Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and ß-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

Successful transplantation of transfected enriched buffalo (Bubalus bubalis) spermatogonial stem cells to homologous recipients.

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ±â€¯4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ±â€¯0.25%) and lipofectamine 3000 (15.57 ±â€¯0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.

Utility of 18 F-FDG PET/CT in pre-surgical risk stratification of patients with breast cancer.

OBJECTIVE: To determine the correlation between fluorine-18-fluorodeoxyglucose (18F-FDG) uptake values and clinicopathological prognostic markers using preoperative 18F-FDG positron emission tomography/computed tomography (PET/CT) in primary breast cancer (BC). SUBJECTS AND METHODS: One hundred and twelve patients with primary BC were studied prospectively. Pretreatment 18F-FDG PET/CT was performed. Maximum standardized uptake values (SUVmax) were compared with various clinicopathological variables. RESULTS: In a univariate analysis, SUVmax correlated well with the following prognostic variables: T stage, absence of progesterone receptor (PR), absence of estrogen receptor (ER), triple negative lesions (ER/PR and Her 2 negative) and high histologic grade. Metastatic lesions and ductal lesions had higher SUVmax than lobular carcinoma. No significant correlation was found between SUVmax,and human epidermal growth factor receptor 2 (Her-2) statusor perineural and lymphovascular invasion. Multivariate analyses showed that breast density, tumor size and PR negativity were significantly correlated with SUVmax (P=0.046 and 0.009, respectively). CONCLUSION: The pre-treatment tumor SUVmax could be utilized as an independent imaging biomarker of the tumor aggressiveness and poor prognosis. Risk stratification based on this index could play a pivotal role in alteration of treatment planning, such as neoadjuvant chemotherapy (precision oncology).

Calcium ionophore enhanced developmental competence and apoptotic dynamics of goat parthenogenetic embryos produced in vitro.

Parthenogenetically developed embryos are efficient sources of in vitro embryo production, having less ethical issue and being useful for investigating culture conditions/treatments, early developmental, genomic studies, and homonymous source of stem cells. Keeping its advantages in mind, we aimed to study the effects of different activating agents on embryo production and its quality and gene expression. In the present study, 1348 immature oocytes recovered were parthenogenetically developed to embryos. Usable-quality immature oocytes were collected by puncturing the surface follicles and matured in in vitro maturation (IVM) medium for 27 h in a humidified 5% CO2 incubator at 38.5°C. The matured oocytes were parthenogenetically activated by exposure to 5 µM calcium ionophore for 5 min or 7% ethanol for 7 min sequentially followed by 4 h incubation in 2 mM 6-DMAP and then in vitro cultured (IVC) in RVCL/G-2 medium for 8 days. Matured oocytes were activated by calcium ionophore, the cleavage rate observed was 76.67 ± 3.47%, and further they developed into 4-cell, 8-16-cell, morula, blastocyst, and hatched blastocyst with 85.30 ± 1.57%, 70.60 ± 2.00%, 45.05 ± 2.66%, 22.89 ± 2.40%, and 5.70 ± 1.97%, respectively. Whereas ethanol-activated oocytes showed cleavage rate of 87.60 ± 1.70% and further culture developed into 4-cell, 8-16 cell, morula, blastocyst, and hatched blastocyst with 86.14 ± 1.03%, 71.56 ± 2.21%, 40.90 ± 2.45%, 19.02 ± 1.26%, and 2.22 ± 0.38%, respectively. Blastocyst developed from calcium ionophore-activated oocytes showed significantly (P < 0.05) higher total cell number (282.25 ± 27.02 vs 206.00 ± 40.46) and a lower apoptotic index (2.42 ± 0.46 vs 4.07 ± 1.44) than blastocyst developed from ethanol-activated oocytes. The relative expression of anti-apoptotic genes (BCL2, BCL2A1, MCL) at different stages of embryos produced by either calcium ionophore or ethanol activation was found to be increased in earlier stages and decreased in later stages of embryonic development. Similarly, when these embryos were subjected to pro-apoptotic genes (BAX, BAD, BAK), expression was found to be slightly higher in blastocysts than other stages. This study shows that calcium ionophore-activated blastocysts were developmentally more competent than the ethanol-activated blastocysts.

Tumor characteristics and metabolic quantification in carcinoma breast: An institutional experience.

BACKGROUND: In India, carcinoma breast is the most common cancer among urban women population and second most common cancer after carcinoma cervix in rural areas. One in 22 women in India develops carcinoma of the breast in their lifetime. Fluorine-18-fluoro-2-deoxy-D-glucose (18F-FDG) uptake in breast cancer usually indicates the degree of tumor metabolism and hence can predict its behavior and prognosis. On the other hand, the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) or neu state of breast cancer is a biomarker that provides important prognostic information in addition to predicting response to therapy. AIMS: The main objective of this study is to assess whether a correlation exists between 18F-FDG uptake in untreated cases of breast cancer, their receptor status (ER, PR, and HER-2 or neu), tumor histology, and tumor size. SUBJECTS AND METHODS: Sixty consecutive female patients, with biopsy-proven primary breast cancer, were enrolled in this prospective study for whom 18F-FDG positron emission tomography-computed tomography scan was done in the Department of Nuclear Medicine. Results obtained were analyzed using appropriate statistical tests (t-test and Pearson Chi-square tests), and interpretation was made with 95% confidence level. RESULTS: In our series, a positive correlation between tumor size, high tumor grade, and standardized uptake value (SUV) was found. Tumors with positive receptor status for estrogen, progesterone, and HER-2/neu receptors had statistically insignificant lower maximum SUV (SUVmax) values than their negative counterparts. Triple-negative breast tumors (ER-, PR-, and no overexpression of HER-2/neu) are currently a subject of major interest because of their aggressiveness, poor prognosis, and lack of targeted therapy. Based on receptor status when the SUVmaxof the group with triple-negative receptor status (ER-/PR-/HER-2/neu-) was compared to rest of the patient group, it was seen that patients with negative receptor status had significantly higher mean SUVmaxvalues. CONCLUSIONS: We have inferred that in patients with breast cancer, various biological parameters such as tumor size, grade, histology, and hormonal receptor status have different impact on tumor metabolic activity.

Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation.

In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

Effect of TGF-ß1 superfamily members on survival of buffalo (Bubalus bubalis) embryonic stem-like cells.

This study examined the effects of supplementation of ES-like cell culture medium with bone morphogenetic protein (BMP)-4 (0, 10, 20 or 100 ng/ml) or Noggin (250, 500 or 750 ng/ml) or TGF-ß1 (0, 0.1, 1 or 10 ng/ml) or SB431542 (0, 10, 25 or 50 µm), an inhibitor of TGF-ß1 signalling, on survival, colony area and expression level of pluripotency genes in buffalo ES-like cells at passage 40-80, under different culture conditions. BMP-4 supplementation significantly reduced (p < 0.05) colony survival rate, percentage increase in colony area and relative mRNA abundance of OCT4, whereas that of NANOG and SOX-2 was increased significantly (p < 0.05). Noggin supplementation did not affect the colony survival rate and percentage increase in colony area in the presence of FGF-2 and LIF. In the presence of FGF-2 alone, it significantly reduced (p < 0.05) the relative mRNA abundance of OCT4 and SOX-2 and increased (p < 0.05) that of NANOG. Supplementation with TGF-ß1 at 1.0 ng/ml but not at other concentrations increased colony survival rate but had no effect on percentage increase in colony area at any concentration. Supplementation with SB-431542 decreased (p < 0.05) colony survival rate at 50 µm but not at other concentrations. The percentage increase in colony area was lower (p < 0.05) with 10 µm SB-431542 than that in the controls, whereas at higher concentrations of 25 or 50 µm, SB-431542 decreased (p < 0.05) the colony size instead of increasing it. In conclusion, these results suggest that BMP-4 induces differentiation in buffalo ES-like cells, whereas TGF-ß/activin/nodal pathway may not be playing a crucial role in maintaining pluripotency in these cells.

Buffalo (Bubalus bubalis) ES cell-like cells are capable of in vitro skeletal myogenic differentiation.

When buffalo embryonic stem (ES) cell-like cells that expressed surface markers SSEA-4, TRA-1-60, TRA-1-81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX-1 and NUCLEOSTEMIN as confirmed by RT-PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three-dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF-68 and NESTIN (ectodermal lineage), BMP-4 and α-skeletal actin (mesodermal lineage), and α-fetoprotein, GATA-4 and HNF-4 (endodermal lineage). When these EBs were cultured on gelatin-coated dishes, they spontaneously differentiated to several cell types such as epithelial- and neuron-like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10(-8) M or 10(-7) M retinoic acid for 25 days, ES cells could be directed to form muscle cell-like cells, the identity of which was confirmed by expression of α-actinin by immunofluorescence and of MYF-5, MYOD and MYOGENIN genes by RT-PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell-like cells to undergo directed differentiation to cells of skeletal myogenic lineage.

Oxygen concentration and cysteamine supplementation during in vitro production of buffalo (Bubalus bubalis) embryos affect mRNA expression of BCL-2, BCL-XL, MCL-1, BAX and BID.

This study examined the effects of O(2) concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 µm, respectively; IVM, IVF and IVC carried out in 20% O(2)), on blastocyst rate and relative mRNA abundance of some apoptosis-related genes measured by real-time qPCR in immature and in vitro-matured buffalo oocytes and in embryos at 2-, 4-, 8- to 16-cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL-positive cells was significantly lower (p < 0.05) under 5% O(2) than that under 20% O(2). The mRNA expression of anti-apoptotic genes BCL-2 and MCL-1 was significantly higher (p < 0.05) and that of pro-apoptotic genes BAX and BID was lower (p < 0.05) under 5% O(2) than that under 20% O(2) concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL-XL and MCL-1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O(2) groups and in cysteamine supplemented vs controls. At the 8- to 16-cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL-2 and MCL-1 was highest under 5% O(2) concentration and that of BAX and BID was highest (p < 0.05) under 20% O(2) concentration. These results suggest that one of the mechanisms through which beneficial effects of low O(2) concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti-apoptotic and a decrease in the expression of pro-apoptotic genes.

Production of nuclear transfer embryos by using somatic cells isolated from milk in buffalo (Bubalus bubalis).

Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p < 0.01), than for MDF-NT embryos (16.44 ± 0.75%, 170.57 ± 4.50 respectively). We conclude that somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos.

Term placenta shows methylation independent down regulation of Cyp19 gene in animals with retained fetal membranes.

Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 gene encodes the aromatase enzyme responsible for catalyzing the rate limiting step in estrogen biosynthesis, an important hormone for placental maturation and expulsion. The present study was aimed for comparative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animals with and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples of RFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides of placenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of both PI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than promoter methylation is responsible for decreased aromatase expression in placental cotyledons of animals suffering from RFM.

Buffalo (Bubalus bubalis) embryonic stem cell-like cells and preimplantation embryos exhibit comparable expression of pluripotency-related antigens.

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin-C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages strongly expressed stage-specific embryonic antigen (SSEA)-4 but lacked expressions of SSEA-1 and SSEA-3. Putative ES cells also expressed tumour rejection antigen (TRA)-1-60, TRA-1-81 and Oct4. Whereas in all early embryonic stages, TRA-1-60 was observed only in the periplasmic space, and TRA-1-81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency-related surface antigen phenotype, which resembles that of the ICM.

Assessment of DNA damage during in vitro development of buffalo (Bubalus bubalis) embryos: effect of cysteamine.

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 µM, respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8- to 16-cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8- to 16- (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2-cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8- to 16-cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2-cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H2O2 caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.

Reproductive biotechniques in buffaloes (Bubalus bubalis): status, prospects and challenges.

The swamp buffalo holds tremendous potential in the livestock sector in Asian and Mediterranean countries. Current needs are the faster multiplication of superior genotypes and the conservation of endangered buffalo breeds. Recent advances in assisted reproductive technologies, including in vitro embryo production methodologies, offer enormous opportunities to not only improve productivity, but also to use buffaloes to produce novel products for applications to human health and nutrition. The use of molecular genomics will undoubtedly advance these technologies for their large-scale application and resolve the key problems currently associated with advanced reproductive techniques, such as animal cloning, stem cell technology and transgenesis. Preliminary success in the application of modern reproductive technologies warrants further research at the cellular and molecular levels before their commercial exploitation in buffalo breeding programmes.

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos.

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 microm cysteamine. Supplementation of IVM medium with 50 microm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 microm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 microm (P < 0.05) cysteamine, whereas 200 microm cysteamine was ineffective. Supplementation of both IVM medium with 50 microm cysteamine and of IVC medium with 100 microm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.

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.

Laboratory production of buffalo (Bubalus bubalis) embryos.

There is an increasing interest in large-scale in vitro production (IVP) of buffalo embryos through in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes for faster multiplication of superior germplasm. The recovery of total and usable quality oocytes from slaughterhouse ovaries is low in this species. The nuclear maturation rates of buffalo oocytes matured in the presence of follicular fluid or serum and hormones like luteinizing hormone, follicle-stimulating hormone and oestradiol vary from 70 to 80% and are comparable to those reported for cattle oocytes. However, with fertilization rates of 40-55%, and the yield of blastocysts at around 10-15%, the efficiency of IVP is much lower than that in cattle. The in vitro sperm preparation procedures and the systems employed for performing IVF and culture of zygotes up to blastocyst stage are suboptimal and need substantial improvements. The quality and viability of blastocysts produced need to be checked by cell count, and after transfer to synchronized recipients, for development of quality control standards.

Enlarging pulmonary histoplasmoma associated with pulmonary tuberculosis.

Histoplasmosis is not a frequently reported infection from India. Similarity of its clinical and radiological features with pulmonary tuberculosis, may cause a difficult diagnostic dilemma when the two occur together. We report a case who initially presented with cavitary lesions over right lung responding well to anti tuberculosis chemotherapy but subsequently a small opacity present over the left upper zone showed a relentless increase till it attained a massive size few years later. Operative removal and histopathological examination of the mass confirmed it to be histoplasmoma. She became sputum AFB smear positive and culture negative for a short while during this period and this is explained on the basis of previous reports.

In vitro capacitation of goat spermatozoa.

Goat epididymal and ejaculated spermatozoa were incubated in Krebs-Ringer bicarbonate buffer containing pyruvate and lactate as energy source. A 3 hr incubation for epididymal and 4 hr for ejaculated spermatozoa was required for the capacitation and acrosome reaction to take place. Calcium is an essential requirement which was needed for motility maintenance/activation and for the initiation of acrosome reaction. A 2-fold increase in cAMP content was measured over 3 hr period of incubation of goat epididymal spermatozoa which was not seen when calcium ions were either omitted or chelated with EGTA. There is thus a definite involvement of Ca2+ ions and cAMP in capacitation and acrosome reaction of goat spermatozoa.