23 Viability staining techniques for cryopreserved spermatozoa in 3 caudata species

2019 ◽  
Vol 31 (1) ◽  
pp. 137
Author(s):  
A. Gillis ◽  
K. Counsell ◽  
A. Julien ◽  
R. Marcec ◽  
A. Kouba ◽  
...  
Keyword(s):  
Cell Death ◽  
Nucleic Acid ◽  
Cell Viability ◽  
Fluorescent Dyes ◽  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Annexin V ◽  
Reproductive Technologies ◽  
Primary Analysis ◽  
Nucleic Acid Stain

Salamanders are the most threatened vertebrate taxa; thus, conservation-based research including spermatozoa cryopreservation and other assisted reproductive technologies is essential to their survival. To determine the effectiveness of sperm cryopreservation, methods for evaluating sperm quality are necessary but underdeveloped in caudate research. Evaluating motility has been the primary analysis for sperm viability but is difficult to perform due to the scythe-like morphology, slow rotating progression, and minute undulations of the tail membrane. Estimating apoptosis is a new approach to evaluating caudate spermatozoa survival through cryostress. Fluorescent dyes, such as SYBR-14, annexin-V, and propidium iodide (PI), are valuable tools for identifying degrees of cell viability, apoptosis, and necrosis. Annexin-V marks the externalization of phosphatidylserine on the cell membrane indicating early steps in the apoptosis signalling cascade. Compromised membranes allow PI, a nucleic acid stain, access to DNA, marking cellular necrosis. The SYBR-14 is a nucleic acid stain that permeatesssss intact membranes, labelling live cells. These fluorescent stains were assessed for marking viability and stages of cell death in post-thaw spermatozoa across 3 caudate species: the Eastern tiger salamander (Ambystoma tigrinum), Kweichow Emperor newt (Tylototriton kweichowensis), and black-spotted newt (Notophthalmus meridionalis). For each species, spermic urine samples were acquired by hormone treatment and frozen based on protocols developed in A. tigrinum, yielding an average of 18.2% relative motility recovered at thaw. Straws were thawed for 5min at 20°C. Viability was tested by staining 5μL 1:1 with a 1:50 dilution of SYBR-14 and 2μL of PI. Stages of cell death were evaluated by staining 10μL with 2μL of annexin-V and 2μL of PI. Cell viability was assessed immediately under a fluorescence microscope. For each of the 3 species, 2 samples were stained with both assays in triplicate. Sperm stained with SYBR-14 alone were considered viable, and sperm stained with any annexin-V or PI were considered not viable. Visible dynamic shifting from SYBR-14 to PI staining was observed in real time, indicating rapid necrosis. Morphological abnormalities, not observed in unstained samples, were prevalent across all species following staining, signifying a possible cytotoxic effect of the dyes. High mortality and abnormality rates suggest that fluorescent dyes have elevated toxicity and permeability in caudate sperm. Caudate spermic urine has a very low osmolality, implying high permeability, which could lead to rapid staining and toxicity effects. Shorter incubation times may be required for accurate staining. Results may also indicate that cryopreservation protocols need to be species specific and do not transfer well across taxa. This is one of the earliest studies to evaluate the use of fluorescent stain protocols on measuring cell viability in caudate sperm and indicates that further refinement is required.

scholarly journals Intrinsic and Extrinsic Factors Predicting the Cumulative Outcome of IVF / ICSI Treatment

2019 ◽  
Vol 9 (2S) ◽  
pp. 269-273 ◽  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Reproductive Technologies ◽  
Process Time ◽  
Success Rates ◽  
Embryo Viability ◽  
Extrinsic Factors ◽  
Response Level ◽  
Female Population ◽  
Incident Cases

Infertility rates in India becoming increased in last decade principally due to the urbanization conditions and the lifestyle habits. It is giving alarm by continuously reporting the progress in incident cases of infertility amongst the young Indian adults of both male and female population. Among the various Assisted Reproductive Technologies (ART) available today in the treatment of infertility, In Vitro Fertilization (IVF) is found to be the most applicable treatment method of choice. This involves the administration of different hormones and drugs to treat infertility. In the present scenario technically IVF treatment process is tedious, laborious, high cost and most importantly success rates reported to be very low (20-30%). The prediction of IVF success rates is becoming an important scientific knowledge and practice, which helps both the doctor and the candidate couple to know about the conditions hence to take the right decision. The accurate prediction of the IVF success rate is really a challenging task in obstetrics and gynecology medicine. The success rates of the IVF depends on the various factors such as Intrinsic factors i.e, Genetic predisposition, Age, Body mass Index, Hormonal balance, Embryo viability, Sperm quality, Endometriosis and overall patient’s response level of the candidate couple and the Extrinsic factors such as Medical equipment technology, Treatment methods, Personal experiences of clinicians and embryologists, Process time, Stress due to the lifestyle etc.

154 A method of oviductal semen deposition for use in the goat

2020 ◽  
Vol 32 (2) ◽  
pp. 203
Author(s):  
N. Buzzell ◽  
S. Blash ◽  
K. Miner ◽  
M. Schofield ◽  
J. Pollock ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
The United States ◽  
Reproductive Technologies ◽  
Poor Quality ◽  
Implant Removal ◽  
Suitable Alternative ◽  
Midline Laparotomy ◽  
Post Surgery

The objective of this study was to investigate a method of oviducal semen deposition as a strategy for producing offspring from poor-quality cryopreserved goat sperm. Invitro fertilisation (IVF) and AI are common assisted reproductive technologies used in small ruminants, but they have varied results in the goat. The use of poor-quality cryopreserved-thawed sperm (<50% live/dead ratio at post-thaw) can decrease the rate of success. These procedures were performed in the month of November in Central Massachusetts in the United States (42° N). Seven 10-year-old dairy goats (Saanen, Toggenburg, and Alpine breeds) were synchronised and superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, two daily injections of 36mg of FSH ~12h apart on Days 12-15, and progesterone implant removal on Day 14 followed by an injection of 50µg of gonadotrophin-releasing hormone. Sperm deposition was performed on Day 17 (72 h after implant removal). The animals were anaesthetised using a standardised protocol, intubated, and maintained using isoflurane, and sterile prep was performed before a midline laparotomy procedure. Straws from a single ejaculate from a transgenic founder that was cryopreserved using a commercial two-step glycerol-egg yolk-based extender were used. A straw from this collection was post-thawed 30 days after collection and, using a commercial live/dead stain, 67% live sperm was determined. The optimal type of sperm prep and sperm concentration is unknown and may be dependent on sperm quality. Therefore, different gradient preps using Vitrolife SpermGrad at three volumes (1.5 (used on two animals), 1.0, and 0.5mL) as well as two volumes of IVF Bioscience Bovine BO-SemenPrep (4.0mL (used on two animals) and 2.0mL) were used. All five pellets were diluted in 1.0mL of IVF Bioscience Bovine BO-IVF media. Sperm concentrations ranging from 75×106 to 27×106 spermmL−1 were deposited into one oviduct; then, a 10:1 dilution was performed and 7.5×106 to 2.7×10 spermmL−1 were deposited into the contralateral oviduct. The depositions were performed just proximal to the uterotubal junction in a volume of 0.1mL of diluent via a tuberculin syringe attached to a 20-gauge needle. Two days following the procedure, oviducts were flushed postmortem from three of the seven randomly selected goats. All three had fertilised embryos, and nineteen 8-cell embryos were retrieved. Three of these embryos were surgically transferred to the distal uterine horn of a suitable recipient. The recipient became pregnant and produced a single offspring. The remaining four of seven goats were killed 41 days post-surgery. Two of the four goats were pregnant, with one carrying one fetus and the other carrying five fetuses. Further studies are needed to optimise this method, but these initial results indicate that oviducal semen deposition directly into the oviduct proximal to the uterotubal junction may be a suitable alternative for producing offspring from suboptimal cryopreserved-thawed goat sperm.

scholarly journals From One Ejaculate to Another: Transference of Sperm Traits via Seminal Plasma Supplementation in the Ram

Biology ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 33
Author(s):  
Christine Green ◽  
Jessica P. Rickard ◽  
Simon P. de Graaf ◽  
Angela J. Crean
Keyword(s):  
Sperm Motility ◽  
Seminal Plasma ◽  
Perceived Risk ◽  
Positive Relationship ◽  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Reproductive Technologies ◽  
Before And After ◽  
The Difference ◽  
Sperm Traits

Males can adjust sperm motility instantaneously in response to the perceived risk of sperm competition. The speed of this response suggests that sperm motility is regulated by changes in seminal plasma rather than changes in the sperm cells themselves. Hence, here we test whether inter-ejaculate variation in seminal plasma can be used to alter sperm quality prior to use in assisted reproductive technologies. We supplemented fresh ejaculates of Merino rams with seminal plasma collected from previous ‘donor’ ejaculates to test whether changes in sperm kinetics were related to the relative quality of donor to focal ejaculates. We found a positive relationship between the change in sperm traits before and after supplementation, and the difference in sperm traits between the donor and focal ejaculate. Hence, sperm motility can be either increased or decreased through the addition of seminal plasma from a superior or inferior ejaculate, respectively. This positive relationship held true even when seminal plasma was added from a previous ejaculate of the same ram, although the slope of the relationship depended on the identity of both the donor and receiver ram. These findings indicate that seminal plasma plays a key role in the control and regulation of sperm kinetics, and that sperm kinetic traits can be transferred from one ejaculate to another through seminal plasma supplementation.

scholarly journals The use of insulin-like growth factor 1 improved the parameters of the seminogram in a patient with severe oligoasthenoteratozoospermia

2019 ◽  
Vol 7 ◽  
pp. 2050313X1983415
Author(s):  
José Arturo Mora Rodríguez ◽  
Leonardo M Porchia ◽  
Felipe Camargo ◽  
Esther López-Bayghen
Keyword(s):  
Growth Factor ◽  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Quality Parameters ◽  
Intradermal Injection ◽  
Sperm Parameters ◽  
Insulin Like Growth Factor ◽  
Progressive Motility

Male patients suffering from oligoasthenoteratozoospermia typically failed to achieve pregnancy, even with assisted reproductive technologies. Growth hormone and insulin-like growth factor 1 have been shown to regulate sperm quality parameters; therefore, the insulin-like growth factor 1 supplement could improve sperm parameters. Here, we determine the effect insulin-like growth factor 1 has on sperm parameters in a patient suffering from oligoasthenoteratozoospermia. A 47-year-old male was administered once a day 1.5 IU of insulin-like growth factor 1 by intradermal injection for 2 months. Seminogram analysis was performed before and after. Treatment with insulin-like growth factor 1 resulted in a 15.5-fold improvement in sperm concentration (1.1 × 106 vs 18.3 × 106 per mL), 71.4% change in volume (0.7 vs 1.2 mL), increased progressive motility (2% vs 43%), and the total volume of sperm with progressive motility (0% vs 23.6%). Here, we show that administering a daily dose of insulin-like growth factor 1 can improve sperm quality parameters.

Sperm output and body condition are maintained independently of hibernation in an endangered temperate amphibian

2019 ◽  
Vol 31 (4) ◽  
pp. 796 ◽  
Author(s):  
Sinlan Poo ◽  
Kristin M. Hinkson ◽  
Edward Stege
Keyword(s):  
Body Condition ◽  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Reproductive Technologies ◽  
Treatment Groups ◽  
History Of ◽  
Unexpected Findings ◽  
Sperm Output ◽  
Wyoming Toad ◽  
Number Of Cells

Hibernation is an integral part of the life history of species living in seasonal environments. However, our knowledge about the link between hibernation and reproductive success in amphibians remains limited, which poses an obstacle for critical conservation efforts. To fill this gap, we quantified the effects of captive hibernation on sperm quality, sperm quantity and body condition in an endangered anuran, the Wyoming toad (Anaxyrus baxteri), and used naturally hibernated wild toads as a standard for comparison. We hypothesised that hibernation is essential for optimal sperm output but is detrimental to body condition. Sperm collection was performed using assisted reproductive technologies for both captive and wild toads. Contrary to our hypotheses, no differences were observed in sperm metrics (total number of cells, concentration, motility and viability) or in body condition across captive treatment groups (0, 30 or 60 days of hibernation). Moreover, no difference was found between sperm metrics of captive toads and wild toads. These unexpected findings suggest that hibernation may not be an essential process for spermiation in A. baxteri while using exogenous hormones, and illustrate the potential of temperate amphibians to adapt to varying environmental conditions during winter months.

scholarly journals Follicular Metabolites-Assisted Clinical Evaluation of IVF/ICSI Outcomes

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Bing Qu ◽  
Yunhe Xiong ◽  
Xiaofan Yu ◽  
Jinli Ding ◽  
Jing Weng ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Sperm Quality ◽  
Public Health Problem ◽  
Decisive Role ◽  
Reproductive Technologies ◽  
Sperm Parameters ◽  
Success Rates ◽  
Vitro Fertilization ◽  
Icsi Outcomes

As infertility became a significant public health problem, assisted reproductive technologies (ARTs) were introduced. However, the fertilization rate of in vitro fertilization (IVF) per cycle varied, and patients needed to repeat IVF or change to intracytoplasmic sperm injection (ICSI). Here, 75 couples suffering from female fallopian tubal blockage (tubal group) and 42 spouses beset by male abnormal sperm status (dysspermia group) were recruited. We comprehensively explored the relationship among couples’ clinical factors, follicular metabolites, and IVF/ICSI stepwise outcomes. IVF/ICSI outcomes were affected by follicular metabolites and physical status in both women and men, regardless of which side infertility came from. Particularly, in the tubal group, the energy supporting pathways—glycolysis and pyruvate metabolism—were most essential in follicles, and IVF/ICSI outcomes were also related to sperm parameters. However, in the dysspermia group, in addition to sperm conditions, oocyte quality acted as a compensation for poor sperm quality, for which aminoacyl-tRNA biosynthesis and the related supporting metabolism were critical in the follicular environment, and ultimately played a decisive role in IVF/ICSI outcomes. The respective logistic regression models in combination with selective male sperm parameters, estradiol (E2), follicular alanine, glutamine, glycoprotein, lipid, and acetic acid, were constructed to predict IVF or ICSI outcomes. No matter which sex infertility comes from, factors from both men and women should be considered. The current study provides a feasible option for pre-IVF evaluation, as well as guidance for follow-up clinical intervention to improve IVF/ICSI success rates.

scholarly journals Zerumbone inhibits prostate cancer cell viability and induces cell death by non-apoptotic pathway

2016 ◽  
Vol 11 (4) ◽  
pp. 771
Author(s):  
Xian-De Cao ◽  
Hui-Min Zheng
Keyword(s):  
Prostate Cancer ◽  
Cell Death ◽  
Cell Viability ◽  
Cancer Cell ◽  
Propidium Iodide ◽  
Prostate Cancer Cell ◽  
Apoptotic Cell ◽  
D1 Protein ◽  
Annexin V ◽  
Apoptotic Pathway

<p class="Abstract">The aim of the present study was to investigate the role of zerumbone on the proliferation, cell cycle arrest and cell death in DU-145 prostate cancer cell lines. The MTT assay revealed that zerumbone (20 µM) reduced proliferation of DU-145 cells to 39.0% at 48 hours. It also increased the proportion of propidium iodide stained cells to 53.4% compared 1.0% in control. However, the population of annexin V-stained cells remained uneffected indicating induction of non-apoptotic cell death by zerumbone. Treatment of DU-145 cells with zerumbone (20 µM) caused 8-fold enhancement in the level of reactive oxygen species (ROS). On the other hand, exposure of the zerumbone treated DU-145 cells to glutathione inhibited the generation of ROS. Fow cytometry using propidium iodide staining revealed that zerumbone treat-ment increased proportion of cells in G1 phase to 71.3% on compared to 34.7% in the control. The results from Western blot analysis revealed a significant increase in the expression of cyclin D1 protein in DU-145 cells on treatment with 20 µM concentration of zerumbone. Thus, zerumbone treatment inhibits prostate cancer cell viability and can be used for its treatment.</p><p> </p>

Unfolded Protein Response is Involved in Trans-Platinum (II) Complex-Induced Apoptosis in Prostate Cancer Cells via ROS Accumulation

2019 ◽  
Vol 19 (9) ◽  
pp. 1184-1195
Author(s):  
Didem Karakas ◽  
Buse Cevatemre ◽  
Arzu Y. Oral ◽  
Veysel T. Yilmaz ◽  
Engin Ulukaya
Keyword(s):  
Prostate Cancer ◽  
Cell Death ◽  
Cell Viability ◽  
Er Stress ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Treatment Modalities ◽  
Drug Candidate ◽  
Ros Generation ◽  
Dependent Manner

Background:Prostate cancer is one of the most common cancer types and it is the sixth leading cause of cancer-related death in men worldwide. Even though novel treatment modalities have been developed, it still a lifethreatening disease. Therefore novel compounds are needed to improve the overall survival.Methods:In our study, it was aimed to evaluate the anti-cancer activity of newly synthesized Platinum (II) [Pt(II)] complex on DU145, LNCaP and PC-3 prostate cancer cell lines. The cytotoxic activity of Pt(II) complex was tested by SRB and ATP cell viability assays. To detect the mode of cell death; fluorescent staining, flow cytometry and western blot analyses were performed.Results:The Pt(II) complex treatment resulted in a decrease in cell viability and increasing levels of apoptotic markers (pyknotic nuclei, annexin-V, caspase 3/7 activity) and a decrease in mitochondrial membrane potential in a dose dependent manner. Among cell types, tested PC-3 cells were found to be more sensitive to Pt(II) complex, demonstrating elevation of DNA damage in this cell line. In addition, Pt(II) complex induced Endoplasmic Reticulum (ER) stress by triggering ROS generation. More importantly, pre-treatment with NAC alleviated Pt(II) complex-mediated ER stress and cell death in PC-3.Conclusion:These findings suggest an upstream role of ROS production in Pt(II) complex-induced ER stressmediated apoptotic cell death. Considering the ROS-mediated apoptosis inducing the effect of Pt(II) complex, it warrants further evaluation as a novel metal-containing anticancer drug candidate.

Specific Cytostatic and Cytotoxic Effect of Dihydrochelerythrine in Glioblastoma Cells: Role of NF-κB/β-catenin and STAT3/IL-6 Pathways

2019 ◽  
Vol 18 (10) ◽  
pp. 1386-1393 ◽  
Author(s):  
Tereza C.C. Silva ◽  
Giselle P. de Faria Lopes ◽  
Noélio de J. Menezes-Filho ◽  
Diêgo M. de Oliveira ◽  
Ezequiel Pereira ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Annexin V ◽  
Experimental Models ◽  
Blue Dye ◽  
Dependent Manner ◽  
Glioblastoma Cells ◽  
U251 Cell ◽  
U251 Cells

Background: A glioblastoma is a primary CNS tumor that is more aggressive and lethal than other brain tumors. Its location, rapid proliferation, invasive growth, angiogenesis and immunosuppression are the main factors that limit its treatment, making it a major challenge to neuro-oncology. Objective: This study investigated the in vitro effects of the alkaloid dihydrochelerythrine (DHC), which is extracted from Zanthoxylum stelligerum, on the viability, proliferation, cell death and β-catenin, NFκB, STAT3/pSTAT3 and interleukins roles. Method: In vitro experimental models of human (U251 and GL-15) and murine (C6) glioblastoma cells were cultured in the presence of DHC at increasing concentrations for MTT assay and exclusion trypan blue dye to determine EC50. Afterward, C6 and U251 cells were treated with 100 µM DHC or DMSO 0.1% for cell cycle, annexin and expression of β-catenin/NFκB/STAT3/pSTAT3 by flow cytometry or immunofluorescence. Interleukin quantification was made by Cytometric Bead Array. Results: A significant decrease was observed in C6 and U251 cell viability in a time and dose-dependent manner. GL-15 cell viability decreased only when treated with 200 µM DHC. This maximum concentration affected neither astrocytes nor microglia viability. A cytostatic effect of DHC was observed in C6 and U251 cells after 48 h of 100 µM DHC treatment. After 72 h of DHC treatment, C6 presented 80% of annexin-V+ cells compared to 10% of annexin-V+ U251 cells. C6 cells demonstrated significant high levels of NFκ B and β-catenin cytoplasmic fraction. Additionally, DHC treatment resulted in higher significant levels of IL-6 than did other interleukins and STAT3 up-regulation in U251 cells. Conclusion: These results demonstrate that DHC acts as a chemosensitizing agent selective for glioma cells not affecting non-tumor cells. Considering tumor heterogeneity, DHC demonstrated an anti-cancer potential to activate different cell death pathways. DHC demonstrated could be used for chemotherapy and immunotherapy applications in glioblastomas in the future.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

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