Influence of semen storage and cryoprotectant on post-thaw viability and fertility of stallion spermatozoa

The aim of the current study was to verify that stallion, spermatoza could be cooled for 24 hours and then frozen. In experiment I, one ejaculate from each of 13 stallions was used. Semen was collected and split into two parts; one part immediately frozen using standard cryo-preservation techniques and the other diluted, stored in an Equitainer for 24 hours, and then frozen. In experiment II, one ejaculate from each of 12 stallions was collected, diluted with Botu-Semen, and split into two parts: one cooled in an Equitainer and the other in Max-Semen Express without prior centrifugation. After 24 hours of cooling, the samples were centrifuged to remove seminal plasma and concentrate the sperm, and resuspended in Botu-Crio (R) extender containing on e of three cryoprotectant treatments (1% glycerol + 4% dimethylformamide, 1% glycerol + 4% dimethylacetamide and 1% glycerol + 4% methylformamide), maintained at 5 degrees C for 20 minutes, then frozen in nitrogen vapour. No difference was observed between the two cooling systems. The association of 1% glycerol and 4% methylformamide provided the best post-thaw progressive motility. For experiment III, two stallions were used for a fertility trial. Forty three inseminations were performed using 22 mares. No differences were seen in semen parameters and pregnancy rates when comparing the two freezing protocols (conventional and cooled/frozen). Pregnancy rates for conventional and cooled/frozen semen were, respectively, 72.7% and 82.3% (stallion A), and 40.0% and 50.0% (stallion B). We concluded that cooling equine-semen for 24 hours before freezing while maintaining sperm viability and fertility is possible.

Viability and cell cycle analysis of equine fibroblasts cultured in vitro

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD(A (R))) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% x 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.

Motility and viability of ram sperm cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of refrigeration systems upon frozen bull sperm viability assessed by computer-assisted sperm analysis and fluorescent probes

Sperm cryopreservation success depends upon the maintenance of spermatozoa fertility potential. Sperm cells must preserve both integrity and functionality of several cell structures. The stabilization phase must allow the exit of water from the sperm cells via osmosis. This study aimed to compare the effect of refrigeration in the commercial refrigerator (CR) and the transport/refrigeration box (TRB) upon the viability of frozen bull sperm diluted in three different extenders (A, B and C). Ten Nellore bulls, Bos taurus indicus maintained in Artificial Insemination Center were used and the spermatozoa samples was assessed for Plasma Membrane Integrity and CASA evaluation. The stabilization phase (5 degrees C/4 hours) was performed in the CR as well as in the TRB, and then samples were exposed to nitrogen vapor during 20 minutes and then plunged into nitrogen. The statistical analysis was done using the variance analysis and the significance level was set at 5%. In the CR the post-thawing parameters for PM and ALH were higher (p < 0.05) in the extender A (glicine egg-yolk) and extender B (glicine egg-free) when compared with extender C (TRIS egg-yolk). As for BCF, STR and LIN, the parameters were higher (p < 0.05) in extender B than in C. Samples that were stabilized in the TRB presented higher post-thawing parameters (p < 0.05) for PM and LIN in extender A and extender B when compared with C. BCF and STR parameters were higher (p < 0.05) in extemder B when compared with C. Extender B samples had higher (p < 0.05) PMI when stabilized in CR. The findings in this experiment enable us to say that both CR and TRB were effective in keeping the viability of post-thawing bull semen.

Clinical Viability for Immediate Loading of Dental Implants: Part II-Treatment Alternatives

The treatment with implants aims to obtain a direct interface between bone and implant. The implant is kept load-free during 4 to 6 months in the 2-stage procedure, which is considered a requisite for osseointegration. However, this period is based on empirical principles and uncomfortable for patient. So, the immediate loading protocol was Suggested to submit implants to occlusal function after placement. This protocol has been applied for several conditions of edentulism. The aim of this study was to evaluate the treatment alternatives for immediate loading of complete and partial edentulous patients. In general, the studies have demonstrated high previsibility for rehabilitation of complete edentulous arches with full-arch, implant-supported prosthesis. The rehabilitation with immediate loading for maxillary overdenture is questionable because there is no longitudinal study in literature. The studies with partial edentulous arches have demonstrated high success rates for implants placed in the mandibular and maxillary anterior region. Additional care is recommended for posterior region mainly in the maxillary arch, and further studies are suggested to corroborate this treatment.

Clinical Viability of Immediate Loading of Dental Implants: Part I-Factors for Success

Two-stage procedure for dental implants presents corroborated clinical success over 40 years. The evolution of surgical techniques, development of diagnostic methods, knowledge about tissue biology, and quality of implants regarding design and surface supported studies with I surgical stage followed by immediate prosthesis placement. However, several factors influence the treatment success with immediate loading. SO, this Study aimed to evaluate some factors regarding the success and characteristics of implants and patients.

Evaluation of immediate bone-cell viability and of drill wear after implant osteotomies: Immunohistochemistry and scanning electron microscopy analysis

Purpose: This study sought to evaluate the effect of repeated implant drilling on the immediate bone-cell viability, and to evaluate drill wear by scanning electron microscopy.Materials and Methods: The tibiae of 10 rabbits were used, divided into 5 groups (G): G1 corresponded to new drills, and G2, G3, G4, and G5 corresponded to drills used 10, 20, 30, and 40 times, respectively. The animals received 10 sequential osteotomies in each tibia. The animals were euthanized immediately after the osteotomies by perfusion with 4% formaldehyde. Samples then underwent immunohistochemistry processing for ordinal qualitative analysis of osteoprotegerin (OPG), the RANK ligant (RANKL; a tumor-related necrosis factor receptor family), and osteocalcin protein immunolabels, as detected by the immunoperoxidase method and revealed with 3,3-diaminobenzidine. Drill wear and plastic deformation were analyzed by scanning electron microscopy (SEM).Results: The proteins were expressed in osteocytes of the superior bone cortical during the 40 drillings. However, in G4 and G5, a discrete increase in the expression of RANKL was observed, when compared with OPG; this increase was statistically significant in G5 (P = .016). The SEM analysis revealed major plastic deformation and drill wear in G4 and G5.Conclusion: Based on the present methodology, it may be concluded that cell viability is preserved if a less traumatic surgical protocol is used. However, the repeated use of drills alters the protein balance as of the thirtieth perforation. (C) 2008 American Association of Oral and Maxillofacial Surgeons.

Effect of Psidium cattleianum leaf extract on Streptococcus mutans viability, protein expression and acid production

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [ 100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production. Copyright (C) 2008 S. Karger AG, Basel.

Increased Viability of Odontoblast-Like Cells Subjected to Low-Level Laser Irradiation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytotoxicity of two novel formulations of calcium phosphate cements: A comparative in vitro study

The purpose was to evaluate the cytotoxicity of two novel formulations (alpha and beta) of calcium phosphate cements. Positive control, represented by a commercial hydroxyapatite cement, and negative control were included for comparative purposes. A continuous lineage of fibroblastic cells was used, and the effect of the tested materials on both cell proliferation and viability was assessed by counting cell number on hemocytometer and by the trypan blue exclusion test, respectively. Study design attempted to simulate clinical use by allowing direct and indirect contact of cells and cements. Results were analyzed by the Kruskal-Wallis test and indicated that the beta formulation was extremely cytotoxic (P < 0.001), because this material induced the greatest reduction on cell proliferation and viability. The alpha formulation behaved similarly to the positive control regarding its effect on cell proliferation and viability. Thus, it is concluded that alpha formulation has promise for further evaluation of its behavior in vivo.

Cytotoxicity of Portland Cement with Different Radiopacifying Agents: A Cell Death Study

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Nutritional stress enhances cell viability of odontoblast-like cells subjected to low level laser irradiation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Low-level laser therapy (670 nm) on viability of random skin flap in rats

This study investigated the effects of 670 nm laser, at different fluences, on the viability of skin flap in rats. One hundred male animals were used. The animals were divided into control group; group treated with 3 J/cm(2); group treated with 6 J/cm(2); group treated with 12 J/cm(2) and group treated with 24 J/cm(2). The skin flap was made on the backs of all animals studied, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was done immediately after the surgery and on days 1, 2, 3 and 4 after surgery. The percentage of necrosis of the flap was calculated at the 7th postoperative day. Additionally, a sample of each flap was collected to enable us to count the blood vessels. Treated animals showed a statistically significant smaller area of necrosis than did the control group. The necrosis in the treated groups was 41.82% (group 2), 36.51% (group 3), 29.45% (group 4) and 20.37% (group 5). We also demonstrated that laser irradiation at 670 nm, at all doses used, had a stimulatory effect on angiogenesis. Our study showed that the 670 nm laser was efficient to increase the viability of the skin flap, at all fluences used, with a tendency of reaching better results at higher doses.