Percentage Of Live Cells Research Articles

Effect of dietary energy intake and somatotropin administration after weaning on growth rate and semen characteristics of Granadina goat bucks

Twenty-eight growing Granadina goat bucks, divided into 4 groups of 7, in a 2 × 2 factorial arranged design, were used to study the effect of post-weaning levels of dietary energy and administration of recombinant bovine somatotropin (rbST) on growth and semen characteristics. Every 14 days, bucks were offered sustenance at rates of either 1.0 (standard diet, 2.36 Mcal EM kg^{-1}; 15% CP) or 1.25 (increased diet, 2.95 Mcal EM kg^{-1}; 18% CP) times the requirements for growth energy and protein, for a total of 99 days and in combination with or without subcutaneous administration of 125 mg rbST. The average daily gains (ADG) were greater (P < 0.01) in bucks fed the high energy diet (133 ± 25 vs. 111 ± 23 g day^{-1}) and the ADG were greater (P < 0.05) in the bucks treated with rbST than in the untreated bucks (130 ± 28 vs. 114 ± 23 g day^{-1}). The percentage of live sperm cells was not different between bucks fed increased diet of NRC recommendations and bucks fed standard diet. Similarly, the sperm output of bucks on the increased diet did not differ from that of bucks on the standard diet (2282 ± 1137 vs. 1946 ± 529 × 10^6/mL). Semen volume (0.51 ± 0.29 vs. 0.55 ± 0.28 mL), sperm concentration (2210 ± 1139 vs. 2055 ± 656 × 10^6/mL), total sperm cells (1233 ± 962 vs. 1014 ± 572 ×x 10^6), and motile sperm cells (67.1 ± 14.5 vs. 60.9 ± 19.3) were not affected by rbST. No significant differences due to dietary and hormonal treatments were observed in scrotal circumference. Dietary and hormonal treatments had no effect on either serum concentration of particular metabolites, except cholesterol. Results from this study indicate that both high energy diets and chronic application of rbST enhanced the growth performance of young Granadina goat bucks; nevertheless, none of these effects altered the final scrotal circumference, particular blood metabolites, and sperm output.

219 ISOLATION AND CHARACTERIZATION OF DOMESTIC CAT SPERMATOGONIAL CELLS

Spermatogonial stem cells (SSC) have the capacity for self-renewal and the potential of producing progenitor spermatogonia that will differentiate into spermatozoa. SSC transplantation may be a valuable alternative for the propagation of genetically important males and preservation of endangered wild felids, as recently demonstrated by the production of ocelot spermatozoa after the xeno-transplantation of a mixed germ cell population into the testis of a domestic cat (Silva et al. 2011 J. Androl.). SSC are in low numbers in the testis and have been isolated in different mammalian species by using specific cell surface markers; however, the expression of SSC-surface markers in feline species has not been characterised. In the present study, testes of domestic cats were obtained from veterinary clinics. The selected testes (n = 4) ranged in size from 1.3 to 2.0 cm in length. To obtain a suspension of a mixed population of spermatogonial cells, seminiferous tubules were enzymatically dissociated using two digestion steps followed by dual filtration through 100-μm and 40-μm nylon mesh filters and a final separation over a 5-layer Percoll™ density gradient (35, 30, 27.5, 25 and 20%). Spermatogonial cells were morphologically identified by their characteristic large round nucleus with a homogenous appearance in the bands formed at the 30%, 27.5% and 25% layers. The mean number of cells/testis collected was ∼13 × 106 ± 12.2, with an overall percentage of live cells of 97%. After isolation, cells were fixed with 4% PFA, blocked overnight and stained with primary antibodies specific for SSC-surface markers (CD49f, CD9, C-Kit, GFRα1, GPR125 and Thy-1) and pluripotent stage-specific embryonic antigen markers (SSEA-1 and SSEA-4). Fluorescence microscopy showed a positive expression of GFRα1, GPR125 and C-Kit, but not for CD49f, CD9, or Thy-1. It also revealed that a low number of cells were positive for SSEA-1 and SSEA-4. For further characterisation, molecular detection of the pluripotent gene Oct-4 and the germ cell-specific genes BOLL, DAZL and VASA was performed in germ cells isolated from one testis of four individuals. For RT-qPCR, the Cells-to-cDNA™ II kit (Ambion) was used to produce cDNA from an aliquot of ∼30 000 cells directly after isolation. RT-qPCR showed that none had detectable levels of Oct-4 within the range of the standard. Three of the four testes expressed all three germ cell-specific genes, BOLL, DAZL and VASA, while only VASA was detected in the remaining testis. These results suggest that cat SSCs and spermatogonial cells express some of the SSC markers tested. However, the positive expression of SSEA-1 and SSEA-4 in a low number of cells further supports the stem cell-like state of cat SSCs and that these markers can be used in dual staining for purifying cat SSCs from a mixed germ cell population by fluorescence-activated cell sorting.

Neuroprotective effects of prostaglandin analogues on retinal ganglion cell death independent of intraocular pressure reduction

Prostaglandin (PG) analogues may have an additional effect to protect neurons independent of IOP reduction. Only a few reports indicated that some PG analogues had neuroprotective effects or increased blood flow in in vivo and in vitro models. However, there is no comparative study using all clinically available PG analogues and also using primary culture of retinal ganglion cell (RGC). Our purpose of study is to investigate the direct neuroprotective effect of PG analogues on glutamate- and hypoxia-induced RGC death using rat purified primary RGC culture with latanoprost acid, travoprost acid, bimatoprost acid, bimatoprost, tafluprost acid, unoprostone, and PGF2α. Purified RGCs cultures were obtained from retinas of 6 days old Wistar rats, following a two-step immuno-panning procedure. After 72 h of cultivation, the neuroprotective effect of PG analogues (1 nM, 10 nM and 100 nM) was investigated by culturing the RGCs in 25 μM glutamate for a further 72 h or 5% O2 hypoxic condition for 24 h. The RGC viability under each condition normalized to that under normal condition without stress was evaluated as live cell percentage based on a total of 15 repeated experiments. As a result, 100 nM of latanoprost acid, tafluprost acid, bimatoprost acid, and bimatoprost significantly increased RGC survival rate by suppressing apoptosis. PG analogues indicated IOP independent neuroprotective effect on glutamate- or hypoxia-induced RGC death using rat primary RGC culture at clinically available intracameral concentration. Since those profiles were different from clinical efficacy in IOP reduction, the mechanism of neuroprotection may be not related to FP receptor stimulation.

Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen

The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry. The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor®488 conjugate, mitochondrial activity was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to 50.4% (P < 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes (P < 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However, after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P < 0.05). Nevertheless, after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after freezing-thawing.

Mannitol-containing macroporous calcium phosphate cement encapsulating human umbilical cord stem cells

Stem cell-based tissue engineering offers immense promise for bone regeneration. The objective of this study was to develop a self-setting, mannitol-containing calcium phosphate cement (CPC) encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs) for bone tissue engineering. hUCMSCs could be an inexhaustible and low-cost alternative to the gold-standard bone marrow MSCs, which require an invasive procedure to harvest. hUCMSCs were encapsulated in alginate beads and mixed into the CPC paste. Water-soluble mannitol porogen was incorporated into CPC to create macropores. The porosity was increased from 49% for the hUCMSC-encapsulating CPC to 64% after adding mannitol and absorbable-fibres (p < 0.05). Flexural strength of the construct was increased from 0.3 MPa to 2.0 MPa via fibres. Live cell percentage was > 80% for all constructs. The ALP and OC gene expressions were low at 1 day and greatly increased at 14 days. The constructs that contained mannitol had significantly higher ALP and OC expressions than that without mannitol. ALP activity of hUCMSCs inside CPC with mannitol and fibre was significantly higher than that without mannitol. At 14 days, mineralization by the encapsulated hUCMSCs was eight-fold higher than that at 1 day. In conclusion, a novel mannitol-containing porous CPC-hUCMSC construct was developed for bone tissue engineering. Its advantages include cell delivery inside a load-bearing CPC that has injectable and in situ setting capabilities. hUCMSCs inside CPC had good viability and successfully osteodifferentiated. The self-setting and strong hUCMSC-encapsulating CPC scaffold is promising for bone tissue engineering in a wide range of orthopaedic and craniofacial applications.

Reduced Chondrocyte Viability Is Associated With the Use of Surgical Marker Pen Ink

Background: Sterile surgical marker pens are commonly used in cartilage repair surgery to aid in the placement of periosteal patches or collagen membranes in autologous chondrocyte implantation. Purpose: To investigate the effects that methylene blue and crystal violet marker pen ink have on human chondrocytes when cultured on collagen membranes in vitro. Study Design: Controlled laboratory study. Methods: Human chondrocytes were applied to Chondro-Gide collagen membranes at a volume of 12 million cells. In the first experiment, 2 sterile marker pens, one containing methylene blue and the other crystal violet inks, were used to mark membranes immediately before the addition of cells. In the second experiment, the same marker pens marked the membranes after 7 days of cell culture. In each experiment, 3 groups of membrane were tested for each pen. Group A consisted of no ink mark, group B had only the uppermost “smooth” layer marked, and group C had the lower “porous” layer marked. All membranes were then cultured in standard growth media for 24 hours. Cell viability was assessed at 24 hours on all membranes using a live/dead-cell viability assay. Cell viability was quantified with florescent microscopy with mean percentage of live cells in each marker pen group compared with control membranes using the Student t test (P < .05). Results: Control membranes (group A) with no ink showed cell viability approaching 100%. A statistically significant reduction in cell viability with both methylene blue (23.1%; P < .0001) and crystal violet (18.9%; P < .0001) was found adjacent to the ink mark on the smooth side (group B) and on the porous side remote from the ink (group C) in both experiments (<30%; P < .0001). Areduction in cell viability was noted on the smooth side remote from the ink mark but did not reach statistical significance. Marked cell death was seen with both dyes (<15%; P < .0001) adjacent to the ink on the porous side. Conclusion: Chondrocyte viability is significantly reduced when cells are cultured in vitro on collagen membranes marked with methylene blue and crystal violet pen ink. Clinical Relevance: Surgeons should be aware of the potential negative effect of marker pens in cell-based therapies.

Induction of apoptosis with diallyl disulfide in AGS gastric cancer cell line

PurposeDiallyl disulfide (DADS) is a major organosulfur compound derived from garlic. It has been reported that DADS is able to inhibit the proliferation of several tumor cells. In this study, the effect of DADS was investigated in terms of the proliferation of AGS, gastric adenocarcinoma cell line at various concentrations.MethodsThe viability of cultured cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. To detect the induction of apoptosis, Annexin V-FITC/propodium iodide (PI) staining assay was performed. Analysis of reactive oxygen species (ROS) and the distribution of cells in the cell cycle were measured by a flow cytometer. And using the Western blot analysis, the change of Fas, caspase-3, Bax, Bcl-2 activity was measured.ResultsThe percentage of live AGS cells was decreased to 23% of that in the control group after 400 µM DADS treatment for 48 hours. The Annexin V positive/PI negative (apoptosis portion) area increased from low concentration of DADS to high concentration. When comparing among the DADS treatment groups, the amount of ROS production increased in a dose dependent manner. The percentage of sub-diploid DNA content increased from 8.71% at 50 µM to 25.74% at 400 µM DADS treatment group. The expressions of Fas, caspase-3, Bax were increased and that of Bcl-2 was decreased in a dose dependent manner.ConclusionDADS decreases the viability of AGS cell lines and induces apoptosis in a dose-dependent manner. But the relationship of the anti-proliferative effect of DADS and related molecular changes were not clearly proportional to the concentration of DADS.

Influence of PGF_{2\alpha} on semen quality and libido in Holstein bulls

The aim of this study was to determine the effects of Cloprostenol (PGF_{2\alpha} analogue) on semen quality and libido in Holstein bulls. Ten low libido Iranian Holstein bulls were randomly allocated to 2 groups and received Cloprostenol (n = 5) or saline (n = 5) 30 min prior to the semen collection 2 days per week for 2 months. Reaction time was significantly decreased in the treatment group. Duration of ejaculation was significantly increased in the treatment group. Semen volume and sperm concentration were higher in treated bulls when compared with controls. The percentage of morphologically normal sperm cells, percentage of live sperm cells, motile sperm cells, and post-thaw motile sperm cells were not affected by the Clopostenol. Plasma testosterone concentrations were increased after Cloprostenol injection. It was concluded that injection of PGF_{2\alpha} improved libido, semen quality, and plasma testosterone concentration in low libido Holstein bulls.

Cytotoxicity of current adhesive systems: In vitro testing on cell cultures of primary murine macrophages

Objectives The aim of this study was to evaluate, in vitro, the potential cytotoxicity of dentinal adhesives on alveolar macrophages of Wistar rats, after diffusion through dentin. Methods The cytotoxicity of adhesives [single bond plus (SB), clearfil SE bond (CF) and Xeno V (XE)] applied to the occlusal surface of human dentin disks adapted to a dentin barrier test device were analyzed. The sets placed on a monolayer of cells were incubated for 24, 48 and 72 h. Culture medium and Escherichia coli lipopolysaccharides (LPS) were used as negative and positive controls, respectively. Cellular cytotoxicity was evaluated by observing the cell survival rate (MTT assay) and nitric oxide production (NO). The data were analyzed by one-way factorial ANOVA and Tukey's and Tamhane's paired comparisons T2 ( α = 0.05). Results All the adhesive systems reduced the percentage of live cells by over 50%, compared with the control group. Within the same period of time, there was a statistically significant difference between the adhesives and LPS compared with the negative control group. SB presented a statistically significant difference between 24 h and 72 h, and XE between 48 h and 72 h. The quantity of NO produced in 24 h did not differ statistically between the NC and adhesive groups. After 48 h there was a significant difference between SB/CF and XE/NC. At 72 h only CF showed a significant difference from each of the other groups. LPS differed statistically from all the other groups at all the evaluation times. Significance Components of the adhesives tested may permeate the dentin in sufficient concentrations to cause death and damage to cell metabolism in the alveolar macrophages of rats, which indicates potential cytotoxicity to pulpal cells.

Effects of nanopesticide chlorfenapyr on mice

The effects of chlorfenapyr nano preparation on mice were examined. The animals were administered with different doses of 4.84, 9.68, or 19.36 mg kg−1 of chlorfenapyr common preparation or nano preparation intraperitoneally (IP). Micronucleus (MN) test and comet assay (CA) experiments revealed that at the same dose, similar DNA damage in the peripheral blood lymphocytes of the mouse and chromosome damage in bone marrow cells of the mouse occurred with chlorfenapyr nano preparation; however, the severity of these effects was less than that found in common preparation. It is noteworthy that there were no differences in apoptotic ratio and live cell percentage in the liver cells of the mouse between these two pesticide formulations using flow cytometry. Data indicate that chlorfenapyr nanoformulation is less toxic to mouse cells than the common formulation.

Effects of benzalkonium chloride-preserved, polyquad-preserved, and sofZia-preserved topical glaucoma medications on human ocular epithelial cells

INTRODUCTION|: To investigate potentially adverse effects of different topical glaucoma medications and preservatives on cultured ocular epithelial cells. METHODS|: Confluent cultures of human corneal (10.014 pRSV-T) and conjunctival cells (1-5c-4) were assayed with 100 μL of different glaucoma medications for 25 minutes at 37°C and 5% CO₂. We also tested the preservative sofZia® (Alcon Laboratories, Fort Worth, TX, USA), as well as a range of concentrations of the preservative benzalkonium chloride (BAK; 0.001% to 0.050%). Balanced salt solution was used as the "live" control and a solution containing 70% methanol and 0.2% saponin was used as a "dead" control. The LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA, USA) was used to determine the percentage of dead and live cells via ethidium homodimer and calcein fluorescence, respectively. RESULTS|: The toxicity of the prostaglandin analogs latanoprost, tafluprost and travoprost preserved with BAK was similar to the toxicity observed in their respective BAK concentrations. The prostaglandin analog travoprost (0.004%) preserved with the oxidizing preservative sofZia had much greater corneal and conjunctival cell survival than travoprost preserved with BAK. Travoprost (0.004%) containing polyquad also performed statistically better than its BAK-preserved formulation. CONCLUSION|: Ocular surface side effects have previously been demonstrated with chronic, long-term exposure to intraocular pressure-lowering medications containing the common preservative BAK. BAK alone has significant in-vitro cytotoxicity to cultured ocular epithelial cells. Substitution of BAK with polyquad or sofZia resulted in significantly higher percentages of live conjunctival and corneal cells. Further studies are needed to understand the- clinical implications of these findings.

Protein profile study in European eel (Anguilla anguilla) seminal plasma and its correlation with sperm quality

Summary Along with sperm quality parameters, the protein profile of European eel seminal plasma was analyzed during induced spermiation (n = 56 samples). Motility, Percentage of live cells, spermatozoa head morphometry and concentration showed low values during the initial weeks of spermiation and maintained high levels throughout the rest of the experiment. The protein profile gradient by SDS-PAGE (4–15%) registered four important electrophoretic bands around 80, 40, 26 and 12 KDa. Three of them showed significant differences in concentration during treatment (80, 40 and 12 KDa), and all of them showed the highest value on the 8th week. Both 80 and 12 KDa bands increased until the 8th week, followed by a progressive decline. One possible explanation for these profiles is that, in the first weeks of treatment, proteins originated from blood plasma are accumulated in the seminal plasma, and from the 8th week some of these proteins are incorporated into the spermatic membranes. The 40 KDa protein band also increased during the first 8 weeks, but maintained high concentrations in the seminal plasma for the rest of the experiment. One result confirms the theory that the presence of proteins in the seminal plasma having a molecular weight lower than 50 KDa increased spermatozoa motility, since the 40 KDa band displayed significantly higher values coinciding with the high percentages of spermatozoa motility. Seminal plasma proteins seem to have an important role in spermatogenesis and spermatozoa movement, but further studies are necessary to discover the identity of these proteins and their precise functions.

A new method to determine initial viability of entrapped cells using fluorescent nucleic acid staining

Entrapped bacterial cells are widely used in several biotechnological applications. Cell entrapment procedures are known to affect the viability of bacterial cells. To determine the effect of entrapment procedures on viability of bacterial cells, dissolution of the entrapment matrices using chelating agents or heat is required immediately after the entrapment is completed. Chelating agents and heat applied in the matrix dissolution reduce cell viability and in turn hinder accurate quantification of viable cells. In this study, a method to determine the effect of entrapment procedure on bacterial cell viability which involves entrapping cells directly onto glass slides was developed. The developed method showed less viability reduction than the methods requiring matrix dissolution. The percentage of live cells in the culture before entrapment ranged from 54% to 74%, while the percent of live cells after entrapment determined by the developed method was 39-62%.

Booster biocides and microfouling

Antifouling (AF) paints are used to prevent the attachment of living organisms to the submerged surfaces of ships, boats and aquatic structures, usually by the release of biocides. Apart from copper, organic booster biocides are the main active components in AF paints, but their use can have a negative impact on the marine environment. The direct effects of biocides on marine bacteria are poorly known. This work investigates the impact of two biocides, viz. diuron and tolylfluanid, on the growth and the viability of marine microorganisms and on their ability to form biofilms. The biocides in solution were found to inhibit growth of two strains of marine bacteria, viz. Pseudoalteromonas and Vibrio vulnificus, at a high concentration (1000 μg ml−1), but only a small effect on viability was observed. Confocal laser scanning microscopy (CLSM) showed that the booster biocides decreased biofilm formation by both bacteria. At a concentration of 10 μg ml−1, the biocides inhibited cell attachment and reduced biofilm thickness on glass surfaces. The percentage of live cells in the biofilms was also reduced. The effect of the biocides on two diatoms, Fragilaria pinnata and Cylindrotheca closterium, was also evaluated in terms of growth rate, biomass, chlorophyll a content and attachment to glass. The results demonstrate that diuron and tolylfluanid are more active against diatoms than bacteria.

Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll ® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL -treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.

Effect of liquid storage on sorted boar spermatozoa

A routine use of boar sexed semen is far from being a reality due to many limiting factors among which is the long sorting time necessary to obtain the adequate number of sexed spermatozoa for artificial insemination and the high susceptibility to damages induced by cryopreservation. The aim of this study was to evaluate the modification induced by 24–26 h storage on sorted boar spermatozoa on the basis of their viability, acrosome status, Hsp70 presence, and in vitro fertilizing ability. The percentage of viable cells, according to SYBR green/PI staining, was negatively affected (P < 0.05) by sorting procedure. Moreover, liquid storage significantly (P < 0.05) reduced membrane integrity of sorted spermatozoa as compared to all the other groups. Neither sorting nor storage influenced the percentage of live cells with reacted acrosome, according to FITC-PNA/PI staining. Sorted samples, after 24–26 h storage, were characterized by an increase (P < 0.05) of sperm cells negative for Hsp70, as observed by immunofluorescence, and by a decrease (P < 0.05) in Hsp70 content, as evidenced by western blot. While sorting procedure did not adversely affect both penetration rate and total efficiency of fertilization, these parameters were negatively (P < 0.05) influenced by storage after sorting. In order to minimize damages that compromise fertility and function of sex-sorted boar spermatozoa, the mechanisms by which sorting and liquid storage cause these injures require further study.

In Vitro Toxicity of Topical Ocular Prostaglandin Analogs and Preservatives on Corneal Epithelial Cells

To determine the effect of 4 formulations of commercially available prostaglandin analogs (PGAs) on human corneal epithelial cells in vitro. The test solutions (PGAs) examined were tafluprost 0.005% with 0.010% benzalkonium chloride (BAK), travoprost 0.004% with 0.015% BAK, travoprost 0.004% with sofZia, and latanoprost 0.005% with 0.020% BAK. Also tested independently were the 4 respective BAK or sofZia concentrations related to each PGA. Balanced salt solution (BSS) was used as the live control, and a fixative solution containing 70% methanol and 0.2% saponin was used as the dead control. Immortalized human corneal epithelial cells were exposed to test or control solution for 25 min at 37 degrees C and 5% CO(2). A live/dead assay was used to measure the toxicity of the PGAs. The percentage of live cells in the PGA groups ranged from 2% to 72% of the BSS group (live control). The PGA with the highest relative live cell percentage, at 72% of the live control, was travoprost with sofZia. The next highest PGA, exhibiting 14% live cells, was the formulation of travoprost containing BAK. The other 2 PGAs, tafluprost and latanoprost, had few surviving cells, with 3% and 2% live cells, respectively. The BAK concentrations exhibited 4%, 3%, and 3% for the 0.01%, 0.015%, and 0.02% concentrations, respectively. The stand-alone sofZia cell survival was 68% of the live control. All 4 PGA formulations tested demonstrated significantly more toxicity in human corneal epithelial cells than the live control, but there were significant differences among the PGAs. Travoprost with sofZia exhibited the least toxicity, followed by travoprost with BAK, and then tafluprost and latanoprost. The stand-alone preservative systems were also tested and showed similar survival percentages to each respective PGA. The true clinical implications of these findings require further investigation.

European Eel Sperm Diluent for Short‐term Storage

The sperm of European eel shows a high density and the time of spermatozoa motility is very short after activation with sea water. These characteristics make difficult the sperm handling and its quality assessment. Several diluents were previously described for the Japanese eel obtaining over 3 weeks' conservation times under refrigeration, but they rendered bad results in the European species. In the present study, several diluents were developed taking as basis the P1 medium, and using different dilution ratios (1 : 50, 1 : 100) and two pH (6.5, 8.5). The effect of the addition of bovine serum albumin (BSA, 2% w/v) was also evaluated. At 24 h, undiluted samples already showed significant lower motility and viability than sperm samples diluted in the different media. The results for diluents with pH 6.5 and 8.5 were different. Spermatozoa diluted in media at pH 6.5 cannot be activated at 24 h, while samples diluted in the diluents with pH 8.5 and added with BSA did not show significant differences with respect to the fresh sperm motility until 48 h. The viability (percentage of alive cells) did not show differences until 1 week, independent of the dilution ratio. After 1 week, the motility was approximately 30% in the media containing BSA, which presented no differences for head size of the spermatozoa (perimeter and area) until 72 h and 1 week, respectively. In conclusion, the combination of one medium having similar physico-chemical characteristics to the seminal plasma, including pH 8.5, and supplemented with BSA can be used in different dilution ratios for the sperm's short-term storage, preserving its motility capacity.

Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC-chitosan and CPC-chitosan-fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC-chitosan-fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC-chitosan-fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.

Ganglioside GT1b protects human spermatozoa from hydrogen peroxide‐induced DNA and membrane damage

We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.