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Biomedical subjects

M S Chauhan

Publications and source records attributed to M S Chauhan.

At least 19 recordsLinked to original sources
PubMed2007

Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos.

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.

Animals↗
PubMed2006

Repeated ultrasound-guided transvaginal oocyte retrieval from cyclic Murrah buffaloes (Bubalus bubalis): oocyte recovery and quality.

The present study was undertaken to explore the potential of the Murrah breed of buffaloes as donors of oocytes and to find out the recovery rate and oocyte quality in cyclic Murrah buffaloes subjected to oocyte recovery once a week. Murrah buffaloes (n = 5) were synchronized for estrus by a single prostaglandin injection schedule. The animals were subjected to transvaginal oocyte retrieval (TVOR) once weekly for 6 weeks, starting from Day 7 of the oestrous cycle (Day 0 = day of oestrus). TVOR was performed using an ultrasound machine with a 5 MHz transvaginal transducer, single lumen 19-gauge, 60 cm long needle and a constant vacuum pressure of 50 mmHg. The number and size of follicles in each ovary was determined before puncture. The follicles were characterized on the basis of their diameter as small (3-5 mm), medium (6-9 mm) and large (> or = 10 mm). The oocytes recovered were classified as grade A, cumulus-oocytes complexes (COCs) with > or = 5 layers of cumulus cells; grade B, those with two to four layers; grade C, partially denuded oocytes; and grade D, completely denuded oocytes. The mean (+/- S.E.M) number of small, medium and large follicles, and the number of total follicles observed per animal per session, which was 2.2 +/- 0.3, 0.6 +/- 0.2, 0.9 +/- 0.1 and 3.7 +/- 0.3, respectively, did not differ between animals or between puncture sessions. Small follicles constituted a major proportion (59%) of the total observed follicles. A mean (+/- S.E.M) number of 3.0 +/- 0.3 follicles were punctured and 2.0 +/- 0.3 oocytes recovered per animal per session, with a recovery rate of 68%. Out of the total 61 oocytes recovered, 36 (59%) were of grades A + B whereas 25 (41%) were of grades C + D. In conclusion, this study describes the potential of cyclic Murrah buffaloes as donors of oocytes collected by repeated TVOR once a week, without any adverse effects on follicular growth and oocyte recovery. It also describes an efficient system for carrying out TVOR in buffaloes.

Animals↗
PubMed2004

Xenogenous fertilization of equine oocytes following recovery from slaughterhouse ovaries and in vitro maturation.

The in vitro production (IVP) of equine embryos using currently available protocols has met limited success; therefore investigations into alternative approaches to IVP are justified. The objective of this study was to evaluate the feasibility of xenogenous fertilization and early embryo development of in vitro matured (IVM) equine oocytes. Follicular aspirations followed by slicing of ovarian tissue were performed on 202 equine ovaries obtained from an abattoir. A total of 667 oocytes (3.3 per ovary) were recovered from 1023 follicles (recovery rate, 65%). Oocytes underwent IVM for 41 +/- 2 h (mean +/- S.D.), before being subjected to xenogenous gamete intrafallopian transfer (XGIFT). An average of 13 +/- 0.8 oocytes and 40x10(3) spermatozoa per oocyte were transferred into 20 oviducts of ewes. Fourteen percent of transferred oocytes (36/259) were recovered between 2 and 7 days post-XGIFT and 36% of those recovered displayed embryonic development ranging from the 2-cell to the blastocyst stage. Fertilization following XGIFT was also demonstrated by the detection of zinc finger protein Y (ZFY) loci. Ligation of the uterotubal junction (UTJ), ovarian structures, or the duration of oviductal incubation did not significantly affect the frequency of embryonic development or recovery of oocytes/embryos after XGIFT. In conclusion, equine embryos can be produced in a smaller non-equine species that is easier for handling.

Animals↗
PubMed2002

Production of buffalo (Bubalus bubalis) embryos in vitro: premises and promises.

Techniques for in vitro production (IVP) of buffalo embryos adopting the procedures developed in cattle have received increasing interest in the recent times. A high oocyte maturation, fertilization and cleavage rate and a low rate of blastocyst yield and calving following transfer of in vitro produced buffalo embryos have been obtained. The efficiency of IVP in buffalo is much lower than that in cattle. Several problems need to be resolved before IVP technology can be used regularly in buffalo breeding. This review attempts to present an overview of the different techniques used in buffalo to produce transferable embryos in vitro, namely in vitro maturation and fertilization of immature oocytes and in vitro development of the resulting cleaved embryos to the blastocyst stage before transfer. The problems associated with IVP, the possible solutions and the new biotechniques linked to IVP are discussed.

Animals↗
PubMed2001

Stent thrombosis in the modern era: a pooled analysis of multicenter coronary stent clinical trials.

BACKGROUND: There are limited studies of stent thrombosis in the modern era of second-generation stents, high-pressure deployment, and current antithrombotic regimens. METHODS AND RESULTS: Six recently completed coronary stent trials and associated nonrandomized registries that enrolled 6186 patients (6219 treated vessels) treated with >/=1 coronary stent followed by antiplatelet therapy with aspirin and ticlopidine were pooled for this analysis. Within 30 days, clinical stent thrombosis developed in 53 patients (0.9%). The variables most significantly associated with the probability of stent thrombosis were persistent dissection NHLBI grade B or higher after stenting (OR, 3.7; 95% CI, 1.9 to 7.7), total stent length (OR, 1.3; 95% CI, 1.2 to 1.5 per 10 mm), and final minimal lumen diameter within the stent (OR, 0.4; 95% CI, 0.2 to 0.7 per 1 mm). Stent thrombosis was documented by angiography in 45 patients (0.7%). Clinical consequences of angiographic stent thrombosis included 64.4% incidence of death or myocardial infarction at the time of stent thrombosis and 8.9% 6-month mortality. CONCLUSIONS: Stent thrombosis occurred in <1.0% of patients undergoing stenting of native coronary artery lesions and receiving routine antiplatelet therapy with aspirin plus ticlopidine. Procedure-related variables of persistent dissection, total stent length, and final lumen diameter were significantly associated with the probability of stent thrombosis. Continued efforts to eliminate this complication are warranted given the serious clinical consequences.

Aspirin↗
PubMed2001

Coronary artery stenting in the aged.

OBJECTIVES: The study compared the safety and efficacy of coronary artery stenting in aged and nonaged patients and identified predictors of adverse clinical outcomes. BACKGROUND: Limited data are available on the outcomes of stenting in the aged (> or = 80 years) compared to nonaged patients. METHODS: The study was a pooled analysis of 6,186 patients who underwent coronary artery stenting in six recent multicenter trials. A clinical events committee adjudicated clinical end points, and quantitative angiography was performed by an independent core laboratory. RESULTS: There were 301 (4.9%) aged patients (> or = 80 years). Compared to nonaged patients, aged patients had a higher prevalence of multivessel disease (16.5% vs. 9.6%, p = 0.001), unstable angina (50.8% vs. 42.1%, p = 0.003), moderate to severe target lesion calcification (30.4% vs. 15.3%, p = 0.001) and smaller reference vessel diameter (2.90 mm vs. 2.98 mm, p = 0.004). Procedural success rate (97.4% vs. 98.5%, p = 0.14) was similar in the two groups. In-hospital mortality (1.33% vs. 0.10%, p = 0.001), bleeding complications (4.98% vs. 1.00%, p < 0.001) and one-year mortality (5.65% vs. 1.41%, p < 0.001) were significantly higher for the aged patients. Clinical restenosis was similar for the two groups (11.19% vs. 11.93%, p = 0.78). Advanced age, diabetes, prior myocardial infarction and presence of three-vessel disease were independent predictors of long-term mortality. CONCLUSIONS: Coronary artery stenting can be performed safely in patients > or = 80 years of age, with excellent acute results and a low rate of clinical restenosis, albeit with higher incidences of in-hospital and long-term mortality, and vascular and bleeding complications compared to nonaged patients.

Age Factors↗
PubMed2000

Percutaneous coronary revascularization in elderly patients: impact on functional status and quality of life.

BACKGROUND: Percutaneous coronary intervention (PCI) is frequently performed in elderly patients, but little is known about its impact on overall health and quality of life. OBJECTIVE: To examine changes in health-related quality of life among elderly patients after PCI. DESIGN: Observational study. SETTING: 75 U.S. hospitals. PATIENTS: Participants in two clinical trials of PCI. MEASUREMENTS: Health-related quality of life was assessed by using the Medical Outcomes Study Short Form (SF-36) survey and the Seattle Angina Questionnaire at baseline, 6 months, and 1 year. RESULTS: Serial data on health-related quality of life were available for 295 elderly (> or =70 years) and 1150 nonelderly (<70 years) patients. At 6 months, physical health had improved in 51% of elderly patients and mental health had improved in 29%. Cardiovascular-specific health status had improved in 58% to 75% of elderly patients. Improvement did not significantly differ between elderly and non-elderly patients at 6 months or 1 year. CONCLUSIONS: Elderly patients selected for participation in a trial of PCI had substantial improvements in health-related quality of life after PCI that were similar to those in younger patients.

Activities of Daily Living↗
PubMed1999

Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct.

The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 +/- 0.1) than for grade B (0.8 +/- 0.1), C (0.6 +/- 0.1), or D (0.3 +/- 0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos.

Animals↗
PubMed1998

Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro.

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.

Animals↗
PubMed1998

In vitro maturation and fertilization, and subsequent development of buffalo (Bubalus bubalis) embryos: effects of oocyte quality and type of serum.

In Experiment 1, to determine the developmental potential of buffalo oocytes of different qualities, compact cumulus-oocyte complexes (COCs) with an unexpanded cumulus mass, and with homogeneous ooplasm were classified as Grade 1 (with > or =5 layers of cumulus cells) and Grade 2 less than 4 layers of cumulus cells). Grade-3 oocytes were either without cumulus cells or with expanded cumulus mass, and with irregular ooplasm. The oocytes were matured for 24 h at 38.5 degrees C, 5% CO2 in air in maturation medium (10% fetal bovine serum (FBS) in TCM-199 supplemented with 5 microg mL(-1) follicle stimulating hormone-P). The nuclear maturation and cleavage rates, and the proportion of cleaved embryos which developed to morula and blastocyst stage were in the order Grade 1>Grade 2>Grade 3 (P < 0.05). For Experiment 2, the maturation medium consisted of TCM-199 supplemented with one of the following sera at 10% concentration: (1) buffalo oestrus serum (BOS), (2) superovulated buffalo serum (SBS), (3) fetal bovine serum (FBS) and (4) steer serum (SS). After in vitro fertilization (IVF), the oocytes were co-cultured with buffalo oviductal epithelial cells in TCM-199 containing the respective sera at 10% concentration for the subsequent 9 days. The extent of cumulus expansion and nuclear maturation were not different among different groups. The cleavage rates were lower (P < 0.05) with FBS than with BOS, SBS and SS. The proportion of cleaved embryos which developed to blastocyst stage was higher (P < 0.05) with SBS than with BOS, FBS and SS.

Animals↗