In-situ thermal conductive heating (TCH) for soil remediation: A review.

This paper provides a comprehensive overview of research works on in-situ thermal conductive heating (TCH), including heat transfer in soil, desorption behavior of pollutants, and mass transfer mechanism within the site. Each stage influences the effectiveness of subsequent stages. Comparison of simulation and experimental results demonstrates that heat transfer and temperature rise in soil are related to the hydrogeological conditions, wells layout and pollutants contents. Thermal desorption of pollutants from soil particles can be influenced by four aspects: energy input, pollutant properties, soil characteristics, and the binding state of pollutant in soil. The exponential decay kinetic model exhibits better applicability for fitting thermal desorption processes. After desorption, the pollutants migrate in soil driven by high temperature and extraction pressure, while hydrogeological conditions of the site determine the actual migration path and rate. Applying convection-dispersion model allows for quantitatively describing the complex migration behavior of pollutants in heterogeneous sites. Future research should focus more on the composite effects of multiple factors in TCH and develop multi-field coupling models through the combination of numerical simulation and in-situ experiments. Accurate characterization and prediction of entire TCH process can improve remediation efficiency, reduce energy costs, and achieve sustainable low-carbon remediation.

Mechanisms and extended kinetic model of thermal desorption in organic-contaminated soil.

Thermal desorption is a preferred technology for site remediation due to its various advantages. To ensure the effective removal of different pollutants in practical applications, it is necessary to understand the kinetic behaviors and removal mechanisms of pollutants in thermal desorption process. This paper explored the thermal desorption processes of five organic pollutants (nitrobenzene, naphthalene, n-dodecane, 1-nitronaphthalene, and phenanthrene) at 50-350 °C in two different subsoils with 6-18% moisture content. The results suggested that the thermal desorption process was well-fitted by the exponential decay model (R2 = 0.972-0.999) and could be divided into two distinct stages. The first stage was relatively fast and highly influenced by soil moisture, while the second stage showed a slower desorption rate due to the constraints imposed by the soil texture and structure. The influence of soil moisture on thermal desorption depended on the octanol/water partition coefficient (KOW) of pollutants. Pollutants with log KOW values lower than the critical value exhibited enhanced thermal desorption, while those with log KOW values higher than the critical value were inhibited. The critical value of log KOW might be between 3.33 and 4.46. Changes in soil texture and structure caused by heating promoted thermal desorption, especially for naphthalene, 1-nitronaphthalene and phenanthrene. The differences in texture and structure between the two soils diminished as the temperature increased. Finally, an extended kinetic model under changing temperature conditions was derived, and the simulation results for the two subsoils were very close to the actual thermogravimetric results, with the differences ranging from -1.28% to 0.94% and from -0.67% to 1.35%, respectively. These findings propose new insights into the influencing mechanisms of soil moisture and structure on the thermal desorption of organic pollutants. The extended kinetic model can provide reference for future kinetic research and guide practical site remediation.

Integrated Analysis of Quantitative Proteome and Transcriptional Profiles Reveals the Dynamic Function of Maternally Expressed Proteins After Parthenogenetic Activation of Buffalo Oocyte.

Maternal-effect genes are especially critical for early embryonic development after fertilization and until massive activation of the embryonic genome occurs. By applying a tandem mass tag (TMT)-labeled quantitative proteomics combined with RNA sequencing approach, the proteome of the buffalo was quantitatively analyzed during parthenogenesis of mature oocytes and the two-cell stage embryo. Of 1908 quantified proteins, 123 differed significantly. The transcriptome was analyzed eight stages (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst) of Buffalo using the RNA sequencing approach, and a total of 3567 unique genes were identified to be differently expressed between all consecutive stages of pre-implantation development. Validation of proteomics results (TUBB3, CTNNA1, CDH3, MAP2K1), which are involved in tight junction and gap junction, revealing that the maternal expression of the proteins possibly plays a role in the formation of cellular junctions firstly after parthenogenetic activation. Correlation and hierarchical analyses of transcriptional profiles and the expression of NPM2 and NLRP5 mRNA of buffalo in vitro developed oocytes and parthenogenetic embryos indicated that the "maternal-to-zygotic transition" (MZT) process might exist in the model of parthenogenesis, which is similar to a normally fertilized embryo, and may occur between the 8-cell to 16-cell stage. These data provide a rich resource for further studies on maternal proteins and genes and are conducive to improving nuclear transfer technology.

Maternal transcription profiles at different stages for the development of early embryo in buffalo.

Maternal mRNAs deposited in the egg during oogenesis are critical during the development of early embryo, before the activation of the embryonic genome. However, there is little known about the dynamic expression of maternally expressed genes in mammals. In this study, we made buffalo parthenogenesis as our research model to analyse maternal transcription profiles of pre-implantation embryo in buffalo using RNA sequencing. In total, 3,567 unique genes were detected to be differentially expressed among all constant stages during early embryo development (FPKM > 0). Interestingly, a total of 10,442 new genes were discovered in this study, and gene ontology analysis of the new differentially expressed genes indicated that the new genes have a wide cellular localization and are involved in many molecular functions and biological processes. Moreover, we identified eight clusters that were only highly expressed in a particular developmental stage and enriched a number of GO terms and KEGG pathways that were related to specific stage. Furthermore, we identified 1,530 hub genes (or key members) from the maternally expressed gene networks, and these hub genes were involved in 11 stage-specific modules. After visualization using Cytoscape 3.2.1 software, we obtained complex interaction network of hub genes, indicating the highly efficient cooperation between genes during the development in buffalo embryos. Further research of these genes will greatly deepen our understanding of embryo development in buffalo. Collectively, this research lays the foundation for future studies on the maternal genome function, buffalo nuclear transfer and parthenogenetic embryonic stem cells.

The functions and mechanisms of sequence differences of DGAT1 gene on milk fat synthesis between dairy cow and buffalo.

In this research communication we describe the DGAT1 sequence and promoter region in dairy cows and buffalo and compare the activities of DGAT1 between the two species in order to increase knowledge of the cause of milk fat variation. pGL-3 basic vectors were used to construct the reporter gene. Based on the predicted promoter region, 4 truncated plasmid vectors were constructed in cow-DGAT1 and 3 plasmid vectors in buffalo-DGAT1. Each reporter plasmid was transfected into the bovine mammary epithelial cell (BMEC), 293T cell, and CHO cells to analyze the activity using Dual-Luciferase Reporter Assay System. The results show that the region between -93 to -556 bp was essential for cow promoter activity while -84 to -590 bp was essential for buffalo promoter activity revealing these regions contain core promoter. The buffalo has higher promoter activity than cow yet it was not statistically significant. Comparison of candidate mutation K232A between cow and buffalo population revealed the presence of both the allelic population in dairy cows (lysine and alanine) however, only K (lysine) allelic amino acid was found in buffalo population. The absence of the alanine allelic population from buffalo explains the higher fat content of buffalo milk.

Proteomics Analysis Reveals that Warburg Effect along with Modification in Lipid Metabolism Improves In Vitro Embryo Development under Low Oxygen.

The molecular mechanism regulating embryo development under reduced oxygen tension remains elusive. This study aimed to identify the molecular mechanism impacting embryo development under low oxygen conditions. Buffalo embryos were cultured under 5% or 20% oxygen and were evaluated according to their morphological parameters related to embryo development. The protein profiles of these embryos were compared using iTRAQ-based quantitative proteomics. Physiological O2 (5%) significantly promoted blastocyst yield, hatching rate, embryo quality and cell count as compared to atmospheric O2 (20%). The embryos in the 5% O2 group had an improved hatching rate of cryopreserved blastocysts post-warming (p < 0.05). Comparative proteome profiles of hatched blastocysts cultured under 5% vs. 20% O2 levels identified 43 differentially expressed proteins (DEPs). Functional analysis indicated that DEPs were mainly associated with glycolysis, fatty acid degradation, inositol phosphate metabolism and terpenoid backbone synthesis. Our results suggest that embryos under physiological oxygen had greater developmental potential due to the pronounced Warburg Effect (aerobic glycolysis). Moreover, our proteomic data suggested that higher lipid degradation, an elevated cholesterol level and a higher unsaturated to saturated fatty acid ratio might be involved in the better cryo-survival ability reported in embryos cultured under low oxygen. These data provide new information on the early embryo protein repertoire and general molecular mechanisms of embryo development under varying oxygen levels.

Molecular characterisation of the buffalo SCAP gene and its association with milk production traits in water buffaloes.

The study reported in this Research Communication was conducted to investigate the molecular characterisation of buffalo SCAP gene, expression analysis, and the association between single nucleotide polymorphisms and milk production traits in 384 buffaloes. Sequence analysis revealed the SCAP gene had an open reading frame of 3837 bp encoding 1279 amino acids. A ubiquitous expression profile of SCAP gene was detected in various tissues with extreme predominance in the mammary gland during early lactation. Moreover, eleven SNPs in buffalo SCAP gene were identified, six of them (g.1717600A>G, g.1757922C>T, g.1758953G>A, g.1759142C>T, g.1760740G>A, and g.1766036T>C) were found to be significantly associated with 305-day milk yield. Thus, buffalo SCAP could sever as a candidate gene affecting milk production traits in buffalo and the identified SNPs might potentially be genetic markers.

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region.

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y-chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y-chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.

Four novel polymorphisms of buffalo INSIG2 gene are associated with milk production traits in Chinese buffaloes.

Insulin-induced genes (INSIGs), including INSIG1 and INSIG2, are important mediators that play a pivotal role in the lipid metabolism and could cause the retention of the SCAP/SREBP complex. Therefore, the objective of this study is to detect the single nucleotide polymorphisms (SNPs) of buffalo INSIG2 gene and evaluate their associations with milk production traits in Chinese buffaloes. A total of four SNPs (g.621272A > G, g.621364A > C, g.632543G > A, and g.632684C > T) were identified using DNA pooled sequencing, and the SNP genotyping for the identified SNPs was performed by using Matrix-assisted laser desorption/ionization time of flight mass spectrometry method from 264 individuals. The results showed that four SNPs were significantly associated with 305-day milk yield or protein percentage in Murrah and crossbred breeds (P < 0.05), but they had no significant effect on milk production traits in Nili-Ravi buffaloes (P > 0.05). Linkage disequilibrium (LD) analysis revealed that one haplotype block was successfully constructed, of which the diplotype H1H1 showed significant association with 305-day milk yield in Murrah buffaloes (P < 0.05). Our findings provide evidence that polymorphisms in buffalo INSIG2 gene are associated with milk production traits, and could be used as a candidate gene for marker-assisted selection in buffalo breeding program.

Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

Effect of donor age and parent-to-child transplant on living-related donor kidney transplantation: a single center's experience of 236 cases.

To study the impact of parent-to-child transplant and older donor age on recipients' post-transplant creatinine levels, a total of 236 patients who received living donor kidney transplantation were evaluated for kidney viability based on creatinine (Cr) level. Of the 236 pairings, 113 (48%) were parent-to-child followed by sibling transplants (66, 30%). Recipient Cr levels were significantly higher at 6 months and 3 years post-transplant in the parent-to-child transplants compared to other donor-recipient relationships. In addition, donor age (average age: 44.1 ± 11.5; range: 19-66) contributed to higher recipient post-transplant Cr levels (p < 0.01). Pre-transplant donor and recipient Cr levels tended to result in higher post-transplant Cr levels in recipients (p < 0.05). Multivariate logistic regression analysis revealed that the presence of both parent-to-child transplant and older donor significantly increased the risk of elevated post-transplant Cr levels in recipients with an estimated odds ratios ranging from 3.46 (95% CI: 1.71-6.98) at 6 months to 8.04 (3.14-20.56) at 3 years post-transplant. Donor age significantly affected transplant survival as measured by higher recipient post-transplant Cr levels. In addition, parent-to-child transplant pairings, along with older donor age, significantly increased the risk of elevated post-transplant Cr levels in recipients.

Molecular cloning and expression analysis of the STAT1 gene in the water buffalo (Bubalus bubalis).

Signal transducer and activator of transcription 1 (STAT1) is a critical component of the transcription factor complex in the interferon (IFN) signaling pathways. Of the seven STAT isoforms, STAT1 is a key mediator of type I and type III IFN signaling, but limited information is available for the STAT genes in the water buffalo. Here, we amplified and identified the complete coding sequence (CDS) of the buffalo STAT1 gene by using reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that the buffalo STAT1 gene length size was 3437 bp, containing an open reading frame (ORF) of 2244 bp that encoded 747 amino acids for the first time. The buffalo STAT1 CDS showed 99, 98, 89, 93, 86, 85, and 87% identity with that of Bos taurus, Ovis aries, Homo sapiens, Sus scrofa, Rattus norvegicus, Mus musculus, and Capra hircus. The phylogenetic analyses revealed that the nearest relationship existed between the water buffalo and B. taurus. The STAT1 gene was ubiquitously expressed in 11 buffalo tissues by real-time PCR, whereas STAT1 was expressed at higher levels in the lymph. The STAT1 gene contained five targeted microRNA sequences compared with the B. taurus by the miRBase software that provide a fundamental for identifying the STAT1 gene function.

Evaluation of corrective effect of 6 degree of freedom couch on setup errors in intensity modulated radiotherapy for postoperative rectal cancer patients.

Objective: To explore the corrective effect of 6 degree of freedom couch on rotation errors in intensity modulated radiotherapy (IMRT) for postoperative rectal cancer patients, further to probe into the clinical application value of 6 degree of freedom couch in radiotherapy. Methods: From January 1, 2020 to December 1, 2020, 30 patients with rectal cancer receiving postoperative intensity modulated radiotherapy in The First Hospital of Hebei Medical University were included in this retrospective study. The setup error values in all direction of patients before and after 6 degree of freedom correction were collected during each radiotherapy session. Results: In this study, a total of 382 data before and after the correction of 6 degree of freedom couch were collected. It was found that the setup errors in the Y direction gradually increased, was maximal in the third week, and then became smaller, and the setup errors in the other directions increased with the extension of radiotherapy time and reached the maximum at the 5th week. In the translation direction, the setup errors value in Z direction occurred more frequently than that in X and Y directions between the range of 0.21-0.80 cm. In the rotation direction, the setup errors value in rotation X direction occurred more frequently than that in rotation Y and Z directions between the range of 0.21°-2.99°. In addition, after the correction of the 6 degree of freedom couch in real time, the setup errors in patients were significantly reduced in all directions (P < 0.05). Conclusion: In summary, it was recommended to clinically use 6 degree of freedom couch combined with IMRT for real-time correction of placement errors in patients with rectal cancer undergoing radiotherapy. At the same time, it was necessary to observe the tumor size and body weight changes of patients on the 5th week. If necessary, radiotherapy positioning and planning should be performed in time.

Thermal Comfort Index for Lactating Water Buffaloes under Hot and Humid Climate.

Heat stress results in serious performance losses and adversely affects animal health and welfare under various production systems. This study was conducted to develop a thermal comfort model for lactating buffaloes under hot and humid climate. Twenty Nili-Ravi buffaloes were randomly enrolled for this one-year study. Physiological parameters including rectal temperature (RT), respiratory rate (RR), and body surface temperature (BST) and environmental variables such as wet bulb temperature (WBT), dew point temperature (DPT), and black globe temperature (BGT) were recorded twice a week on each Tuesday and Thursday (n = 1602 and 1560, respectively) at 8:00 am and 2:30 pm. Moreover, ambient temperature (AT, °C) and relative humidity (RH, %), at an interval of every 30 min were recorded. We used a typical correlation analysis to build the index models for thermal comfort. The results revealed that AT positively correlated with BGT, WBT, DPT, BST, RT, and RR, while RH negatively correlated with RT. Moreover, a physiological index model consisting of BST, RT and RR (P1 = 0.578 × BST + 0.047 × RT + 0.429 × RR) and an environmental index model (E1 = 0.881 × AT + 0.194 × RH + 0.455 × BGT - 0.347 × WBT + 0.032 × DPT) proved to be a more accurate index as a pair to reveal the state of thermal comfort in lactating buffaloes. Moreover, these models correlated well with physiological variables, indicating that this this pair of index models can be used to effectively evaluate the thermal comfort in buffaloes.

Transcriptomic in silico analysis of bovine Escherichia coli mastitis highlights its immune-related expressed genes as an effective biomarker.

BACKGROUND: Mastitis is one of the major diseases causing economic loss to the dairy industry by reducing the quantity and quality of milk. Thus, the objective of this scientific study was to find new biomarkers based on genes for the early prediction before its severity. METHODS: In the present study, advanced bioinformatics including hierarchical clustering, enrichment analysis, active site prediction, epigenetic analysis, functional domain identification, and protein docking were used to analyze the important genes that could be utilized as biomarkers and therapeutic targets for mastitis. RESULTS: Four differentially expressed genes (DEGs) were identified in different regions of the mammary gland (teat cistern, gland cistern, lobuloalveolar, and Furstenberg's rosette) that resulted in 453, 597, 577, and 636 DEG, respectively. Also, 101 overlapped genes were found by comparing 27 different expressed genes. These genes were associated with eight immune response pathways including NOD-like receptor signaling pathway (IL8, IL18, IL1B, PYDC1) and chemokine signaling pathway (PTK2, IL8, NCF1, CCR1, HCK). Meanwhile, 241 protein-protein interaction networks were developed among overlapped genes. Fifty-seven regulatory events were found between miRNAs, expressed genes, and the transcription factors (TFs) through micro-RNA and transcription factors (miRNA-DEG-TF) regulatory network. The 3D structure docking model of the expressed genes proteins identified their active sites and the binding ligands that could help in choosing the appropriate feed or treatment for affected animals. CONCLUSIONS: The novelty of the distinguished DEG and their pathways in this study is that they can precisely improve the detection biomarkers and treatments techniques of cows' Escherichia coli mastitis disease due to their high affinity with the target site of the mammary gland before appearing the symptoms.

Comparative transcriptome analysis reveals potential testosterone function-related regulatory genes/pathways of Leydig cells in immature and mature buffalo (Bubalus bubalis) testes.

Leydig cells (LCs) are testosterone-generating endocrine cells that are located outside the seminiferous tubules in the testis, and testosterone is fundamental for retaining spermatogenesis and male fertility. In buffalo, adult Leydig cells (ALCs) are developed by immature Leydig cells (ILCs) in the postnatal testes. However, the genes/pathways associated to the regulation of testosterone secretion function during the development of postnatal LCs remains comprehensively unidentified. The present study comparatively analyzed the transcriptome profiles of ILC and ALC in buffalo with significant differences in testosterone secretion. Differentially expressed genes (DEGs) analysis identified 972 and 1,091 annotated genes that were significantly up- and down-regulated in buffalo ALC. Functional enrichment analysis showed that cAMP signaling being the most significantly enriched pathway, and testosterone synthesis and lipid transport-related genes/pathways were upregulated in ALC. Furthermore, gene set enrichment analysis (GSEA) shows that cAMP signaling and steroid hormone biosynthesis were activated in ALC, demonstrating that cAMP signaling may serve as a positive regulatory pathway in the maintenance of testosterone function during postnatal development of LCs. Protein-protein interaction (PPI) networks analysis highlighted that ADCY8, ADCY2, POMC, CHRM2, SST, PTGER3, SSTR2, SSTR1, NPY1R, and HTR1D as hub genes in the cAMP signaling pathway. In conclusion, this study identified key genes and pathways associated in the regulation of testosterone secretion function during the ILC-ALC transition in buffalo based on bioinformatics analysis, and these key genes might be deeply involved in cAMP generation to influencing testosterone levels in LCs. The results suggest that ALCs might increase testosterone levels by enhancing cAMP production than ILCs. Our data will enhance the understanding of developmental mechanism studies related to testosterone function and provide preliminary evidence for molecular mechanisms of LCs regulating spermatogenesis.

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration.

Sex control technology is of great significance in the production of domestic animals, especially for rapidly breeding water buffalo (bubalus bubalis), which served as a research model in the present study. We have confirmed that a fluorescence protein integrated into the Y chromosome is fit for sexing pre-implantation embryos in the mouse. Firstly, we optimized the efficiency of targeted integration of exogenous gene encoding enhanced green fluorescent protein (eGFP) and mCherry in Neuro-2a cells, mouse embryonic stem cells, mouse embryonic cells (NIH3T3), buffalo fetal fibroblast (BFF) cells. The results showed that a homology arm length of 800 bp on both sides of the target is more efficient that 300 bp or 300 bp/800 bp. Homology-directed repair (HDR)-mediated knock-in in BFF cells was also significantly improved when cells were supplemented with pifithrin-µ, which is a small molecule that inhibits the binding of p53 to mitochondria. Three pulses at 250 V resulted in the most efficient electroporation in BFF cells and 1.5 µg/mL puromycin was found to be the optimal concentration for screening. Moreover, Y-Chr-eGFP transgenic BFF cells and cloned buffalo embryos were successfully generated using CRISPR/Cas9-mediated gene editing combined with the somatic cell nuclear transfer (SCNT) technique. At passage numbers 6-8, the growth rate and cell proliferation rate were significantly lower in Y-Chr-eGFP transgenic than in non-transgenic BFF cells; the expression levels of the methylation-related genes DNMT1 and DNMT3a were similar; however, the expression levels of the acetylation-related genes HDAC1, HDAC2, and HDAC3 were significantly higher (p < 0.05) in Y-Chr-eGFP transgenic BFF cells compared with non-transgenic cells. Y-Chr-eGFP transgenic BFFs were used as donors for SCNT, the results showed that eGFP reporter is suitable for the visualization of the sex of embryos. The blastocyst rates of cloned buffalo embryos were similar; however, the cleavage rates of transgenic cloned embryos were significantly lower compared with control. In summary, we optimized the protocol for generating transgenic BFF cells and successfully generated Y-Chr-eGFP transgenic embryos using these cells as donors.

Developmental potential of buffalo embryos cultured in serum free culture system.

The presence of serum in embryo culture medium has been implicated for increased embryo's sensitivity to cryopreservation, compromised viability, abnormal embryo and fetal development. Hence, designing a serum free culture system is indispensable. The present study aims to compare the efficiency of the serum and granulosa cells monolayer free commercial culture system (SFCS) with the conventional serum supplemented co-culture system (SSCS) and optimized culture system (OCS). Generally, SFCS is designed explicitly for bovine oocyte maturation and embryo culture (SF-IVM and SF-IVC), and SSCS (based on M199, SS-IVM, and SS-IVC) is utilized for buffalo in vitro embryo production. However, OCS is a newly designed culture system in which oocyte maturation is performed in serum supplemented maturation medium, and the subsequent embryos are co-cultured with granulosa cells in serum free culture medium. To evaluate the effect of serum on buffalo embryo production, buffalo oocytes, and their subsequent embryos were cultured in SSCS, SFCS, and OCS, simultaneously. The percentage of cleaved embryos cultured in SSCS and OCS was approximately 4% higher as compared to SFCS. However, OCS significantly showed the maximum proportion of embryos that developed to the blastocyst stage (7d) and hatched (6d) as compared to the SFCS and SSCS. Additionally, OCS promoted the expression of developmentally important genes (BCL2-L1 and VEGF-A), cell number, and cryo-survival ability of blastocysts in comparison with SSCS. Taken together, OCS is more suitable for the oocyte maturation and culture of buffalo embryos. However, to design the serum free culture system, it is recommended to find suitable serum alternatives for in vitro oocyte maturation.

Ubiquitome analysis reveals the involvement of lysine ubiquitination in the spermatogenesis process of adult buffalo (Bubalus bubalis) testis.

Protein ubiquitination, a major and conserved post-translational modification, is known to play a critical regulatory role in many biological processes in eukaryotes. Although several ubiquitinated proteins have been found in buffalo (Bubalus bubalis) testis in our previous studies, large-scale profiling of buffalo testis ubiquitome has not been reported to date. In the present study, we first identified a global profiling of lysine ubiquitination of adult buffalo testis using a highly sensitive LC-MS/MS coupled with immune-affinity enrichment of ubiquitinated peptides. In total, 422 lysine ubiquitination sites were identified in 262 proteins in adult buffalo testis tissue. Bioinformatics analysis showed that the ubiquitinated proteins are involved in a variety of biological processes and diverse subcellular localizations. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein interaction network analysis indicated that proteasome, glycolysis/gluconeogenesis and gap junction pathways are modulated by protein ubiquitination in testis. Besides, 44 ubiquitinated proteins may involve in spermatogenesis according to the SpermatogenesisOnline database, of which, the ubiquitination of HSPA2 and UCHL1 were confirmed by Immunoprecipitation (IP)/Western blot analysis. Taken together, these data provide a global view of ubiquitome in buffalo testis for the first time, and serve as an important resource for exploring the physiological role especially spermatogenesis of lysine ubiquitination in testis in mammals.