scholarly journals 209FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 226 ◽  
Author(s):  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
L. Anel ◽  
P. Bartels
Keyword(s):  
Plasma Membrane ◽  
Assisted Reproduction ◽  
Phase Contrast ◽  
Egg Yolk ◽  
Phase Contrast Microscopy ◽  
White Rhinoceros ◽  
Contrast Microscopy ◽  
Before And After ◽  
Epididymal Spermatozoa

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4h. The cauda epididymis was dissected out and flushed with 2mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1mL aliquot was removed for analysis and the balance (9mL; 2mL fraction A+7mL sperm sample) mixed with an additional 27.2mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2mL of cooled fraction B were added (final sperm concentration=150×106mL−1). Sperm samples were loaded into 0.25-mL straws and frozen over LN2 vapor (4cm for 20min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH) and HEPES saline (197mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5min at 600×g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50ngmL−1 in HEPES saline;; 10min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5μM in HEPES saline;; 30min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100mOsm/kg HEPES saline (1:20; 15min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.

In vitro Maturation of the Exoerythrocytic Stage of Plasmodium knowlesi Observed under Phase Contrast Microscopy

1990 ◽  
Vol 76 (6) ◽  
pp. 923 ◽  
Author(s):  
Pascal Millet ◽  
William E. Collins ◽  
Claude E. Monken ◽  
Bobby G. Brown
Keyword(s):  
Phase Contrast ◽  
In Vitro Maturation ◽  
Plasmodium Knowlesi ◽  
Phase Contrast Microscopy ◽  
Contrast Microscopy

Counting dead spermatozoa in frozen semen

1965 ◽  
Vol 7 (1) ◽  
pp. 59-65 ◽  
Author(s):  
H. R. L. Buttle ◽  
J. L. Hancock ◽  
A. F. Purser
Keyword(s):  
Analysis Of Variance ◽  
Sodium Fluoride ◽  
Phase Contrast ◽  
Egg Yolk ◽  
The Other ◽  
Phase Method ◽  
Phase Contrast Microscopy ◽  
Frozen Semen ◽  
Contrast Microscopy ◽  
Differential Counts

SUMMARYThree methods were used to make differential counts of living and dead bull spermatozoa in samples of frozen semen in an egg-yolk citrate medium containing glycerol.With two methods (‘phase’ and ‘nigrosin’ methods) dead spermatozoa were identified by their altered structure. With a third method (nigrosineosin) dead spermatozoa were identified by their staining affinity. With the phase method spermatozoa were immobilised by treatment with approximately M/40 sodium fluoride and were examined by phase-contrast microscopy in unfixed wet preparations. With the other two methods the spermatozoa were examined in smears stained either with nigrosin alone (nigrosin method) or with nigrosin and eosin (nigrosin-eosin method).Analysis of variance of the results of a factorial experiment involving fluoride-treated and untreated samples from 6 bulls with the three methods showed that differences between semen samples contributed 66% of the variation. A defect of the phase method was that the variance between counts was greater than the theoretically expected value.All spermatozoa in nigrosin-eosin stained preparations were stained with eosin within a few days of the smears being made, so that living and dead spermatozoa could not be distinguished by their differing affinities for eosin. Repeat counts on nigrosin-stained preparations did not differ significantly from counts made several days previously. Sodium fluoride in the concentration used here (M/40) tended to reduce the percentage of dead spermatozoa.

Microtubules and parental genome organisation during abnormal fertilisation in humans

Zygote ◽  
2002 ◽  
Vol 10 (3) ◽  
pp. 223-228 ◽  
Author(s):  
Vanesa Yanina Rawe ◽  
Santiago Brugo Olmedo ◽  
Florencia Noemí Nodar ◽  
Alfredo Daniel Vitullo
Keyword(s):  
Phase Contrast ◽  
Polar Body ◽  
Phase Contrast Microscopy ◽  
Genome Organisation ◽  
Contrast Microscopy ◽  
Chromatin Configuration ◽  
Second Polar Body ◽  
Main Pattern ◽  
Human Zygotes

We analysed the distribution of β-tubulins, acetylated α-tubulins and chromatin configuration in 113 human zygotes showing abnormal fertilisation, 16-18 h after conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). After a first characterisation using phase contrast microscopy, immunofluorescence staining was performed in 67 IVF and 46 ICSI zygotes that developed one, three or more pronuclei and/or subnuclei, with or without extrusion of the second polar body. Independently of the number of pronuclei found, β-tubulins were uniformly distributed throughout the cytoplasm of the abnormal zygotes. We did not observe any kind of microtubule alteration with respect of the ploidy level and/or its origin. The most frequent abnormal fertilisation pattern found after IVF was the presence of three or four pronuclei (74.6%). On the other hand, the presence of one pronucleus (63.0%) was the main pattern found after ICSI. No differences between the two groups were seen in terms of development of subnuclei. Anamolies detected after IVF and ICSI showed different aetiologies such as parthenogenetic activation, gynogenetic or androgenetic development, as well as digynic or diandric fertilisation.

scholarly journals Comparative Morphologic and Morphometric Study on the Developmental Aspects of In Vitro and In Vivo Reared Echino-coccus granulosus Sensu Stricto Using Differential Interference Contrast (DIC)/Nomarski and Phase Contrast Microscopy

2019 ◽  
Author(s):  
Seyedeh Faezeh SADJJADI ◽  
Mina MOTAMEDI ◽  
Tahereh MOHAMMADZADEH ◽  
Seyed Mahmoud SADJJADI
Keyword(s):  
Phase Contrast ◽  
Sensu Stricto ◽  
Whole Body ◽  
Phase Contrast Microscopy ◽  
Differential Interference Contrast ◽  
Morphometric Characters ◽  
Reproductive Structures ◽  
Contrast Microscopy

Background: Echinococcus granulosus is a zoonotic parasite with worldwide distribution. The present study focused on comparative morphologic and morphometric observations on the developmental aspects of whole body, more special the reproductive structures of in vitro reared adult worms (RAW) and in vivo reared adult worms in definitive host (AWIDH) using differential interference contrast (DIC)/Nomarski, phase contrast and routine optical microscopy. Methods: A total number of 10 in vitro and 10 in vivo reared adult worms of E. granulosus sensu stricto, G1 strain were selected. The worms were processed by Formaldehyde-Alcohol-Azocarmine-Lactophenol (FAAL). The details of morphological factors and reproductive structures of each worm including 25 biometrical parameters were studied by routine optical, phase contrast and Nomarski microscopy. The details of the samples were photographed, measured and analyzed. The fine structures of the parasite including the details of cirrus sac and developmental stages in different strobila were more obvious observing by Nomarski microscopy.  Results: The morphometric characters in the RAW and AWIDH showed that length of immature proglottid, length of mature proglottid, length of suckers are larger in RAW than AWIDH worms with statistical difference. Characters in E. granulosus of RAW and AWIDH showed that total number of segments, number of mature segments and the total number of testes were greater in RAW than AWIDH worms; while only the number of mature segments was statistically different is two groups. Conclusion: Application of DIC/Nomarski and phase contrast microscopy together with morphometric criteria are useful means for comparing the developmental aspects of in vitro and in vivo reared adults of E. granulosus.

scholarly journals Evaluation of Roflumilast (BCRP inhibitor) and Methotrexate (BCRP substrate) on Viability of Primary Squamous Cell Carcinoma – An in vitro Study

2019 ◽  
Vol 12 (2) ◽  
pp. 683-687
Author(s):  
Miss Pamila ◽  
Ramya Sugumar ◽  
Darling Chellathai David
Keyword(s):  
Cell Cycle ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Line ◽  
Phase Contrast ◽  
Phase Contrast Microscopy ◽  
Primary Squamous Cell Carcinoma ◽  
Cycle Arrest ◽  
Contrast Microscopy

In this study we evaluated the possible beneficial drug- interaction between Roflumilast (BCRP inhibitor) and Methotrexate (BCRP substrate) on viability of primary squamous cell carcinoma cell line using an in vitro technique. The KB cell line was treated with Roflumilast and Methotrexate to evaluate its anticancer activity using MTT assay. Image analysis under phase contrast microscopy was performed and flow-cytometry was done to see for cell cycle arrest as a result of drug treatment. Cell viability gradually decreased with the increasing concentrations of roflumilast, methotrexate and the cytotoxic effect with the combination of roflumilast and methotrexate also increased proportionally. Phase contrast microscopy indicated characteristic features of apoptosis which was confirmed in flow cytomtery and indicated cell cycle arrest in M phase. Efflux pump mediated multidrug resistance being a common feature among all cancers, the results of our study evidence the use of combined methotrexate and roflumilast to overcome drug resistance by exploiting the fact that the former is a BCRP substrate and latter a BCRP inhibitor. By combining the two drugs, it allows optimization of therapy by dose reduction of methotrexate and roflumilast and thereby resulting in better efficacy.

scholarly journals Pixantrone, a new anticancer drug with the same old cardiac problems? An in vitro study with differentiated and non-differentiated H9c2 cells

2018 ◽  
Vol 11 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Ana Reis-Mendes ◽  
Marisa Alves ◽  
Félix Carvalho ◽  
Fernando Remião ◽  
Maria Lourdes Bastos ◽  
...  
Keyword(s):  
Anticancer Drug ◽  
Phase Contrast ◽  
Published Data ◽  
Phase Contrast Microscopy ◽  
H9c2 Cells ◽  
Nuclear Condensation ◽  
Reduction Assay ◽  
Contrast Microscopy ◽  
Mtt Reduction

Abstract Pixantrone (PIX) is an anticancer drug approved for the treatment of multiple relapsed or refractory aggressive B-cell non-Hodgkin’s lymphoma. It is an aza-anthracenedione synthesized to have the same anticancer activity as its predecessors, anthracyclines (e.g. doxorubicin) and anthracenediones (e.g. mitoxantrone), with lower cardiotoxicity. However, published data regarding its possible cardiotoxicity are scarce. Therefore, this work aimed to assess the potential cytotoxicity of PIX, at clinically relevant concentrations (0.1; 1; and 10 µM) in both non-differentiated and 7-day differentiated H9c2 cells. Cells were exposed to PIX for 48 h and cytotoxicity was evaluated through phase contrast microscopy, Hoescht staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. Cytotoxicity was observed in differentiated and non-differentiated H9c2 cells, with detached cells and round cells evidenced by phase contrast microscopy, mainly at the highest concentration tested (10 µM). In the Hoechst staining, PIX 10 µM showed a marked decrease in the number of cells when compared to control but with no signs of nuclear condensation. Furthermore, significant concentration-dependent mitochondrial dysfunction was observed through the MTT reduction assay. The NR assay showed similar results to those obtained in the MTT reduction assay in both differentiated and non-differentiated H9c2 cells. The differentiation state of the cells was not crucial to PIX effects, although PIX toxicity was slightly higher in differentiated H9c2 cells. To the best of our knowledge, this was the first in vitro study performed with PIX in H9c2 cells and it discloses worrying cytotoxicity at clinically relevant concentrations.

Cryopreservation of feline epididymal spermatozoa from dead and alive animals and its use in assisted reproduction

Zygote ◽  
2009 ◽  
Vol 18 (1) ◽  
pp. 1-8 ◽  
Author(s):  
N. Cocchia ◽  
F. Ciani ◽  
R. El-Rass ◽  
M. Russo ◽  
G. Borzacchiello ◽  
...  
Keyword(s):  
Genetic Resources ◽  
Assisted Reproduction ◽  
Genetic Material ◽  
Egg Yolk ◽  
Domestic Cat ◽  
Long Term Storage ◽  
Sperm Sample ◽  
Wild Felid ◽  
Epididymal Spermatozoa

SummaryCryopreservation of gametes is an important tool in assisted reproduction programmes; long-term storage of oocytes or spermatozoa is necessary when in vitro fertilization (IVF) or artificial insemination is to be performed at a future date. Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of endangered populations. The objectives of this work were to: (1) examine sperm motility, viability, abnormality and acrosome integrity of frozen–thawed domestic cat epididymal spermatozoa; and (2) evaluate the same cryopreservation method on wild feline spermatozoa, needed to preserve their genetic resources. Epididymides were collected from 20 domestic cats during routine neutering procedure and from two wild felines at autopsy. The sperm samples, diluted with 4% glycerol/Tris/egg yolk, were loaded into 0.25 ml mini-straws, exposed to nitrogen vapour and stored in liquid nitrogen. After 4 weeks, samples were thawed and re-evaluated. The quality of each fresh and frozen–thawed sperm sample was tested by determining the motility (54.7 ± 11.3% and 32 ± 13.1% respectively for cat spermatozoa; 38.3 ± 18.7% and 21.5 ± 16.8% respectively for tiger spermatozoa), viability (74.3 ± 8.6% and 45.2 ± 9.4% respectively for cat spermatozoa; 42.4 ± 14.5% and 33.5 ± 12.9% respectively for wild felid spermatozoa), morphology and acrosomal status. The present study showed that feline epididymal spermatozoa can be frozen in egg-yolk extender with 4.0% glycerol in 0.25 ml straws. The procedure used in the present study for epididymal cat sperm cryopreservation may be applied to bank the genetic resources of wild felid species.

Fine structure of the walls of the conidia of various fungi and of the conidiophore of Fomes annosus

1969 ◽  
Vol 17 (1) ◽  
pp. 13 ◽  
Author(s):  
G Scurfield ◽  
Costa EWB Da
Keyword(s):  
Fine Structure ◽  
Phase Contrast ◽  
Phase Contrast Microscopy ◽  
Type Ii ◽  
Surface Electron ◽  
Opposite Pole ◽  
Conidium Formation ◽  
Contrast Microscopy ◽  
Before And After ◽  
Circular Arrangement

Light and phase contrast microscopy were used to investigate the structure and mode of formation of the conidia of various fungi. Conidial walls were examined with the electron microscope before and after chemical treatment of the walls to remove surface electron-opaque material. Two sorts of microfibrillar arrangement occurred: (i) a circular arrangement about an electron-opaque papilla at one pole which gives way to a more disperse arrangement at the opposite pole (Fomes annosus); (ii) a disperse arrangement (Pithomyces chartarum, Hormodendron resinae, Aschochyta pisi, Colleto- trichum lindemuthianurn). Conidial wall texture did not appear to be related to conidial ontogeny. Type (ii) conidia had perforate end walls. The occurrence of such walls is discussed in relation to spore formation and germination. The possibility that conidium formation involves the sequence: arrestment of growth →altered chemical composition and/or microfibrillar wall texture → swelling → conidium formation - is discussed on the basis of the microfibril arrangements in the conidiophore of Fomes annosus.

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