Flow investigations were made on the slender body having a 3.5 calibre ogive nose and a length-to-diameter (
L
/
D
) ratio of 16 at an angle of attack of 50° and a freestream velocity of 17 m/s which ...corresponds to a diametric Reynolds number of 29,000. Experiments consisted of the measurement of velocity and pressure fluctuations at different stations of the slender body. Commercial software ANSYS Fluent was utilized for computations. The literature indicated that a rectangular cross-sectioned ring placed at a distance of 3.5 times the diameter from the tip was used to reduce the side force of the slender body at higher angles of attack. The present work aims to investigate the effect of the ring on the unsteadiness in the flow behaviour of the slender body at a high angle of attack. Results indicated that the use of a ring leads to the onset of a localized wake disturbance downstream of the ring. The existence of moderate and higher-order frequencies could be observed in the wake. Computed results too indicated the change in the flow behaviour due to the presence of rings.
Computational investigations were carried out on an ogive-cylinder configuration having a slenderness ratio of 7.5. The geometry of the nose tip was generated based on the physical geometry of the ...nose tip observed under a scanning electron microscope. Time-dependent simulations were performed on the slender body at a diameter Reynolds number of 3.0×104. Results indicated that the onset of vortex asymmetry was mainly due to the micro-tip imperfection existing at the tip. It was also observed that the variation in the roll angle of the model affected the flow physics largely which has been experimentally observed by several researchers in the past. The computed results were in better agreement with the referenced experimental data.
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.
Buffalo embryos were produced by handmade cloning using peripheral blood-derived lymphocytes as donor cells. Although the blastocyst rate was lower (P < 0.01) for lymphocyte- than control skin ...fibroblast-derived embryos (6.6 ± 0.84% vs. 31.15 ± 2.97%), the total cell number (152.6 ± 23.06 vs. 160.1 ± 13.25) and apoptotic index (6.54 ± 0.95 vs. 8.45 ± 1.32) were similar. The global level of H3K9ac was higher (P < 0.05) in lymphocyte- than that in skin-derived blastocysts; whereas in IVF blastocysts, the level was not significantly different from the two cloned groups. The level of H3K27me3 was similar among the three groups. The expression level of DNMT1, DNMT3a, HDAC1, and IGF-1R was higher (P < 0.01) in lymphocytes than that in skin fibroblasts. The expression level of CDX2 was higher (P < 0.05) than that of DNMT3a, IGF-1R, OCT4, and NANOG was lower (P < 0.05) in lymphocyte-derived than in IVF blastocysts; that of DNMT1 and HDAC1 was similar in the two groups. The expression level of all these genes, except that of NANOG, was lower (P < 0.05) in lymphocyte- than in skin fibroblast-derived blastocysts. It is concluded that, peripheral blood-derived lymphocytes can be used for producing handmade cloning embryos in bubaline buffaloes.
We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB+) or colorless cytoplasm (BCB−). The ...blastocyst rate was higher (p<0.001) for BCB+ than for BCB− oocytes (43.41±2.54 vs. 22.74±1.76%). BCB+ blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher (p<0.05) than that of BCB− blastocysts. The global level of H3K9me2, which was similar in BCB+ and BCB− blastocysts, was higher (p<0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher (p<0.05) and that of GATA2 was lower (p<0.05) in BCB+ than that in BCB− blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower (p<0.05) and that of CDX2 was higher (p<0.05) in BCB+ than in IVF blastocysts. In conclusion, because BCB+ blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB− blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.
We compared the cloning efficiency of buffalo embryos produced by handmade cloning (HMC) using ear skin- and milk-derived donor cells. The blastocyst rate was lower (p < 0.05) for milk-derived than ...that for skin-derived embryos, whereas the total cell number and apoptotic index were similar. The global level of H3K9ac was higher (p < 0.05) in skin- than in milk-derived cells, whereas the level of H3K27me3 was similar in the two groups. The global level of H3K9ac was similar between milk-derived and in vitro-fertilized (IVF) blastocysts, which was higher (p < 0.05) than that in skin-derived blastocysts. The level of H3K27me3 was similar among the three groups. The expression level of IGF-1R and G6PD was higher (p < 0.05) in skin- than in milk-derived cells, whereas DNMT1, DNMT3a, and HDAC1 expression level was similar. In the blastocysts, the expression level of DNMT1, HDAC1, OCT4, and CDX2 was higher (p < 0.05) in skin-derived than that in IVF blastocysts. The expression level of DNMT3a and IGF-1R, was in the order (p < 0.05) skin-derived and IVF > milk-derived blastocysts and that of NANOG was (p < 0.05) IVF-> milk-derived > skin-derived blastocysts. The expression level of all these genes, except NANOG, was lower (p < 0.05) in milk- than in skin-derived or IVF blastocysts. In conclusion, milk-derived cells can be used for producing HMC embryos of quality similar to that of skin-derived embryos, although with a lower blastocyst rate.
Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear ...transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.
The present study evaluated the effects of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2 and epidermal growth factor (EGF) on proliferation and the expression ...of some genes in spermatogonial cells. Spermatogonial cells were isolated from prepubertal buffalo testes and enriched by double enzyme treatment, filtration through 80- and 60-μm nylon mesh filters, differential plating on lectin-coated dishes and Percoll density gradient centrifugation. Cells were then cultured on a buffalo Sertoli cell feeder layer and formed colonies within 15–18 days. The colonies were found to predominantly contain undifferentiated Type A spermatogonia because they bound Dolichos biflorus agglutinin and did not express c-kit. The colonies expressed alkaline phosphatase, NANOG, octamer-binding transcription factor (OCT)-4 and tumour rejection antigen (TRA)-1–60. Cells were subcultured for 15 days, with or without growth factor supplementation. After 15 days, colony area and the relative mRNA abundance of PLZF were higher (P<0.05) following supplementation with 40ngmL-1 GDNF+10ngmL-1 EGF+10ngmL-1 FGF2 than with the same concentrations of GDNF alone or GDNF plus either EGF or FGF2. Expression of TAF4B was higher (P<0.05) in the presence of FGF2, whereas the expression of THY1 was not affected by growth factor supplementation. In the Sertoli cell feeder layer, EGF and FGF2 decreased (P<0.05), whereas GDNF increased (P<0.05), the relative mRNA abundance of ETV5 compared with control. In conclusion, an in vitro culture system that incorporates various growth factors was developed for the short-term culture of buffalo spermatogonia.
In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited ...dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.
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