Chinese Swamp Buffalo Research Articles

Activation of Wnt/β-Catenin Signaling Pathway Enhances the Derivation of Buffalo (Bubalus bubalis) Embryonic Stem Cell-Like Cells.

Wnt/β-Catenin signaling pathway plays an important role in maintaining self-renewal and pluripotency of human and mouse embryonic stem cells (ESCs). Activation of Wnt/β-Catenin signaling pathway by glycogen synthase kinase-3 (GSK3) inhibitor, the Wnt signaling agonist, could maintain the pluripotency of human and mouse ESCs in the presence of serum. However, the role of signaling pathway in the derivation of buffalo ESCs remains unclear. In this study, we used GSK3 inhibitors (6-bromoindirubin-3'-oxime [BIO] and CHIR99021) and investigated the effect of Wnt/β-Catenin activation on colony formation, proliferation, self-renewal, and pluripotency of Chinese swamp buffalo (buffalo) embryonic stem cell-like cells (ES-like cells), which were isolated from blastocysts. The results showed that buffalo ES-like cells displayed typical morphological characteristics of pluripotent stem cells: positive for alkaline phosphatase staining, expression of pluripotent markers, including OCT4, SOX2, SSEA-1, SSEA-4, LIN28, CH1, NANOG, and the proliferative markers, PCNA and C-MYC. Furthermore, activation of Wnt/β-Catenin signaling pathway by GSK3 inhibitors could promote colony formation and proliferation of buffalo ES-like cells and maintain their undifferentiated state, and upregulate the expression levels of pluripotent-related genes and proliferation-related genes. These results indicated that Wnt/β-Catenin signaling pathway plays an important role in the derivation and pluripotency of buffalo ES-like cells.

Mitochondrial sequence-based evolutionary analysis of riverine-swamp hybrid buffaloes of India indicates novel maternal differentiation and domestication patterns.

In this study, mitochondrial D-loop sequence data on riverine, swamp and hybrid buffaloes from India have been generated and compared with other reported Indian riverine, Chinese and Bangladeshi swamp buffalo populations. Sequence analysis revealed the presence of 132 haplotypes, with a haplotype diversity of 0.9611±0.0045 and a nucleotide diversity of 0.04801±0.00126. For the first time, the existence of riverine-swamp hybrids among the Indian Chilika buffalo population has been recorded, having 49 chromosomes, which was also confirmed by mitochondrial haplotype sharing between Chilika and Indian swamp as well as Chinese swamp buffalo populations in the network analysis. Phylogenetic analysis documents the sharing of reported pre-domestication haplogroups 'SA1', 'SA2', 'SA3' and 'SB1' between the Chilika and swamp buffalo populations of India, China and Bangladesh, an indication of the migration of swamp buffaloes towards Bangladesh and adjoining lower parts of India and north towards Chinese domestication sites. The results have also been supplemented by multidimension scaling, grouping Indian and Chinese swamp buffaloes more closely together with Bangladeshi buffaloes, but into a separate quadrant, whereas Chilika grouped away from other riverine as well as swamp buffaloes. These findings thus confirm the previous reports that the northeast region of India, close to the Indo-China border, is the point of evolution of swamp buffaloes with multiple sites of domestication.

MiRNA transcriptome comparison between muscle and adipose tissues indicates potential miRNAs associated with intramuscular fat in Chinese swamp buffalo.

The amount of intramuscular fat (IMF) affects the tenderness and juiciness of beef and is an important indicator of beef quality. A few miRNAs involved in IMF deposition have been identified in other livestock. However, in the buffalo, the association between miRNA and IMF has not been reported and the miRNA expression profile remains poorly understood. In this study, small RNA sequencing was performed to characterize the miRNA expression pattern in muscle and adipose tissues using the Illumina platform. A total of 108 differentially expressed (DE) miRNAs were identified, including 98 known miRNAs and 10 novel miRNAs. A qRT-PCR experiment confirmed the quality of the DE analysis. Eight DE miRNAs showed high expression in adipose tissue and a considerable expression level in muscle tissue. Functional enrichment indicated that bta-miR-148a, bta-miR-143, bta-miR-10b, bta-let-7i, bta-let-7f, bta-let-7b, bta-miR-30a-5p, and bta-miR-100 were significantly associated with adipogenesis, suggesting these as candidate regulators for IMF deposition in buffalo. However, further functional validation is required. This is the first characterization of the miRNA expression profile in the muscle and adipose tissues of buffalo. These results provide information for the identification of miRNAs with potential effects on IMF deposition in buffalo.

Identification and analysis of the expression of microRNA from lactating and nonlactating mammary glands of the Chinese swamp buffalo

To study the role of microRNA (miR) in the lactation physiology of water buffalo, 2 multiparous dairy buffaloes (including an 8-yr-old buffalo that had been lactating for 3 mo, as well as a 10-yr-old nonlactating, nonpregnant buffalo) were used for miR library construction. The profile of differentially expressed miR in lactating and nonlactating mammary gland tissues of these water buffalo were investigated using Illumina-Solexa high-throughput sequencing technology (Illumina, San Diego, CA). The data identified 259 miR families, 359 mature miR, 363 pre-miR, 230 novel buffalo miR, and 5 buffalo-specific miR that were expressed in mammary tissues. Some highly significantly differentially expressed miR were explored, including bbu-miR-497, bbu-miR-30a-5p, bbu-miR-148a, bbu-miR-29a, bbu-miR-125a, bbu-miR-125b, and bbu-miR-103. The expression patterns of 18 miR were confirmed by quantitative real-time PCR in both tissues, and the expression of bbu miR-103 and novel miR-57 constituted the largest differences between lactating and nonlactating tissues. Further functional analysis indicated that the overexpression or suppression of miR-103 in buffalo mammary epithelial cells downregulated or upregulated the expression of pantothenate kinase 3, and also significantly increased the transcription factor steroid regulatory element binding protein, followed by the acceleration of de novo synthesis of fatty acids by upregulation of acetyl coenzyme A carboxylase α expression. The expression levels of 34 predicted target genes of novel-miR-57 in lactating and nonlactating mammary gland tissues were all analyzed by quantitative real-time PCR. Finally, only the expression of docking protein 4 could be upregulated or downregulated selectively by bbu-novel-miR-57 in buffalo mammary epithelial cells and the Bcap-37 cell line. This study provides an overview of the miR expression profile of water buffalo and the interaction between some key miR and their target genes, which may improve understanding of the important roles of miR in buffalo milk fat synthesis.

De Novo Transcriptome Assembly of the Chinese Swamp Buffalo by RNA Sequencing and SSR Marker Discovery.

The Chinese swamp buffalo (Bubalis bubalis) is vital to the lives of small farmers and has tremendous economic importance. However, a lack of genomic information has hampered research on augmenting marker assisted breeding programs in this species. Thus, a high-throughput transcriptomic sequencing of B. bubalis was conducted to generate transcriptomic sequence dataset for gene discovery and molecular marker development. Illumina paired-end sequencing generated a total of 54,109,173 raw reads. After trimming, de novo assembly was performed, which yielded 86,017 unigenes, with an average length of 972.41 bp, an N50 of 1,505 bp, and an average GC content of 49.92%. A total of 62,337 unigenes were successfully annotated. Among the annotated unigenes, 27,025 (43.35%) and 23,232 (37.27%) unigenes showed significant similarity to known proteins in NCBI non-redundant protein and Swiss-Prot databases (E-value < 1.0E-5), respectively. Of these annotated unigenes, 14,439 and 15,813 unigenes were assigned to the Gene Ontology (GO) categories and EuKaryotic Ortholog Group (KOG) cluster, respectively. In addition, a total of 14,167 unigenes were assigned to 331 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, 17,401 simple sequence repeats (SSRs) were identified as potential molecular markers. One hundred and fifteen primer pairs were randomly selected for amplification to detect polymorphisms. The results revealed that 110 primer pairs (95.65%) yielded PCR amplicons and 69 primer pairs (60.00%) presented polymorphisms in 35 individual buffaloes. A phylogenetic analysis showed that the five swamp buffalo populations were clustered together, whereas two river buffalo breeds clustered separately. In the present study, the Illumina RNA-seq technology was utilized to perform transcriptome analysis and SSR marker discovery in the swamp buffalo without using a reference genome. Our findings will enrich the current SSR markers resources and help spearhead molecular genetic research studies on the swamp buffalo.

Genetic analysis of river, swamp and hybrid buffaloes of north‐east India throw new light on phylogeography of water buffalo (Bubalus bubalis)

This study analysed buffaloes from north-east India and compared their nuclear and mitochondrial DNA variations with buffaloes of mainland India, China, Mediterranean and South-East Asia. Microsatellite genotypes of 338 buffaloes including 210 from six north-east Indian buffalo populations and three mainland Indian breeds were analysed to evaluate their genetic structure and evolutionary relationships. Phylogenetic analysis and multidimensional scaling plot of pairwise FST revealed the clustering of all swamp-type buffaloes of north-east India with Lower Assamese (significantly hybrid type) buffaloes in one plane and all the mainland river buffaloes in another plane while the upper Assamese buffaloes being distinct from both these clusters. Analysis of mtDNA D-loop region of 530-bp length was performed on 345 sequences belonging to 23 buffalo populations from various geographical regions to establish the phylogeography of Indian water buffalo. The swamp buffaloes of north-east India clustered with both the lineages of Chinese swamp buffalo. Multidimensional scaling display of pairwise FST derived from mitochondrial DNA data showed clustering of upper Assamese, Chilika and Mediterranean buffaloes distinctly from all the other Indian buffalo populations. Median-joining network analysis further confirmed the distinctness and ancestral nature of these buffaloes. The study revealed north-east region of India forming part of the wider hybrid zone of water buffalo that may probably extend from north-east India to South-East Asia.

Role of linker histone H1c during the reprogramming of Chinese swamp buffalo (Bubalus Bubalis) embryos produced by somatic cell nuclear transfer.

During reprogramming, there is exchange of histone H1c and the oocyte-specific linker histone, and H1c may play a critically important role in the reprogramming process of somatic cell nuclear transfer (SCNT). The aim of the present study was to investigate the role of the H1c gene in SCNT reprogramming in Chinese swamp buffalo (Bubalus bubalis) using RNA interference (RNAi). Chinese swamp buffalo H1c gene sequences were obtained and H1c-RNAi vectors were designed, synthesised and then transfected into a buffalo fetal skin fibroblast cell line. Expression of H1c was determined by real-time polymerase chain reaction to examine the efficiency of vector interference. These cells were then used as a nuclear donor for SCNT so as to observe the further development of SCNT embryos. Inhibition of H1c gene expression in donor cells significantly improved the developmental speed of embryos from the 1-cell to 8-cell stage. Furthermore, compared with the control group, inhibition of H1c gene expression significantly reduced the blastocyst formation rate. It is concluded that linker histone H1c is very important in SCNT reprogramming in Chinese swamp buffalo. Correct expression of the H1c gene plays a significant role in preimplantation embryonic development in B. bubalis.

Establishment and biological characteristics comparison of Chinese swamp buffalo (Bubalus bubalis) fibroblast cell lines

To establish fibroblast cell lines from different tissues and to compare the biological characteristics of those cell lines, five fibroblast cell lines derived from Chinese swamp buffalo (Bubalus bubalis) were selected for comparative assays. Cell style and survival rate (before cryogenic preservation and after recovery) were tested, and karyotype, patterns of isoenzymes of lactic dehydrogenase, malic dehydrogenase, and cell cycle were analyzed. These cell lines had a healthy morphology with a typical spindle shape, and assessment of cell style showed these cells to be very pure fibroblasts. Cell growth curves showed a typical "S" shape. Results of microorganism contamination assays were negative, and isoenzyme analysis showed no cross-contamination. The number of chromosomes (2n) of swamp buffalo is 48. Between 28% and 46% of the cells were 2n, and cell apoptosis was not pronounced at 20th generation. Results showed that skin fibroblasts were more adaptable to tissue culture conditions than the ones from kidneys and ear margin, and they are more suitable for cellular manipulation in Chinese swamp buffalo.

Phylogeography and Domestication of Chinese Swamp Buffalo

To further probe into whether swamp buffaloes were domesticated once or multiple times in China, this survey examined the mitochondrial DNA (mtDNA) Control Region (D-loop) diversity of 471 individuals representing 22 populations of 455 Chinese swamp buffaloes and 16 river buffaloes. Phylogenetic analysis revealed that Chinese swamp buffaloes could be divided into two distinct lineages, A and B, which were defined previously. Of the two lineages, lineage A was predominant across all populations. For predominant lineage A, Southwestern buffalo populations possess the highest genetic diversity among the three hypothesized domestication centers (Southeastern, Central, and Southwestern China), suggesting Southwestern China as the most likely location for the domestication of lineage A. However, a complex pattern of diversity is detected for the lineage B, preventing the unambiguous pinpointing of the exact place of domestication center and suggesting the presence of a long-term, strong gene flow among swamp buffalo populations caused by extensive migrations of buffaloes and frequent human movements along the Yangtze River throughout history. Our current study suggests that Southwestern China is the most likely domestication center for lineage A, and may have been a primary center of swamp buffalo domestication. More archaeological and genetic evidence is needed to show the process of domestication.

DGAT1, GH, GHR, PRL and PRLR Polymorphism in Water Buffalo (Bubalus bubalis)

The polymorphism of several genes has been shown to affect the milk composition traits in dairy cattle, including DGAT1-exon8 K232A, GH-intron3 MspI, GH-exon5 AluI, GHR-exon8 F279Y, PRL-exon3 RsaI and PRLR-exon3 S18N. However, the polymorphism and effects of these genes on the milk traits of water buffalo are still unclear. In this study, four DNA pooling samples from Murrah, Nili-ravi, Murrah-Nili-Swamp crossbreed and Chinese swamp buffalo were constructed, respectively, and polymorphism of these sites was investigated using PCR-Single-strand conformation polymorphism and sequencing. Twenty-eight inter-specific single-nucleotide polymorphism (SNPs) were found in these six assayed gene fragments between buffalo and dairy cattle, including nine intra-specific SNPs among buffalo groups. All buffalo fixed a K allele genotype in DGAT1-exon8, MspI(+) restriction site(c nucleotide) and AluI(+) site(c nucleotide) at intron3 and exon5 of GH gene, F allele genotype of F279Y mutation in GHR gene, RsaI(-) restriction site at PRL-exon3/exon4 and N allele genotype of S18N mutation at PRLR-exon3. It provides an indirect evidence that water buffalo have fixed alleles with genotypes reported in dairy cattle, which is thought to be responsible for high milk fat, high protein content and low milk yield. Moreover, three new intra-specific SNPs were found including 275th bp (c/t) in DGAT1 of Murrah buffalo, 109th bp (t/a) in PRL-exon3/exon4 and 43rd bp (c/t) in PRLR-exon3 of Chinese swamp buffalo. Information provided in this study will be useful in further studies to improve buffalo breeding for better lactation performances.

Population structure and phylogeography of Toda buffalo in Nilgiris throw light on possible origin of aboriginal Toda tribe of South India

We report the genetic structure and evolutionary relationship of the endangered Toda buffalo of Nilgiris in South India with Kanarese and two other riverine buffalo breeds. The upgma phylogeny drawn using Nei's distance grouped South Kanara and Toda buffaloes at a single node while Marathwada and Murrah together formed a separate node. Principal component analysis was performed with pairwise interindividual chord distances which revealed clustering of Murrah and Marathwada buffaloes distinctly, while individuals of Toda and South Kanara breeds completely intermingled with each other. Furthermore, there were highly significant group variances (p < 0.01) when the breeds were grouped based on phylogeny, thus revealing the existence of cryptic genetic structure within these buffalo breeds. To know the evolutionary relationship among these breeds, 537-bp D-loop region of mitochondrial DNA was analysed. The phylogenetic analysis of mtDNA haplotypes following NJ algorithm with Chinese swamp buffalo as outgroup revealed a major cluster that included haplotypes from all the four investigated breeds and two minor clusters formed by South Kanara and Toda haplotypes. Reduced median network analysis revealed haplotypes of South Kanara and Toda to be quite distinct from the commonly found haplotypes indicating that these might have been ancestral to all the present-day haplotypes. Few mutations in two of the haplotypes of South Kanara buffalo were found to have contributed to ancestral haplotypes of Toda buffalo suggesting the possible migration of buffaloes from Kanarese region towards Nilgiris along the Western Ghats. Considering the close social, economic and cultural association of Todas with their buffaloes, the present study supports the theory of migration of Toda tribe from Kanarese/Mysore region along with their buffaloes.

Isolation and characterization of EG‐like cells from Chinese swamp buffalo (Bubalus bubalis)

There have been few studies done on the isolation and characterization of Chinese swamp buffalo embryonic germ cells (EG cells). Here, we first report on EG-like cells isolated from Chinese swamp buffalo fetuses. The results showed the cells grew in large, multilayered colonies, which were densely packed with an obvious border resembling mouse embryonic stem cells (ES cells) and EG cells. The buffalo EG-like cells expressed AP, SSEA-1, SSEA-3, SSEA-4 and OCT-4. By RT-PCR, we found that undifferentiated swamp buffalo EG-like cells expressed the OCT-4, NANOG, SOX2, FOXD3, GP130, STAT3, and HEB gene mRNA, but not Fgf4. When these cells were cultured for more than 2weeks without passage, they could differentiate into several types of cells including fibroblast-like, neuron-like, smooth muscle-like, and epithelial-like cells. Some cells formed simple embryoid bodies (EBs) and cystic EBs by suspension culture. By RT-PCR, we found cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA, but not OCT-4, NANOG, and SOX2 gene mRNA, which could be detected in undifferentiated buffalo EG-like cells. At the same time, the expression of KERATIN-14 (Endoderm), GATA4, ACTA2 (Mesoderm) and TUBB3 (Ectoderm) gene mRNA were also detected in cystic EBs. The results suggested that these cells were capable of forming three germ layers in in vitro differentiation. The expression of OCT-4, NANOG and SOX2 might be essential for Chinese swamp buffalo EG-like cells in a pluripotent state. During the isolation and culture of Chinese swamp buffalo EG-like cells, we found the fetuses that were at 30-80days post-coitus were more efficient than others; and the mechanical method was better than trypsin digestion. The maximal passage of the mechanical method was eight, but the trypsin digestion was just three passages. So it seemed like that the buffalo EG-like cells were sensitive to trypsin. In summary, we were the first to isolate and characterize Chinese swamp buffalo EG-like cells that had morphology and characterization similar to those of established EG/EG-like cells in mouse and human.

Independent maternal origin of Chinese swamp buffalo (Bubalus bubalis)

To obtain more knowledge on the origin and genetic diversity of the swamp buffalo (Bubalus bubalis) in China, the complete mitochondrial D-loop sequences of 119 samples representing seven native types were compared. Two mitochondrial DNA (mtDNA) lineages (lineages A and B) were determined for the Chinese swamp buffalo. Examination of the diversity patterns suggest that lineage A has undergone a population expansion event. Divergence of lineages A and B was estimated at 18,000 years ago. Combined analyses of mtDNA sequences from Chinese, Indian, Brazilian/Italian and Southeast Asian/Australian buffalo samples showed independent domestication events in the swamp buffalo from China and the river buffalo from the India subcontinent. The spread of swamp and river buffalo from China and India respectively to mainland Southeast Asia suggests that Southeast Asia is a hybrid zone for buffalo. Our data support the hypothesis of the evolution of domesticated swamp and river buffalo from ancestral swamp-like animals. These ancestral animals were extensively distributed across mainland Asia and most likely are represented today by the wild Asian buffalo (Bubalus arnee).

A Y-linked SNP in SRY Gene Differentiates Chinese Indigenous Swamp Buffalo and Introduced River Buffalo

The complete coding region sequence of the SRY gene in Chinese swamp buffalo was determined by PCR product sequencing. Comparison of swamp and river buffalo SRY gene sequences revealed a single nucleotide polymorphism (SNP, C/G) at the 202 bp site of the coding region. Further, a total of 124 male domestic buffaloes were genotyped at this SNP site using the PCR-SSCP method, and it was found that all Chinese indigenous swamp buffaloes had a guanine (G) at this site, while introduced river buffaloes and crossbred buffaloes showed a cytosine (C). Our findings suggested that this Y-linked SNP displayed type-specific alleles differentiating swamp and river buffaloes, and could be used as an effective marker to detect crossbreeding of swamp buffaloes with introduced river buffaloes in native buffalo populations, and thereby assess genetic diversity status and make proper conservation decisions for indigenous swamp buffaloes. In addition, this SNP can be potentially applied in the study of Asian water buffalo phylogeny from a male perspective.

Pharmacokinetics of radiolabelled buffalo follicle-stimulating hormone (125I-buFSH) in female buffalo calves (Bubalus bubalis).

Journal of Veterinary Pharmacology and TherapeuticsVolume 19, Issue 2 p. 158-160 Pharmacokinetics of radiolabelled buffalo follicle-stimulating hormone (125I-buFSH) in female buffalo calves (Bubalus bubalis) N. PRAKASH, N. PRAKASH Department of Pharmacology Veterinary College University of Agricultural Sciences, Bangalore-560 024, India.Search for more papers by this authorK. NARAYANA, K. NARAYANA Department of Pharmacology Veterinary College University of Agricultural Sciences, Bangalore-560 024, India.Search for more papers by this author N. PRAKASH, N. PRAKASH Department of Pharmacology Veterinary College University of Agricultural Sciences, Bangalore-560 024, India.Search for more papers by this authorK. NARAYANA, K. NARAYANA Department of Pharmacology Veterinary College University of Agricultural Sciences, Bangalore-560 024, India.Search for more papers by this author First published: April 1996 https://doi.org/10.1111/j.1365-2885.1996.tb00031.xAboutRelatedInformationPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume19, Issue2April 1996Pages 158-160 RelatedInformation RecommendedIndependent maternal origin of Chinese swamp buffalo (Bubalus bubalis)C. Z. Lei, W. Zhang, H. Chen, F. Lu, R. Y. Liu, X. Y. Yang, H. C. Zhang, Z. G. Liu, L. B. Yao, Z. F. Lu, Z. L. Zhao, Animal GeneticsSinus Paranasalis of the Egyptian Buffalo, Bos (Bubalus) bubalis L.M. S. El Din Moustafa, Sh. H. Kamel, Zentralblatt für Veterinärmedizin Reihe ADGAT1, GH, GHR, PRL and PRLR Polymorphism in Water Buffalo (Bubalus bubalis)D-S Shi, J Wang, Y Yang, F-H Lu, X-P Li, Q-Y Liu, Reproduction in Domestic AnimalsIsolation and characterization of EG-like cells from Chinese swamp buffalo (Bubalus bubalis)Ben Huang, Ti-San Xie, De-Shun Shi, Tong Li, Xiao-Li Wang, Yi Mo, Zhi-Qiang Wang, Meng-Mei Li, Cell Biology InternationalToxocara vitulorum: treatment based on the duration of the infectivity of buffalo cows (Bubalus bubalis) for their calvesJ. A.ROBERTS, Journal of Veterinary Pharmacology and Therapeutics

Ultrastructure and chemical composition of calcite urinary calculi in Chinese swamp buffalo

Four urinary calculi, derived from Chinese swamp buffalo, were studied by using qualitative chemical analysis, X-ray diffraction, scanning electron microscopy and qualitative energy dispersive (electron probe) microanalysis. Qualitative chemical analysis showed that the predominant ions were calcium and carbonate with small amounts of magnesium and ammonium. X-ray diffraction confirmed that the calculi were primarily composed of calcium carbonate (calcite). On ultrastructural examination, three apparently distinct structural regions were identified in the calculi: outer large laminations; cavities containing variable numbers of small spheres and rods; and large spheres. There did not appear to be material that acted as a nidus and all regions, on qualitative electron probe analysis, contained primarily calcium with trace amounts of magnesium, phosphorus, potassium and chloride. It was concluded that calcite calculi in Chinese swamp buffalo are probably formed through a process of asynchronous layering and that nidus formation may not be necessary. Moreover, the ultrastructure of the calcite calculi is similar to that reported for siliceous calculi in ruminants and this suggests that similar factors may be involved in their formation.