The study examined the effects of different environmental stress on developmental competence and the relative abundance (RA) of various gene transcripts in oocytes and embryos of buffalo. Oocytes collected during cold period (CP) and hot period (HP) were matured, fertilized and cultured in vitro to blastocyst hatching stage. The mRNA expression patterns of genes implicated in developmental competence (OCT-4, IGF-2R and GDF-9), heat shock (HSP-70.1), oxidative stress (MnSOD), metabolism (GLUT-1), pro-apoptosis (BAX) and anti-apoptosis (BCL-2) were evaluated in immature and matured oocytes as well as in pre-implantation stage embryos. Oocytes reaching MII stage, cleavage rates, blastocyst yield and hatching rates increased (P \ 0.05) during the CP. In MII oocytes and 2-cell embryos, the RA of OCT-4, IGF-2R, GDF-9, MnSOD and GLUT-1 decreased (P \ 0.05) during the HP. In 4-cell embryos, the RA of OCT-4, IGF-2R and BCL-2 decreased (P \ 0.05) in the HP, whereas GDF-9 increased (P \ 0.05). In 8-to 16-cell embryos, the RA of OCT-4 and BCL-2 decreased (P \ 0. 05) in the HP, whereas HSP-70.1 and BAX expression increased (P \ 0.05). In morula and blastocyst, the RA of OCT-4, IGF-2R and MnSOD decreased (P \ 0.05) during the HP, whereas HSP-70.1 was increased (P \ 0.05). In conclusion, deleterious seasonal effects induced at the GVstage carry-over to subsequent embryonic developmental stages and compromise oocyte developmental competence and quality of developed blastocysts.

The objective of this study was to compare effects of in vitro culture systems on embryonic development and expression patterns of developmentally important genes in preimplantation buffalo embryos. After IVM/IVF presumptive zygotes were cultured in one of three systems: undefined TCM-199, mCR2aa medium supplemented with 10 % FBS and defined PVA-myo-inositol-phosphate-EGF medium. No (P [ 0.05) differences at 2-cell, 4-cell and 8-cell to 16-cell stages were observed among the three cultured media used, however, increased (P \ 0.05) blastocyst yield, cell number and hatching rate were found in defined medium compared to undefined media. The expression patterns of genes implicated in embryo metabolism (GLUT-1), anti-apoptosis (BCL-2), imprinting (IGF-2R), DNA methylation (DNMT-3A) and maternal recognition of pregnancy (IFNT) were increased (P \ 0.05) in hatched blastocysts derived from defined medium compared to undefined media. In conclusion, serum-free, defined medium improved developmental competence of in vitro cultured buffalo embryos. Whether these differences in morphological development and gene expression have long-term effects on buffalo calves born after embryo transfer remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development.

This study examined the effects of buffalo oocyte extracts (BOE) on donor cells reprogramming and molecular characterisation of oocytes screened via brilliant cresyl blue (BCB) staining and comparison of gene expression profiles of developmentally important genes in blastocysts from IVF and cloned derived from BOE treated donor cells with BCB selected recipient cytoplasts. Relative abundance (RA) of OCT4 and NANOG was increased (P < 0.05) and HDAC-1, DNMT-1, and DNMT-3A decreased (P < 0.05) in extract treated cells (ETCs). This ETCs dedifferentiated into neuron-like lineage under appropriate induction condition. The RA of NASP, EEF1A1, DNMT1, ODC1 and RPS27A was increased (P < 0.05) in BCB+ oocytes, whereas ATP5A1 and S100A10 increased (P < 0.05) in BCB- oocytes. Total cell number and RA of OCT4, NANOG, SOX2, DNMT1, IGF2, IGF2R, MNSOD, GLUT1, BAX and BCL2 in cloned blastocysts derived from BCB+ oocytes with ETC more closely followed that of IVF counterparts compared to BCB+ oocytes with extract untreated cell and BCB- oocytes with ETC derived blastocysts. In conclusion, BOE influenced epigenetic reprogramming of buffalo fibroblasts making them suitable donors for nuclear transfer (NT). BCB staining can be effectively used for selection of developmentally competent oocytes for NT. The combined effects of epigenetic reprogramming of donor nuclei by BOE and higher nuclear reprogramming capacity of BCB+ oocytes improve developmentally important gene expression in cloned blastocysts. Whether these improvements have long-term effects on buffalo calves born following embryo transfer remains unknown.

Background Cellular metabolism is most invariant processes, occurring in all living organisms which involve mitochondrial proteins from both nuclear and mitochondrial genome. Mitochondrial genome and gene expression has played a central role in the oxidative phosphorylation system biogenesis and metabolism of energy. The mitochondrial DNA (mtDNA) copy number, its protein genes expression and activity in tissues vary between various tissues to fulfill specific energy demands across the tissues. To the yet, this tissue-specific diversity is unaware in terms of mitochondrial biogenesis and protein-coding gene expression in the metabolically active tissue of buffalo. Thus, we assessed the variations in mitochondrial functional assay, mtDNA cellular number, and protein gene expression by investigating six bovine tissues. Materials & methods The liver, kidney, heart, muscle, ovary and brain of the same freshly slaughtered buffaloes (n = 3) were investigated for their differences in mitochondrial bioenergetics by measuring the individual OXPHOS complexes and enzymatic activity of citrate synthase in isolated mitochondria. The evaluation of tissue-specific diversity based on the quantification of mitochondrial DNA copy numbers was performed and also comprised an expression study of 13 protein genes encoded by mitochondrial genome. Results The investigated tissues showed striking differences in OXPHOS activities and CS-specific activities. The functional activity of individual OXPHOS complex I was significantly higher in the liver compared to muscle and brain. Tissue-dependent differences again reflected on OXPHOS complex III and V activities, with the liver showing significantly the highest specific activities compared to the heart, ovary, and brain. Additionally, there are considerable differences in the CS-specific activity between tissues, with the ovary, kidney, and liver having significantly greater values. Furthermore, we observed the mtDNA copy number was strictly tissue-specific, indicating the distinct bioenergetics and metabolic requirements of various tissues, with muscle and brain tissues exhibiting the highest levels. Moreover, the CS-specific activity also differs markedly between tissues, with significantly higher values for the ovary, kidney, and liver. Further, we observed a strict tissue specificity of mtDNA copy number, reflecting the specific energy and metabolic demands of different tissues, with brain and muscle tissues showing the highest values. Among 13 PCGs expression analyses, mRNA abundances in all genes were differentially expressed among the different tissue. Conclusion Overall, our results indicate the existence of a tissue-specific variation in mitochondrial activity, bioenergetics, and protein gene expression of mitochondria among various types of buffalo tissues. This study serves as a critical first stage in gathering vital comparable data about the physiological function of mitochondria in energy metabolism in distinct tissues, laying the groundwork for future mitochondrial based diagnosis and research.

BackgroundCellular metabolism is most invariant processes, occurring in all living organisms which involve mitochondrial proteins from both nuclear and mitochondrial genome. Mitochondrial genome and gene expression has played a central role in the oxidative phosphorylation system biogenesis and metabolism of energy. The mitochondrial DNA (mtDNA) copy number, its protein genes expression and activity in tissues vary between various tissues to ful ll speci c energy demands across the tissues. To the yet, this tissue-speci c diversity is unaware in terms of mitochondrial biogenesis and protein-coding gene expression in the metabolically active tissue of buffalo. Thus, we assessed the variations in mitochondrial functional assay, mtDNA cellular number, and protein gene expression by investigating six bovine tissues. Materials & methodsThe liver, kidney, heart, muscle, ovary and brain of the same freshly slaughtered buffaloes (n = 3) were investigated for their differences in mitochondrial bioenergetics by measuring the individual OXPHOS complexes and enzymatic activity of citrate synthase in isolated mitochondria. The evaluation of tissuespeci c diversity based on the quanti cation of mitochondrial DNA copy numbers was performed and also comprised an expression study of 13 protein genes encoded by mitochondrial genome. ResultsThe investigated tissues showed striking differences in OXPHOS activities and CS-speci c activities. The functional activity of individual OXPHOS complex I was signi cantly higher in the liver compared to muscle and brain. Tissue-dependent differences again re ected on OXPHOS complex III and V activities, with the liver showing signi cantly the highest speci c activities compared to the heart, ovary, and brain.Additionally, there are considerable differences in the CS-speci c activity between tissues, with the ovary, kidney, and liver having signi cantly greater values. Furthermore, we observed the mtDNA copy number was strictly tissue-speci c, indicating the distinct bioenergetics and metabolic requirements of various tissues, with muscle and brain tissues exhibiting the highest levels. Moreover, the CS-speci c activity also differs markedly between tissues, with signi cantly higher values for the ovary, kidney, and liver. Further, we observed a strict tissue speci city of mtDNA copy number, re ecting the speci c energy and metabolic demands of different tissues, with brain and muscle tissues showing the highest values. Among 13 PCGs expression analyses, mRNA abundances in all genes were differentially expressed among the different tissue. ConclusionPage 3/22 Overall, our results indicate the existence of a tissue-speci c variation in mitochondrial activity, bioenergetics, and protein gene expression of mitochondria among various types of buffalo tissues. This study serves as a critical rst stage in gathering vital comparable data about the physiological function of mitochondria in energy metabolism in distinct tissues, laying the groundwork for future mitochondrial based diagnosis and research.