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Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine.

ABSTRACT: This study was aimed at isolation of cells from urine and skin on the ventral part of the tails of healthy adult female buffaloes (Bubalus bubalis), an area rarely exposed to solar radiation, establishment of the cells in culture, and their use as donor cells for production of buffalo embryos by handmade cloning (HMC). The blastocyst rate and total cell number of urine- and tail skin-derived embryos were similar to those of control embryos derived from ear skin cells; however, their apoptotic index was lower (p<0.05) than that of control blastocysts. The global level of histone H3 acetylated at lysine 9 (H3K9ac) was similar in the three types of donor cells and in urine- and tail skin-derived HMC blastocysts and in vitro-fertilized (IVF) blastocysts (controls). The global level of histone H3 trimethylated at lysine 27 (H3K27me3) in the cells was in the order (p<0.05) urine?tail skin>ear skin-derived cells, whereas in blastocysts, it was higher (p<0.05) in urine- and tail skin-derived HMC blastocysts than that in IVF blastocysts. The expression level of CASPASE3, CASPASE9, P53, DNMT1, DNMT3a, OCT4, and NANOG, which was similar in HMC blastocysts of three the groups, was lower (p<0.05) than that in IVF blastocysts, whereas that of HDAC1 was similar among the four groups. Following transfer of urine-derived embryos (n=10) to five recipients (two embryos/recipient), one of the recipients delivered a normal calf that is now 5 weeks old.

SUBMITTER: Madheshiya PK

PROVIDER: S-EPMC4487680 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine.

Madheshiya Pankaj K PK Sahare Amol A AA Jyotsana Basanti B Singh Karn P KP Saini Monika M Raja Anuj K AK Kaith Sakshi S Singla Suresh K SK Chauhan Manmohan S MS Manik Radhey S RS Palta Prabhat P

Cellular reprogramming 20150601 3

This study was aimed at isolation of cells from urine and skin on the ventral part of the tails of healthy adult female buffaloes (Bubalus bubalis), an area rarely exposed to solar radiation, establishment of the cells in culture, and their use as donor cells for production of buffalo embryos by handmade cloning (HMC). The blastocyst rate and total cell number of urine- and tail skin-derived embryos were similar to those of control embryos derived from ear skin cells; however, their apoptotic in ...[more]

PMID: 26053516

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Project description:The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical "S" shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10-15 and 8-12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.

Project description:BackgroundSimple sequence repeats (SSRs) of GACA/GATA have been implicated with differentiation of sex-chromosomes and speciation. However, the organization of these repeats within genomes and transcriptomes, even in the best characterized organisms including human, remains unclear. The main objective of this study was to explore the buffalo transcriptome for its association with GACA/GATA repeats, and study the structural organization and differential expression of the GACA/GATA repeat tagged transcripts. Moreover, the distribution of GACA and GATA repeats in the prokaryotic and eukaryotic genomes was studied to highlight their significance in genome evolution.ResultsWe explored several genomes and transcriptomes, and observed total absence of these repeats in the prokaryotes, with their gradual accumulation in higher eukaryotes. Further, employing novel microsatellite associated sequence amplification (MASA) approach using varying length oligos based on GACA and GATA repeats; we identified and characterized 44 types of known and novel mRNA transcripts tagged with these repeats from different somatic tissues, gonads and spermatozoa of water buffalo Bubalus bubalis. GACA was found to be associated with higher number of transcripts compared to that with GATA. Exclusive presence of several GACA-tagged transcripts in a tissue or spermatozoa, and absence of the GATA-tagged ones in lung/heart highlights their tissue-specific significance. Of all the GACA/GATA tagged transcripts, approximately 30% demonstrated inter-tissue and/or tissue-spermatozoal sequence polymorphisms. Significantly, approximately 60% of the GACA-tagged and all the GATA-tagged transcripts showed highest or unique expression in the testis and/or spermatozoa. Moreover, approximately 75% GACA-tagged and all the GATA-tagged transcripts were found to be conserved across the species.ConclusionPresent study is a pioneer attempt exploring GACA/GATA tagged transcriptome in any mammalian species highlighting their tissue, stage and species-specific expression profiles. Comparative analysis suggests the gradual accumulation of these repeats in the higher eukaryotes, and establishes the GACA richness of the buffalo transcriptome. This is envisaged to establish the roles of integral simple sequence repeats and tagged transcripts in gene expression or regulation.

Project description:Toxoplasmosis and neosporosis are diseases with worldwide distribution that are associated with reproductive problems in livestock and responsible for economic losses. This review presents an overview of the current knowledge relative to these diseases in water buffalo (Bubalus bubalis). In general, buffalo are considered resistant to clinical toxoplasmosis because there are studies only reporting serological evidence of natural infection in these animals. Studies have described age, poor hygienic status of the farm, and presence of cats as risk factors for the development of Toxoplasma gondii infection in buffalo. It must be highlighted that buffalo meat, which does not receive adequate freezing treatment, could be a potential source for toxoplasmic human infection as well as the importance of raw buffalo milk in the transmission of toxoplasmosis to human beings. Neospora caninum is considered one of the major causes of abortion and responsible for huge economic losses in cattle. Vertical transmission is the main route to infect calves, and is responsible for maintaining the parasite within a herd. In buffalo, vertical transmission is also described; moreover, although there are indications that N. caninum may be associated with abortion in dairy buffalo, the reproductive importance of neosporosis is apparently lower in buffalo relative to cattle. Most studies have identified a higher time of exposition to N. caninum oocysts relative to age. The household system was also described as a risk factor for infection, possibly due to persistent contact between the home-raised buffalo and canids. The fetal immune competence of buffalo is similar to bovine, and buffalo fetus are highly susceptible to infection during the first trimester of pregnancy, indicating that N. caninum may be an abortigenic agent in buffaloes. Alternatively, it is interesting to note there is evidence that the inflammatory response in pregnant buffalo infected with N. caninum is mild enough to avoid abortion in most cases. It is proposed that the possible transmission of toxoplasmosis through unprocessed milk and buffalo meat may occur, which is important in terms of public health. Additionally, there is strong evidence to suggest that N. caninum may be associated with abortion in buffalo.

Dataset Information

Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine.

Publications

Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine.

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