High-Quality Genomes of Pangolins: Insights into the Molecular Basis of Scale Formation and Adaption to Myrmecophagous Diet.

Pangolins are one of nature's most fascinating species being scales covered and myrmecophagous diet, yet relatively little is known about the molecular basis. Here, we combine the multi-omics, evolution, and fundamental proteins feature analysis of both Chinese and Malayan pangolins, highlighting the molecular mechanism of both myrmecophagous diet and scale formation, representing a fascinating evolutionary strategy to occupy the unique ecological niches. In contrast to conserved organization of epidermal differentiation complex, pangolin has undergone large scale variation and gene loss events causing expression pattern and function conversion that contribute to cornified epithelium structures on stomach to adapt myrmecophagous diet. Our assemblies also enable us to discover large copies number of high glycine-tyrosine keratin-associated proteins (HGT-KRTAPs). In addition, highly homogenized tandem array, amino content, and the specific expression pattern further validate the strong connection between the molecular mechanism of scale hardness and HGT-KRTAPs.

Transcriptome analyses reveal transcriptional profiles of horse oocytes before and after in vitro maturation.

Oocyte in vitro maturation is necessary for the study and application of animal-assisted reproduction technology in animal reproduction and breeding. The comprehensive transcriptional profile of equine oocyte maturated in vitro has not been fully mined yet, which makes many key transcriptional events still unidentified. Here, Smart-seq2 was performed to analyse the gene expression pattern and the underlying regulatory mechanism of horse germinal vesicle (GV) and in vitro metaphase II (MII) oocytes. The results showed that 6402 genes (2640 up-regulated and 3762 down-regulated in MII samples compared to GV) and 4021 lncRNA transcripts (1210 up-regulated and 2811 down-regulated in MII samples compared to GV) were differentially expressed in GV and MII oocytes. Further, GO and KEGG analysis found that differentially expressed mRNAs and lncRNAs were mainly enriched in the pathways related to energy and lipid metabolism. In addition, LGALS3 was found a key gene in mediating the regulation of oocyte meiosis recovery and fertilization ability. This study provides novel knowledge about gene expression and energy metabolism during equine oocyte maturation and a reference for the further study and application of assisted reproductive technology in horse reproduction and breeding.

Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is characterized by follicular dysplasia. An insufficient glycolysis-derived energy supply of granulosa cells (GCs) is an important cause of follicular dysplasia in PCOS. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been proven to regulate the function of GCs. In this study, exosomes extracted from clinical FF samples were used for transcriptome sequencing (RNA-seq) analysis, and a human ovarian granulocyte tumour cell line (KGN cells) was used for in vitro mechanistic studies. METHODS AND RESULTS: In FF exosomal RNA-seq analysis, a decrease in glycolysis-related pathways was identified as an important feature of the PCOS group, and the differentially expressed miR-143-3p and miR-155-5p may be regulatory factors of glycolysis. By determining the effects of miR-143-3p and miR-155-5p on hexokinase (HK) 2, pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate, lactate and apoptosis in KGN cells, we found that upregulated miR-143-3p expression in exosomes from the PCOS group inhibited glycolysis in KGN cells; knockdown of miR-143-3p significantly alleviated the decrease in glycolysis in KGN cells in PCOS. MiR-155-5p silencing attenuated glycolytic activation in KGN cells; overexpression of miR-155-5p significantly promoted glycolysis in KGN cells in PCOS. In this study, HK2 was found to be the mediator of miR-143-3p and miR-155-5p in FF-derived exosome-mediated regulation of glycolysis in KGN cells. Reduced glycolysis accelerated apoptosis of KGN cells, which mediated follicular dysplasia through ATP, lactate and apoptotic pathways. CONCLUSIONS: In conclusion, these results indicate that miR-143-3p and miR-155-5p in FF-derived exosomes antagonistically regulate glycolytic-mediated follicular dysplasia of GCs in PCOS. Video Abstract.

Role of Peroxisome Proliferator-Activated Receptors (PPARs) in Energy Homeostasis of Dairy Animals: Exploiting Their Modulation through Nutrigenomic Interventions.

Peroxisome proliferator-activated receptors (PPARs) are the nuclear receptors that could mediate the nutrient-dependent transcriptional activation and regulate metabolic networks through energy homeostasis. However, these receptors cannot work properly under metabolic stress. PPARs and their subtypes can be modulated by nutrigenomic interventions, particularly under stress conditions to restore cellular homeostasis. Many nutrients such as polyunsaturated fatty acids, vitamins, dietary amino acids and phytochemicals have shown their ability for potential activation or inhibition of PPARs. Thus, through different mechanisms, all these nutrients can modulate PPARs and are ultimately helpful to prevent various metabolic disorders, particularly in transition dairy cows. This review aims to provide insights into the crucial role of PPARs in energy metabolism and their potential modulation through nutrigenomic interventions to improve energy homeostasis in dairy animals.

Male fertility in Mus musculus requires the activity of TRYX5 in sperm migration into the oviduct.

Nowadays, abnormal loss of serine proteases appears very frequently in male patients with unexplained sterility. In fact, many testis-specific serine proteases, the largest family among the four protease families implicated in murine spermatogenesis, are indispensable for reproduction. In the present study, we demonstrate that the previously uncharacterized testis-specific serine protease TRYX5 (1700074P13Rik) is required for male fertility in mice. Tryx5-/- male mice are sterile, yet they have normal spermatogenesis and normal sperm parameters. In vivo fertilization experiments showed that the fertilization rate of Tryx5-/- sperm was almost zero. Sperm counting and analysis of paraffin sections of oviducts revealed that Tryx5-/- sperm were unable to migrate into the oviduct, which is likely the cause of the observed infertility of the Tryx5-/- male mice. Importantly, we also found that there was almost no mature ADAM3 present in Tryx5-/- sperm and almost no ADAM3 precursor in Tryx5-/- elongated spermatids of S13-16 stage, even though testes of Tryx5-/- and wild type mice had the same amount of the total precursor ADAM3. Collectively, our results demonstrate that Tryx5 is essential for male fertility in mice and suggest that TRYX5 functions in the stability or localization of ADAM3 precursor in elongated spermatids S13-16 stage, thereby regulating the ability of sperm to migrate from the uterus into the ampulla of the oviduct, the site of fertilization.

Fatty acid biosynthesis and transcriptional regulation of Stearoyl-CoA Desaturase 1 (SCD1) in buffalo milk.

BACKGROUND: Buffalo milk is considered as a highly nutritious food owing to its higher contents of fatty acids (FA) and rich nutrient profile. Higher fat contents of buffalo milk make it suitable for processing to develop various healthy and nutritious products. Moreover, buffalo milk contains more unsaturated FAs (UFA) such as oleic and linolenic acid, which are important from the human health point of view owing to their desirable physiological effects. However, inadequate information is available about the chemical composition and mechanism of FA synthesis in buffalo milk. In this study, we hypothesized that expression of SCD1 gene could alter the biosynthesis of FA in epithelial cells of mammary gland and subsequently affect the FA contents in buffalo milk. We investigated the transcriptional and biological role of Stearoyl-CoA Desaturase 1 (SCD1) in the buffalo mammary epithelial cells (BMECs) during FA and triacylglycerol (TAG) synthesis. RESULTS: Results revealed that unsaturated fatty acid contents were much higher in concentration in buffalo milk as compared to Holstein cow. Significant increase in the expression level of FAS, ACACA, SREBP1, PPARG, GPAT, and AGPAT genes was observed in response to altered expression of SCD1 in buffalo milk. Moreover, change in SCD1 gene in BMECs also mediated the expression of genes related to FA biosynthesis subsequently leading to alter the FA composition. Overexpression of SCD1 significantly increased the expression of genes associated with FA and TAG synthesis leading to enhance FA and unsaturated FA contents in BMECs. However, down-regulation of SCD1 exhibited opposite consequences. CONCLUSION: Our study provides mechanistic insights on transcriptional regulation of SCD1 to alter FA and TAG synthesis through directly or indirectly mediating biosynthesis and metabolic pathways in BMECs. We provide preliminary findings regarding engineering of FA contents in buffalo milk through SCD1 signaling.

A milk-based self-assemble rotavirus VP6-ferritin nanoparticle vaccine elicited protection against the viral infection.

BACKGROUND: Rotavirus is the leading cause of severe dehydrating diarrhea in young children and the inner capsid protein VP6 is a potential vaccine candidate that can induce cross-protective immune responses against different Rotavirus strains. The use of ferritin nanoparticles as the scaffold of the antigen can improve the immunogenicity of the subunit vaccines and provide broader protection. We here present a non-live and self-assemble recombinant rotavirus VP6-ferritin (rVP6-ferritin) nanoparticle vaccine. RESULTS: The rVP6-ferritin nanoparticles were expressed in E. coli and self-assembled to uniform spherical structure which similar to ferritin, and oral administration of them induced efficient humoral and mucosal immunogenicity in mice. The nanoparticles were further transgenically expressed in the milk of mice, and pup mice breastfed by transgenic rVP6-ferritin mothers had strongly induced immunogenicity and-compared to pups breastfed by wild type mothers-the proportion of rotavirus challenged pups with diarrhea symptoms, the duration and intensity of the diarrhea, and the deleterious effects on overall growth resulting from the diarrhea were all significantly reduced. CONCLUSIONS: These results suggest that this recombinant VP6-ferritin nanoparticle vaccine can efficiently prevent the death and malnutrition induced by the rotavirus infection in infants and is a promising candidate vaccine for rotavirus.

Molecular Cloning, Identification, and Expression Patterns of Myostatin Gene in Water Buffalo (Bubalus Bubalis).

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-ß (TGF-ß) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-ß propeptide, and a TGF-ß domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted. ABBREVIATIONS: MSTN, myostatin; ORF, open reading frame.

Identification and characterization of buffalo 7SK and U6 pol III promoters and application for expression of short hairpin RNAs.

RNA polymerase III (pol III) type 3 promoters, such as 7SK and U6, are routinely used to induce short hairpin RNAs (shRNAs) to knockdown gene expression by RNA interference (RNAi). To extend the application of RNAi to studies of buffalo, an shRNAs expressing system using the buffalo pol III promoters was developed. Buffalo 7SK promoter (bu7SK) and U6 promoter (buU6) sequences upstream of the full-length 7SK and U6 small nuclear RNA sequence in the buffalo genome were identified and characterized, respectively. To determine the functionality of these promoters in constructs driving shRNA expression, anti-EGFP shRNAs (shEGFP) cassettes under the direction of bu7SK and buU6 were constructed. We further compared the EGFP knockdown efficiency of constructs using bu7SK and buU6 with that of promoters of human and bovine origins in BFF cells and mouse PT67 cells by flow cytometry and quantitative real-time PCR assays. We found that the bu7SK and buU6 promoters induced the greatest level of suppression in homologous and heterologous cells relative to promoters derived from other species. Taken together, functional bu7SK and buU6 promoters were identified and characterized, thus laying the groundwork for future development of RNAi therapeutics and gene modification in buffalo species.

Construction and Identification of a Breast Bioreactor for Human-Derived Hypoglycemic Protein Amylin.

The mammary gland of mammals can generate numerous bioactive proteins. To express the human amylin protein in the mammary glands of domestic animals, we engineered a transgenic mammary gland bioreactor. For this study, we produced transgenic mice through prokaryotic microinjection. RT-PCR, qPCR, and Western blotting confirmed the presence of transgenes in the mice. The ELISA assay indicated an amylin yield of approximately 1.44 µg/mL in the mice milk. Further research revealed that consuming milk containing amylin resulted in a slight, but insignificant enhancement in food consumption, blood sugar equilibrium, and glucose tolerance. The influence of amylin-fortified milk on the abundance of fecal strains in mice was examined, and a significant difference in the quantity of strains needed for fatty acid synthesis and metabolism was discovered. The amylin protein gathered from humans is safe to consume, as no harmful effects were detected in the mice. Our study examined the production of human amylin using a new safety strategy that could potentially alleviate diabetic symptoms in the future through oral administration of milk containing amylin.

A Characterization and Functional Analysis of Peroxisome Proliferator-Activated Receptor Gamma Splicing Variants in the Buffalo Mammary Gland.

Peroxisome proliferator-activated receptor γ (PPARG) has various splicing variants and plays essential roles in the regulation of adipocyte differentiation and lipogenesis. However, little is known about the expression pattern and effect of the PPARG on milk fat synthesis in the buffalo mammary gland. In this study, we found that only PPARG-X17 and PPARG-X21 of the splicing variant were expressed in the buffalo mammary gland. Amino acid sequence characterization showed that the proteins encoded by PPARG-X17 and PPARG-X21 are endonuclear non-secreted hydrophilic proteins. Protein domain prediction found that only the PPARG-X21-encoded protein had PPAR ligand-binding domains (NR_LBD_PPAR), which may lead to functional differences between the two splices. RNA interference (RNAi) and the overexpression of PPARG-X17 and PPARG-X21 in buffalo mammary epithelial cells (BMECs) were performed. Results showed that the expression of fatty acid synthesis-related genes (ACACA, CD36, ACSL1, GPAT, AGPAT6, DGAT1) was significantly modified (p < 0.05) by the RNAi and overexpression of PPARG-X17 and PPARG-X21. All kinds of FAs detected in this study were significantly decreased (p < 0.05) after RNAi of PPARG-X17 or PPARG-X21. Overexpression of PPARG-X17 or PPARG-X21 significantly decreased (p < 0.05) the SFA content, while significantly increased (p < 0.05) the UFA, especially the MUFA in the BMECs. In conclusion, there are two PPARG splicing variants expressed in the BMECs that can regulate FA synthesis by altering the expression of diverse fatty acid synthesis-related genes. This study revealed the expression characteristics and functions of the PPARG gene in buffalo mammary glands and provided a reference for further understanding of fat synthesis in buffalo milk.

Age-related compositional and functional changes in the adult and breastfed buffalo rumen microbiome.

Introduction: The buffalo is an important domestic animal globally, providing milk, meat, and labor to more than 2 billion people in 67 countries. The rumen microorganisms of buffaloes play an indispensable role in enabling the healthy functionality and digestive function of buffalo organisms. Currently, there is a lack of clarity regarding the differences in the composition and function of rumen microorganisms among buffaloes at different growth stages. Methods: In this study, metagenomics sequencing technology was applied to examine the compositional and functional differences of rumen microorganisms in adult and breastfed buffaloes. Results: The results revealed that the rumen of adult buffaloes had significantly higher levels of the following dominant genera: Prevotella, UBA1711, RF16, Saccharofermentans, F23-D06, UBA1777, RUG472, and Methanobrevibacter_A. Interestingly, the dominant genera specific to the rumen of adult buffaloes showed a significant positive correlation (correlation>0.5, p-value<0.05) with both lignocellulose degradation-related carbohydrate-active enzymes (CAZymes) and immune signaling pathways activated by antigenic stimulation. The rumen of breastfed buffaloes had significantly higher levels of the following dominant genera: UBA629, CAG- 791, Selenomonas_C, Treponema_D, Succinivibrio, and RC9. Simultaneously, the rumen-dominant genera specific to breastfed buffaloes were significantly positively correlated (correlation>0.5, p-value<0.05) with CAZymes associated with lactose degradation, amino acid synthesis pathways, and antibiotic-producing pathways. Discussion: This indicates that rumen microorganisms in adult buffaloes are more engaged in lignocellulose degradation, whereas rumen microorganisms in breastfed buffaloes are more involved in lactose and amino acid degradation, as well as antibiotic production. In conclusion, these findings suggest a close relationship between differences in rumen microbes and the survival needs of buffaloes at different growth stages.

Revealing microbial diversity in buffalo milk with high somatic cell counts: implications for mastitis diagnosis and treatment.

Mastitis is one of the most serious diseases that threatens the health of dairy animals. The somatic cell count (SCC) in milk is widely used to monitor mastitis. This study aimed to reveal the diversity of microorganisms in buffalo milk with high somatic cell count (SCC ≥ 3 × 105 cells/mL, n = 30) and low somatic cell count (SCC ≤ 5 × 104 cells/mL, n = 10), and identify the dominant bacteria that cause mastitis in a local buffalo farm. We also investigated the potential method to treat bacterial mastitis. The V3-V4 region of 16 S rDNA was sequenced. Results showed that, compared to the milk with low SCC, the high SCC samples showed lower microbial diversity, but a high abundance of bacteria and operational taxonomic units (OTUs). By in vitro isolation and culture, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were found to be the leading pathogens, which is consistent with the 16 S rDNA sequencing data. We further isolated 3 of the main pathogens and established a pathogen detection method based on ELISA. In addition, the antibacterial effects of 10 antimicrobials and 15 Chinese herbal extracts were also investigated. Results showed that the microbial has developed tolerance to several of the antimicrobials. While the water extracts of Chinese herbal medicine such as Galla Chinensis, Coptis chinensis Franch, Terminalia chebula Retz, and Sanguisorba officinalis L can effectively inhibit the growth of main pathogens. This study provides novel insight into the microbial diversity in buffalo milk and a reference for the prevention, diagnosis, and treatment of mastitis.

Multi-omics reveal the effects and regulatory mechanism of dietary neutral detergent fiber supplementation on carcass characteristics, amino acid profiles, and meat quality of finishing pigs.

This study aimed to reveal the effects and regulatory mechanism of dietary NDF on the performance of pigs by multi-omics analysis. Results showed that 16 % dietary NDF significantly improved meat quality, increased flavor amino acid content, and reduced backfat thickness and the feed-to-gain ratio. 16S rDNA sequencing showed that 16 % NDF significantly increased the abundance of Akkermansia, Lachnoclostridium, and Ruminococcus. Transcript analysis showed that genes related to muscle development and lipid metabolism were significantly modified. Metabonomic analysis showed that 16 % NDF significantly increased amino and fatty acid related metabolites. Correlation analysis suggested that 16 % NDF treatment may alter the gut microbiota and metabolites, regulate the expression of genes related to lipid and amino metabolism, and ultimately affect the flavor and performance of pigs. This study provides a novel understanding about the effect and regulatory mechanism of NDF supplements on the finishing pigs and a relevant reference for the improvement of diet formulation.

A Circular RNA Generated from Nebulin (NEB) Gene Splicing Promotes Skeletal Muscle Myogenesis in Cattle as Detected by a Multi-Omics Approach.

Cattle and the draught force provided by its skeletal muscle have been integral to agro-ecosystems of agricultural civilization for millennia. However, relatively little is known about the cattle muscle functional genomics (including protein coding genes, non-coding RNA, etc.). Circular RNAs (circRNAs), as a new class of non-coding RNAs, can be effectively translated into detectable peptides, which enlightened us on the importance of circRNAs in cattle muscle physiology function. Here, RNA-seq, Ribosome profiling (Ribo-seq), and peptidome data are integrated from cattle skeletal muscle, and detected five encoded peptides from circRNAs. It is further identified and functionally characterize a 907-amino acids muscle-specific peptide that is named circNEB-peptide because derived by the splicing of Nebulin (NEB) gene. This peptide localizes to the nucleus and cytoplasm and directly interacts with SKP1 and TPM1, key factors regulating physiological activities of myoblasts, via ubiquitination and myoblast fusion, respectively. The circNEB-peptide is found to promote myoblasts proliferation and differentiation in vitro, and induce muscle regeneration in vivo. These findings suggest circNEB-peptide is an important regulator of skeletal muscle regeneration and underscore the possibility that more encoding polypeptides derived by RNAs currently annotated as non-coding exist.

Expression of biologically active human interferon alpha 2b in the milk of transgenic mice.

Interferon alpha 2b (IFNα-2b) is an important immune regulator widely used in clinic, for the treatment of chronic hepatitis, hairy cell leukemia, chronic myelogenous leukemia and multiple myeloma, etc. The clinically used IFNα-2b is generally produced by E.Coli, which lacks the post-translational O-glycosylation presents on naturally synthesized protein, and has a short serum half-life. In this study, a transgenic cassette pBCN-IFN-pA-CMV-EGFP was constructed, with a 5.2 kb beta-casein regulation fragment from Jersey cow and a 6×His tagged human Interferon alpha 2b (hIFNα-2b) gene fragment. By using pronuclear microinjection technique, transgenic mice were generated and the expression of IFNα-2b in the milk was assayed. The hIFNα-2b was correctly translated in milk of transgenic mice according to Western blot analysis. The expression level of hIFNα-2b was varied among the transgenic mice, and the highest one was about 29.71 µg/L. The recombinant protein exhibited biological activity in vitro by increasing the luminescence value and the MxA gene expression in established WISH cells, and the specific activity is approximately 2.8 × 10(7 )IU/mg. The expression of recombinant hIFNα-2b in mammary glands of transgenic mice constitutes an important step towards low-cost and full biological activity production of this protein drug in mammary gland bioreactor.

Comparative evolutionary and molecular genetics based study of Buffalo lysozyme gene family to elucidate their antibacterial function.

Lysozyme is used as a food preservative, biological medicine, and infant food additive as a natural anti-infective chemical having bactericidal activity and abundantly secreted in mammals' milk, saliva, etc. We systematically analyzed the 16 coding LYZ genes (C and G-type) in buffalo and cattle to elucidate their evolutionary perspective thoroughly by evaluating an evolutionary relationship, motif patterning, physicochemical attributes, gene, and protein structure, as well as the functional role of the mammary gland-specific expressed buffalo and cattle LYZ genes precisely while considering expression levels difference and the interaction sites variation with bacteria envisaged the potential ability of buffalo LYZ protein with enhanced antibacterial effect. Thus, we speculated that the buffalo mammary glands expressed lysozyme has good antibacterial activity. This study on the buffalo lysozyme gene family not only provides comprehensive insights into the genetic architecture and their antibacterial effect but also offers a theoretical basis for the development of new veterinary drugs and animal health care for mastitis, as well as a new molecular genetic basis to study food or medical lysozyme.

Role of Different Members of the AGPAT Gene Family in Milk Fat Synthesis in Bubalus bubalis.

During triacylglycerol synthesis, the acylglycerol-3-phosphate acyltransferase (AGPAT) family catalyzes the conversion of lysophosphatidic acid to phosphatidic acid and the acylation of sn-2 fatty acids. However, the catalytic activity of different AGPAT members is different. Therefore, this study aimed to investigate the mechanism through which different AGPATs affect the efficiency of TAG synthesis and fatty acid composition. The conservation of amino acid sequences and protein domains of the AGPAT family was analyzed, and the functions of AGPAT1, AGPAT3, and AGPAT4 genes in buffalo mammary epithelial cells (BMECs) were studied using RNA interference and gene overexpression. Prediction of the protein tertiary structure of the AGPAT family demonstrated that four conservative motifs (motif1, motif2, motif3, and motif6) formed a hydrophobic pocket in AGPAT proteins, except AGPAT6. According to cytological studies, AGPAT1, AGPAT3, and AGPAT4 were found to promote the synthesis and fatty acid compositions of triacylglycerol, especially UFA compositions of triacylglycerol, by regulating ACSL1, FASN, GPAM, DGAT2, and PPARG gene expression. This study provides new insights into the role of different AGPAT gene family members involved in TAG synthesis, and a reference for improving the fatty acid composition of milk.

Whole-genome transcriptome and DNA methylation dynamics of pre-implantation embryos reveal progression of embryonic genome activation in buffaloes.

BACKGROUND: During mammalian pre-implantation embryonic development (PED), the process of maternal-to-zygote transition (MZT) is well orchestrated by epigenetic modification and gene sequential expression, and it is related to the embryonic genome activation (EGA). During MZT, the embryos are sensitive to the environment and easy to arrest at this stage in vitro. However, the timing and regulation mechanism of EGA in buffaloes remain obscure. RESULTS: Buffalo pre-implantation embryos were subjected to trace cell based RNA-seq and whole-genome bisulfite sequencing (WGBS) to draw landscapes of transcription and DNA-methylation. Four typical developmental steps were classified during buffalo PED. Buffalo major EGA was identified at the 16-cell stage by the comprehensive analysis of gene expression and DNA methylation dynamics. By weighted gene co-expression network analysis, stage-specific modules were identified during buffalo maternal-to-zygotic transition, and key signaling pathways and biological process events were further revealed. Programmed and continuous activation of these pathways was necessary for success of buffalo EGA. In addition, the hub gene, CDK1, was identified to play a critical role in buffalo EGA. CONCLUSIONS: Our study provides a landscape of transcription and DNA methylation in buffalo PED and reveals deeply the molecular mechanism of the buffalo EGA and genetic programming during buffalo MZT. It will lay a foundation for improving the in vitro development of buffalo embryos.