LPIN1, PPARGC1A, INSIG1, and SREBF2 genes and significantly downregulated the expression of INSIG2 gene but had no significant effect on the expression of ACSL1, GPAM, ABCG2, FASN, and SREBF1 genes. In addition, knockdown/overexpression of PPARG gene significantly decreased/increased triacylglycerol (TAG) content in BMECs. This study revealed that buffalo PPARG gene is a key gene regulating buffalo milk fat synthesis.

The presence of synthetic polymers in the maturation medium affects the cryotolerance and developmental capacity after parthenogenic activation of vitrified goat oocytes.

The purpose of this present study is to assess if addition of the synthetic polymers in maturation medium can influence cryotolerance and subsequently embryonic development of mammalian oocytes. We examined the roles of two polymers, including polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), on in vitro maturation (IVM), embryonic developmental capacity, and cryotolerance of goat oocytes. The present study includes two parts. At first, goat cumulus-oocyte complexes (COCs) were matured in a medium supplemented with 10% fetal bovine serum (FBS), 3 mg/ml PVP, or 1 mg/ml PVA, respectively. Data of oocyte with first polar body, cleavage, and blastocyst following parthenogenetic activation (PA) were recorded. Secondly, after maturation in the above medium, oocytes were vitrified using the Cryotop technique and then the morphology, cleavage and blastocyst formation of vitrified oocytes have been checked. The results demonstrated that the adding of PVP or PVA in maturation medium can't affect IVM of goat oocytes in comparison with FBS, as concern cumulus cell expansion, first polar body formation, and embryonic development. Additionally, without plunging into liquid nitrogen, only exposure to the vitrification and warming solutions cannot also influence the quality of oocytes, in terms of morphology, cleavage, and blastocyst formation. However, after IVM with synthetic polymers and vitrification, the ratio of oocytes with standard morphology in PVP or PVA group was only 59.47% ± 3.56% or 54.86% ± 5.19%, respectively, and was significantly less than that in the FBS group (89.37% ± 4.52%, P < 0.05). Furthermore, the cleavage ratio of oocytes in PVP or PVA group was 37.41% ± 4.17% or 27.71% ± 3.91% and was considerably less than that in the FBS group (64.97% ± 4.69%, P < 0.05). In addition, the cleavage ratio in PVP group was statistically higher than that in PVA group (P < 0.05). In terms of blastocyst development, a significant difference was observed between the synthetic polymer group and the FBS group (24.96% ± 3.62%, P < 0.05). However, the blastocyst ratio in the PVA group (7.51% ± 1.68%) was statistically less than the PVP groups (13.20% ± 4.59%, P < 0.05) and the FBS group (P < 0.05). In conclusion, two potential serum replacements, either PVP or PVA, can support IVM and embryonic development of goat oocytes at the concentration used in this study. But IVM with synthetic polymers supplemented to maturation medium may reduce the cryotolerance of oocytes. Additionally, the supportive function of PVP on embryonic development of vitrified oocytes might be better than that of PVA.