Super-enhancers conserved within placental mammals maintain stem cell pluripotency.

Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in placental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.

Effect of Disulfiram on the Reproductive Capacity of Female Mice.

In the process of assisted reproduction, the high-oxygen in vitro environment can easily cause oxidative damage to oocytes. Disulfiram (DSF) can play an anti-oxidant or pro-oxidant role in different cells, and the effect of DSF on oocytes remains unclear. Moreover, it remains unclear whether the use of DSF in the early stages of pregnancy has a negative impact on the fetus. In this study, we found that DSF increased serum FSH levels and increased the ovulation rate in mice. Moreover, DSF enhanced the antioxidant capacity of oocytes and contributed to the success rate of in vitro fertilization. Moreover, the use of DSF in early pregnancy in mice increased the uterine horn volume and the degree of vascularization, which contributed to a successful pregnancy. In addition, it was found that DSF regulated the mRNA expression of angiogenesis-related genes (VEGF), follicular development-related genes (C1QTNF3, mTOR and PI3K), ovulation-related genes (MAPK1, MAPK3 and p38 MAPK) and antioxidant-related genes (GPX4 and CAT). These results indicate that DSF is helpful for increasing the antioxidant capacity of oocytes and the ovulation rate. In early pregnancy in mice, DSF promotes pregnancy by increasing the degree and volume of uterine vascularization.

Low-Dose Sodium Salicylate Promotes Ovulation by Regulating Steroids via CYP17A1.

To meet the current demand of assisted reproduction and animal breeding via superovulation and reduce the impact of hormone drugs, it is necessary to develop new superovulation drugs. This study examined the role of inflammation and steroids in ovulation. Sodium salicylate can regulate inflammation and steroids. However, the effect of sodium salicylate on ovulation has not been studied. In this study, mice were intraperitoneally injected with different concentrations of sodium salicylate for four consecutive days. The effects of sodium salicylate on oocyte quality and on the number of ovulations were examined, and these effects were compared with those of pregnant horse serum gonadotropin (PMSG)/follicle-stimulating hormone (FSH) treatment. We found that low-dose sodium salicylate increased the levels of ovulation hormones and inflammation by promoting the expression of CYP17A1. Sodium salicylate had the same effect as the commonly used superovulation drug PMSG/FSH and reduced the histone methylation level. Sodium salicylate can promote ovulation in mice and Awang sheep. It can greatly decrease the use of hormone drugs, reduce breeding costs and physical impacts, and can thus be used for livestock breeding.

Scd1 Deficiency in Early Embryos Affects Blastocyst ICM Formation through RPs-Mdm2-p53 Pathway.

Embryos contain a large number of lipid droplets, and lipid metabolism is gradually activated during embryonic development to provide energy. However, the regulatory mechanisms remain to be investigated. Stearoyl-CoA desaturase 1 (Scd1) is a fatty acid desaturase gene that is mainly involved in intracellular monounsaturated fatty acid production, which takes part in many physiological processes. Analysis of transcripts at key stages of embryo development revealed that Scd1 was important and expressed at an increased level during the cleavage and blastocyst stages. Knockout Scd1 gene by CRISPR/Cas9 from zygotes revealed a decrease in lipid droplets (LDs) and damage in the inner cell mass (ICM) formation of blastocyst. Comparative analysis of normal and knockout embryo transcripts showed a suppression of ribosome protein (RPs) genes, leading to the arrest of ribosome biogenesis at the 2-cell stage. Notably, the P53-related pathway was further activated at the blastocyst stage, which eventually caused embryonic development arrest and apoptosis. In summary, Scd1 helps in providing energy for embryonic development by regulating intra-embryonic lipid droplet formation. Moreover, deficiency activates the RPs-Mdm2-P53 pathway due to ribosomal stress and ultimately leads to embryonic development arrest. The present results suggested that Scd1 gene is essential to maintain healthy development of embryos by regulating energy support.

Sperm-derived RNAs improve the efficiency of somatic cell nuclear transfer (SCNT) through promoting R-loop formation.

Mammalian sperm and oocytes are haploid cells that carry parental genetic and epigenetic information for their progeny. The cytoplasm of oocytes is also capable of reprograming somatic cells to establish totipotency through somatic cell nuclear transfer (SCNT). However, epigenetic barriers seriously counteract SCNT reprogramming. Here, we found that sperm-derived RNAs elevated chromatin accessibility of nuclear donor cells concurrent with the appearance of increased RNA amount and decreased cell proliferation, instead of activating DNA damage response. Additionally, tri-methylation of lysine 9 on histone H3 (H3K9me3) and the H3K9 methyltransferase SUV39H2 were significantly downregulated by the sperm-derived RNA treatment. Our findings thus raise a fascinating possibility that sperm RNA-induced R-loops may activate gene expression and chromatin structure, thereby promoting SCNT reprogramming.

Asymmetric expression of maternal mRNA governs first cell-fate decision.

The goal of preimplantation development is to establish the fates of the embryonic and extra-embryonic cells. However, when and how cell fates are determined during early mammalian embryonic development remains unclear. We report that the high mobility group (HMG) protein family member HMGA1 was distributed differentially in mouse two-cell blastomeres. Knockdown of Hmga1 expression in one of the two cells reduced the number of cells contributing to the inner cell mass (ICM), suggesting that differential distribution of HMGA1 in the blastomeres in two-cell mouse embryos affected the selection of embryonic cell lineages. Mechanistically, HMGA1 promotes the expression of the ICM-specific gene Sox2. The results of this study show that mouse embryos demonstrate heterogeneity as early as the two-cell stage, and that these differences are related to cell-fate differentiation in early mouse embryos.

Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network.

The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.

Early cleavage of preimplantation embryos is regulated by tRNAGln-TTG-derived small RNAs present in mature spermatozoa.

tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle-associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.

Coping with DNA Double-Strand Breaks via ATM Signaling Pathway in Bovine Oocytes.

As a common injury almost all cells face, DNA damage in oocytes-especially double-strand breaks (DSBs), which occur naturally during the first meiosis phase (meiosis I) due to synaptic complex separation-affects the fertilization ability of oocytes, instead of causing cancer (as in somatic cells). The mechanism of oocytes to effectively repair DSB damage has not yet been clearly studied, especially considering medically induced DSBs superimposed on naturally occurring DSBs in meiosis I. It was found that maturation rates decreased or increased, respectively corresponding with overexpression or interference of p21 in bovine oocytes. At the same time, the maturation rate of bovine oocytes decreased with a gradual increase in Zeocin dose, and the p21 expression in those immature oocytes changed significantly with the gradual increase in Zeocin dose (same as increased DSB intensity). Same as p21, the variation trend of ATM expression was consistent with the gradual increase in Zeocin dose. Furthermore, the oocytes demonstrated tolerance to DSBs during meiosis I, while the maturation rates decreased when the damage exceeded a certain threshold; according to which, it may be that ATM regulates the p53-p21 pathway to affect the completion of meiosis. In addition, nonhomologous recombination and cumulus cells are potentially involved in the process by which oocytes respond to DSB damage.

Generation of gene-edited sheep with a defined Booroola fecundity gene (FecBB) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9.

Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q>R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.

miR-544 Regulates Dairy Goat Male Germline Stem Cell Self-Renewal via Targeting PLZF.

The balance between the self-renewal and differentiation of male germline stem cells (mGSCs) is critical for the initiation and maintenance of mammalian spermatogenesis. The promyelocytic leukemia zinc finger (PLZF), a zinc finger protein, is a critical factor for maintaining the self-renewal of mGSCs, so, evaluation of the PLZF pathway in mGSCs may provide a deeper insight into mammalian spermatogenesis. miRNA was also an important regulating factor for the self-renewal and differentiation of mGSCs; however, there is currently no data indicating that which miRNA regulate the self-renewal and differentiation of mGSCs via PLZF. Here, we predicted the prospective miRNA targeting to PLZF using the online Bioinformatics database-Targetscan, and performed an analysis of the dual-luciferase recombinant vector, psiCHCEKTM-2-PLZF-3'UTR. miR-544 mimics (miR-544m), miR-544 inhibitors (miR-544i), Control (NC, scrambled oligonucleotides transfection), pPLZF-IRES2-EGFP or PLZF siRNA were transfected into mGSCs; the cells proliferation was evaluated by BRDU incorporation assay and flow cytometry, and the mGSC marker, GFRa1, PLZF, KIT, DAZL, and VASA expression were analyzed by RT-qPCR, immunofluorescence and Western blot. The results showed that miR-544 regulates dairy goat male germline stem cell self-renewal via targeting PLZF. Our study identifies a new regulatory pathway for PLZF and expands upon the PLZF regulatory network in mGSCs.

H1foo is essential for in vitro meiotic maturation of bovine oocytes.

Oocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

Allogenic Follicular Fosterage Technology: Problems, Progress and Potential.

The allogeneic follicular fosterage (AFF) technique transfers cumulus-oocyte complexes (COCs) from pubertal female animals to the dominant follicles of adult female animals for further development, allowing the COCs to further develop in a completely in vivo environment. This article reviews the history of AFF and JIVET and their effects on oocyte and embryo development as well as freezing resistance. Improving the efficiency and reproducibility of AFF technology is crucial to its clinical application. This article discusses factors that affect the success rate of AFF, including differences in specific technical procedures and differences between pubertal and adult follicles. Designing standardized procedures and details to improve the synchronization of donor COCs and recipient follicle maturity and reducing the damage to COCs caused by follicular aspiration may be the direction for improving the success rate of AFF in the future.

Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

Dynamic role of Scd1 gene during mouse oocyte growth and maturation.

Mammalian reproductive ability is regulated by many factors, among which the fatty acid metabolism network provides energy for oocyte growth and primordial follicle formation during early mouse oogenesis. But the mechanism behind that is still unknown. Stearoyl-CoA desaturase 1 (Scd1) gene expression is increased during the oogenesis process, supporting the oocyte's healthy growth. Taking advantage of gene-edited mice lacking stearoyl-Coenzyme A desaturase 1 gene (Scd1-/-), we analyzed relative gene expression in perinatal ovaries from wildtype, and Scd1-/- mice. Scd1 deficiency dysregulates expression of meiosis-related genes (e.g., Sycp1, Sycp2, Sycp3, Rad51, Ddx4) and a variety of genes (e.g., Nobox, Lhx8, Bmp15, Ybx2, Dppa3, Oct4, Sohlh1, Zp3) associated with oocyte growth and differentiation, leading to a lower oocyte maturation rate. The absence of Scd1 significantly impedes meiotic progression, causes DNA damage, and inhibits damage repair in Scd1-/- ovaries. Moreover, we find that Scd1 absense dramatically disrupts the abundance of fatty acid metabolism genes (e.g., Fasn, Srebp1, Acaca) and the lipid droplet content. Thus, our findings substantiate a major role for Scd1 as a multifunctional regulator of fatty acid networks necessary for oocyte maintenance and differentiation during early follicular genesis.

Genomic selection analysis of morphological and adaptation traits in Chinese indigenous dog breeds.

The significant morphological differences and abundant germplasm resources of Chinese indigenous dog breeds can be attributed to the diverse geographical environment, including plateaus, mountains, and a long history of raising dogs. The combination of both natural and artificial selection during the past several thousand years has led to hundreds of dog breeds with distinct morphological traits and environmental adaptations. China is one of the earliest countries to domesticate dogs and there are more than 50 ancient indigenous dog breeds. In this study, the run of homozygosity (ROH) and proportion of the autosomal genome covered by ROHs (FROH) were calculated for 10 dog breeds that are the most representative Chinese indigenous dogs based on 170K SNP microarray. The results of FROH showed that the Chuandong hound dogs (HCSSC) have the highest level of inbreeding among the tested breeds. The inbreeding in HCSSC occurred more recently than the Liangshan dogs (SCLSQ) dogs because of more numbers of long ROHs in HCSSC dogs, and the former also have higher inbreeding degree. In addition, there are significant differences in the inbreeding degree among different subpopulations of the same breed, such as the Thin dogs from Shaanxi and Shandong province. To explore genome-wide selection signatures among different breeds, including coat color, ear shape, and altitude adaptability, we performed genome selection analyses of FST and cross population extended haplotype homozygosity (XP-EHH). For the coat color, the FST analysis between Xiasi dogs (XSGZ) and HCSSC dogs was performed and identified multiple genes involved in coat color, hair follicle, and bone development, including MC1R, KITLG, SOX5, RSPO2, and TBX15. For the plateau adaptability, we performed FST and XP-EHH analyses between dogs from Tibet (Tibetan Mastiffs and Nyingchi dogs) and plain regions (Guangxi Biwei dogs GXBWQ and Guandong Sharpei dogs). The results showed the EPAS1 gene in dogs from Tibet undergo strong selection. Multiple genes identified for selection signals based on different usage of dogs. Furthermore, the results of ear shape analyses showed that MSRB3 was likely to be the main gene causing the drop ear of domestic dogs. Our study provides new insights into further understanding of Chinese indigenous dogs.

α-Ketoglutarate Improves Meiotic Maturation of Porcine Oocytes and Promotes the Development of PA Embryos, Potentially by Reducing Oxidative Stress through the Nrf2 Pathway.

α-Ketoglutarate (α-KG) is a metabolite in the tricarboxylic acid cycle. It has a strong antioxidant function and can effectively prevent oxidative damage. Previous studies have shown that α-KG exists in porcine follicles, and its content gradually increases as the follicles grow and mature. However, the potential mechanism of supplementation of α-KG on porcine oocytes during in vitro maturation (IVM) has not yet been reported. The purpose of this study was to explore the effect of α-KG on the early embryonic development of pigs and the mechanisms underlying these effects. We found that α-KG can enhance the development of early pig embryos. Adding 20 µM α-KG to the in vitro culture medium significantly increased the rate of blastocyst formation and the total cell number. Compared with to that of the control group, apoptosis in blastocysts of the supplement group was significantly reduced. α-KG reduced the production of reactive oxygen species and glutathione levels in cells. α-KG not only improved the activity of mitochondria but also inhibited the occurrence of apoptosis. After supplementation with α-KG, pig embryo pluripotency-related genes (OCT4, NANOG, and SOX2) and antiapoptotic genes (Bcl2) were upregulated. In terms of mechanism, α-KG activates the Nrf2/ARE signaling pathway to regulate the expression of antioxidant-related targets, thus combating oxidative stress during the in vitro culture of oocytes. Activated Nrf2 promotes the transcription of Bcl2 genes and inhibits cell apoptosis. These results indicate that α-KG supplements have a beneficial effect on IVM by regulating oxidative stress during the IVM of porcine oocytes and can be used as a potential antioxidant for IVM of porcine oocytes.

Salidroside improves porcine oocyte maturation and subsequent embryonic development by promoting lipid metabolism.

An optimal lipid droplet (LD) content is essential for successful mammalian embryonic development. Salidroside (SAL) is a traditional Chinese medicine and one of the important active components of the Rhodiola plant. SAL possesses antioxidative, anti-aging, and cardiovascular properties. Here, we studied the effects of SAL on in vitro maturation (IVM) of porcine oocytes and the subsequent embryonic development after parthenogenetic activation (PA). We found that 100 µM of SAL had no effect on the extrusion rate of the first polar body of porcine oocytes but significantly improved the subsequent blastocyst formation rate and embryo quality. Our study further revealed that SAL treatment altered the morphology, increased the lipid content in oocytes, increased mitochondrial number. Further analysis revealed that SAL upregulated the expression of genes related to lipid metabolism (FASN, FADS1, HSL, and CPT1a) and the mitochondria function-related genes (PGC-1α). These results suggest that SAL supplementation enhances oocyte maturation and subsequent embryonic development by promoting lipid metabolism, providing the necessary energy for the aforementioned processes.

Effects of CRISPR/Cas9-mediated stearoyl-Coenzyme A desaturase 1 knockout on mouse embryo development and lipid synthesis.

Background: Lipid synthesis is an indispensable process during embryo and growth development. Abnormal lipid synthesis metabolism can cause multiple metabolic diseases including obesity and hyperlipidemia. Stearoyl-Coenzyme A desaturase 1 (SCD1) is responsible for catalyzing the synthesis of monounsaturated fatty acids (MUFA) and plays an essential role in lipid metabolism. The aim of our study was to evaluate the effects of SCD1 on embryo development and lipid synthesis in a knockout mice model. Methods: We used the CRISPR/Cas9 system together with microinjection for the knockout mouse model generation. Ten-week-old female C57BL/6 mice were used for zygote collection. RNase-free water was injected into mouse zygotes at different cell phases in order to select the optimal time for microinjection. Five sgRNAs were designed and in vitro transcription was performed to obtain sgRNAs and Cas9 mRNA. RNase-free water, NC sgRNA/Cas9 mRNA, and Scd1 sgRNA/Cas9 mRNA were injected into zygotes to observe the morula and blastocyst formation rates. Embryos that were injected with Scd1 sgRNA/Cas9 mRNA and developed to the two-cell stage were used for embryo transfer. Body weight, triacylglycerol (TAG), and cholesterol in Scd1 knockout mice serum were analyzed to determine the effects of SCD1 on lipid metabolism. Results: Microinjection performed during the S phase presented with the highest zygote survival rate (P < 0.05). Of the five sgRNAs targeted to Scd1, two sgRNAs with relatively higher gene editing efficiency were used for Scd1 knockout embryos and mice generation. Genome sequence modification was observed at Scd1 exons in embryos, and Scd1 knockout reduced blastocyst formation rates (P < 0.05). Three Scd1 monoallelic knockout mice were obtained. In mice, the protein level of SCD1 decreased (P < 0.05), and the body weight and serum TAG and cholesterol contents were all reduced (P < 0.01).

Knockout of Stearoyl-CoA Desaturase 1 Decreased Milk Fat and Unsaturated Fatty Acid Contents of the Goat Model Generated by CRISPR/Cas9.

Goat milk contains a rich source of nutrients, especially unsaturated fatty acids. However, the regulatory mechanism of milk fat and fatty acid synthesis remains unclear. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme catalyzing monounsaturated fatty acid synthesis and is essential for milk lipid metabolism. To explore milk lipid synthesis mechanism in vivo, SCD1-knockout goats were generated through CRISPR/Cas9 technology for the first time. SCD1 deficiency did not influence goat growth or serum biochemistry. Plasma phosphatidylcholines increased by lipidomics after SCD1 knockout in goats. Whole-blood RNA-seq indicated alterations in biosynthesis of unsaturated fatty acid synthesis, cAMP, ATPase activity, and Wnt signaling pathways. In SCD1-knockout goats, milk fat percentage and unsaturated fatty acid levels were reduced but other milk components were unchanged. Milk lipidomics revealed decreased triacylglycerols and diacylglycerols levels, and the differential abundance of lipids were enriched in glycerolipid, glycerophospholipids, and thermogenesis metabolism pathways. In milk fat globules, the expression levels of genes related to fatty acid and TAG synthesis including SREBP1 were reduced. ATP content and AMPK activity were promoted, and p-p70S6K protein level was suppressed in SCD1-knockout goat mammary epithelial cells, suggesting that SCD1 affected milk lipid metabolism by influencing AMPK-mTORC1/p70S6K-SREBP1 pathway. The integrative analysis of gene expression levels and lipidomics of milk revealed a crucial role of SCD1 in glycerolipids and glycerophospholipids metabolism pathways. Our observations indicated that SCD1 regulated the synthesis of milk fat and unsaturated fatty acid in goat by affecting lipid metabolism gene expression and lipid metabolic pathways. These findings would be essential for improving goat milk nutritional value which is beneficial to human health.