NONINVASIVE MONITORING OF ANDROGENS IN MALE AMAZONIAN MANATEE (TRICHECHUS INUNGUIS): BIOLOGIC VALIDATION

The Amazonian manatee (Trichechus inunguis) is endemic in the Amazonian basin and is the only exclusively fresh water sirenian. Historically hunted on a large scale, this species is now considered endangered, and Studies on the reproductive physiology are critical for the improvement of reproductive management of captive and wild Populations of manatees. The aim of this Study was to verify the viability of androgen measurement in saliva, lacrimal, urine, and fecal samples of the Amazonian manatee by conducting a hormone challenge. Two adult male manatees (A-1 and A-2) were Submitted to an experimentation protocol of 12 day (D1 to D10). On D0, the animals received an intramuscular injection of gonadotropin-releasing hormone (GnRH)-analogue. Salivary, lacrimal, urinary, and fecal samples were collected daily (between 0800 hours and 0900 hours) and frozen at -20 degrees C until assayed. Fecal samples were lyophilized, extracted with 80% methanol, and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and were diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without the extraction step. Hormonal assays were conducted with a commercial testosterone RIA kit. An androgen peak (>median + 2 interquartile range [IQR]) was observed in all matrices of both animals, although it was less prominent in the lacrimal samples of A-2. However, the fecal androgen peak (A-1 peak = 293.78 ng/g dry feces, median [IQR] = 143.58 [32.38] ng/g dry feces; A-2 peak = 686.72 ng/g dry feces, median [IQR] = 243.82 [193.16] ng/g dry feces) occurred later than urinary (A-1 peak = 648.16 ng/mg creatinine [Cr], median [IQR] = 23.88 [30.44] ng/mg Cr; A-2 peak = 370.44 ng/mg Cr, median [IQR] = 113.87 [117.73] ng/mg Cr) and salivary (A-1 peak = 678.89 pg/ml, median [IQR] = 103.69 [119.86] pg/ml; A-2 peak = 733.71 pg/ml, median [IQR] = 262.92 [211.44] pg/ml) androgen peaks. These intervals appear to be correlated with the long digesta passage time in this species. The salivary and urinary peaks were closely associated. These results demonstrate that androgen concentrations in saliva, urine, or feces samples reflect reliably physiologic events and are a powerful tool for noninvasive reproductive monitoring of Amazonian manatees.

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 mM and Glu 5 mM. In study II, semen was collected twice and divided into three aliquots; control, AA 50 mu M and AA 250 mu M. Initial sperm motility was significantly higher in sperm diluted with AA 50 mu M; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 mM.

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the ""Simple"" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the ""Simple"" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the ""Simple"" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the ""Simple"" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species. (c) 2008 Elsevier Inc. All rights reserved.

Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin

The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma membranes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls (n = 56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate-Pisum sativum agglutinin, propidium iodide or 5,5`,6,6`-tetrachloro-1,1`,3,3`-tetraethylbenzimidazolearbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell(R) or Botu-Bov(R)), after which both were packaged in 0.5 mL straws and frozen using an automated system. Two straws of semen from each treatment were thawed, and the semen parameters were evaluated as described above. Cryopreservation of sperm reduced motility, damaging plasma and acrosomal membranes, as well as decreasing mitochondrial function. The Botu-Bov(R) extender was more effective in preserving sperm motility and membrane integrity than was the Bioxcell(R) extender. (C) 2007 Elsevier B.V. All rights reserved.

Vitrification with Glutamine Improves Maturation Rate of Vitrified/Warmed Immature Bovine Oocytes

Contents The current study examined the protective effects of l-glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 mu g/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. l-glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).

Comparative Analysis between Chamomilla recutita and Corticosteroids on Wound Healing. An In Vitro and In Vivo Study

The comparison of chamomile and corticosteroids for treating ulcers was done in vitro and in vivo. The experimental groups were: control; chamomile recutita; triamcinolone acetonide and clobetasol propionate. For the in vitro study the cell viability of fibroblasts cultured for 24 h in media conditioned by the substances was obtained by the MITT reduction analysis. For the in vivo study, 125 male rats were submitted to experimental ulcers treated or not (control) by the substances tested. At 1, 3, 5, 7 and 14 days later 5 animals of each group were sacrificed. The lesions were analyzed by means of clinical observation and histological wound-healing grading. Data were compared by ANOVA (p <= 0.05). All experimental groups presented positive cell viability in 24 h. The cultures treated with chamomile presented the smallest cell viability. All animals of the chamomile group exhibited complete wound healing 9 days before the other groups. Complete repaired lesions were observed after 5 days of treatment only in the chamomile group. Animals treated with chamomile presented significantly faster wound healing in comparison to those treated with corticosteroids. Based on the conditions of this study, we concluded that chamomile in comparison to corticosteroids promotes faster wound healing process. Copyright (C) 2008 John Wiley & Sons, Ltd.

The neuroprotective effect of dental pulp cells in models of Alzheimer`s and Parkinson`s disease

Aim of the present study was to investigate the neuroprotective effect of dental pulp cells (DPCs) in in vitro models of Alzheimer and Parkinson disease. Primary cultures of hippocampal and ventral mesencephalic neurons were treated for 24 h with amyloid beta (A beta(1-42)) peptide 1-42 and 6-OHDA, respectively. DPCs isolated from adult rat incisors were previously cultured in tissue culture inserts and added to the neuron cultures 2 days prior to neurotoxin treatment. Cell viability was assessed by the MTT assay. The co-culture with DPCs significantly attenuated 6-OHDA and A beta(1-42)-induced toxicity in primary cultures of mesencephalic and hippocampal neurons, and lead to an increase in neuronal viability in untreated cultures, suggesting a neurotrophic effect in both models. Furthermore, human dental pulp cells expressed a neuronal phenotype and produced the neurotrophic factors NGF, GDNF, BDNF, and BMP2 shown by microarray screening and antibody staining for the representative proteins. DPCs protected primary neurons in in vitro models of Alzheimer`s and Parkinson`s disease and can be viewed as possible candidates for studies on cell-based therapy.

Tooth Replantation After Use of Euro-Coffins Solution or Bovine Milk as Storage Medium: A Histomorphometric Analysis in Dogs

Purpose: Euro-Collins solution was developed for the preservation of organs for transplantation, whose characteristics have raised interest for its use as a storage medium for avulsed teeth before replantation. This study evaluated histologically and morphometrically the healing process of dog teeth replanted after storage in Euro-Collins solution or bovine milk. Materials and Methods: Eighty roots of 4 young adult mongrel clogs were randomly assigned to 4 groups (n = 20) and the root canals were instrumented and obturated with gutta-percha and a calcium hydroxide-based sealer. After 2 weeks, the teeth were extracted and subjected to the following protocols: GI (negative control), replantation immediately after extraction; GII (positive control), bench-drying for 2 hours before replantation; GIII and GIV, immersion in 10 mL of whole bovine milk and Euro-Collins solution at 4 C, respectively, for 8 hours before replantation. The animals were sacrificed 90 days postoperatively. The pieces containing the replanted teeth were subjected to routine processing for histologic and histometric analyses under light microscopy and polarized light microscopy. Results: Root resorption was observed in all groups. GII exhibited the greatest loss of dental structure (P < .01), and inflammatory resorption was predominant in this group. Storage in milk showed poorer results than immediate replantation and storage in Euro-Collins solution (P < .01). The teeth stored in Euro-Collins solution presented similar extension of root resorption and periodontal ligament reorganization to those of immediately replanted teeth. Conclusions: The findings of this study suggest that the Euro-Collins solution is an adequate storage medium for keeping avulsed teeth for up to 8 hours before replantation. Crown Copyright (C) 2010 Published by Elsevier Inc on behalf of American Association of Oral and Maxillofacial Surgeons. All rights reserved. Oral Maxillofac Surg 68:111-119, 2010

Antimicrobial Effects of Calcium Hydroxide and Chlorhexidine on Enterococcus faecalis

Introduction: Endodontic treatment is commonly based on nonspecific elimination of intraradicular micro-organisms. Although some authors prefer single-visit root canal operations for endodontic treatment, several studies have shown the importance of intracanal medication between sessions to kill microorganisms that biomechanical preparations alone cannot achieve. The purpose of this study was to evaluate the efficacy of calcium hydroxide Ca(OH)2 and chlorhexidine gel on the elimination of intratubular Enterococcus faecalis. Methods: Human uniradicular teeth contaminated with E. faecalis were treated with Ca(OH)(2), 2% chlorhexidine gel, Ca(OH)(2) plus 2% chlorhexidine gel, or saline (0.9% NaCl) as a negative control. Samples obtained at a depth of 0 to 100 mu m and 100 to 200 mu m from these root canal preparations were analyzed for bacterial load by counting the number of colonyforming units (CFUs) and bacterial viability using fluorescence microscopy. Results: A significant decrease in the number of CFUs and the percentage of viable E. faecalis was observed after treatment with either Ca(OH)(2) or chlorhexidine when compared with the control group. Additionally, chlorhexidine gel had a significantly higher antimicrobial efficacy as measured by the number of CFUs and the percentage of viable cells than Ca(OH)(2). No differences were observed between the antimicrobial properties of chlorhexidine gel with and without the addition of Ca(OH)(2). Conclusion: Both Ca(OH)(2) and chlorhexidine have antimicrobial effects on E. faecalis. Chlorhexidine had increased antimicrobial activity when compared with Ca(OH)(2.) Ca(OH)(2) combined with chlorhexidine showed similar antimicrobial activity to chlorhexidine alone. (J Endod 2010;36:1389-1393)

Propolis as storage media for avulsed teeth: microscopic and morphometric analysis in rats

The maintenance of the avulsed teeth in appropriate media for preserving the cellular viability has been important for repairing the periodontal ligament and preventing the root resorption after tooth reimplantation. Propolis is a substance capable of preserving cellular viability. This study aimed to analyze the propolis substance as a storage media for maintaining the avulsed teeth, besides to determine the ideal time period for keeping the tooth inside it. Thus, 60 maxillary right central incisors of rats were extracted and divided into five groups. In groups I and II, teeth were kept in propolis for 60 min and 6 h, respectively; in group III, teeth were kept in milk for 6 h; in group IV, teeth were kept dry for 60 min; and in group V, they were immediately reimplanted. All teeth had their root canals filled with calcium hydroxide paste. Following, teeth were reimplanted in their sockets. After 15 and 60 days, animals were killed and the obtained samples were processed in laboratory for microscopic and morphometric analyzing. The results showed that the occurrence of inflammatory resorption, dental ankylosis and the formation of the connective tissue parallel to the root surface were similar among groups. It could be verified a greater occurrence of replacement resorption in group IV when comparing to other groups. In groups I and IV, the presence of periodontal ligament-like connective tissue was substantially smaller than the other groups. Regarding to the cementum amount over the root, it could be observed that this was present in smaller amount in groups I and IV. Group II was similar to groups III and IV. Therefore, according to the results of this study, the use of propolis as a storage media for maintaining avulsed teeth could be highlighted, and the 6-h period was more appropriate than the 60-min period.

Heat-killed Enterococcus faecalis Alters Nitric Oxide and CXCL12 Production but not CXCL8 and CCL3 Production by Cultured Human Dental Pulp Fibroblasts

Introduction: Fibroblasts are the most abundant cells in dental pulp. To investigate their capacity to produce the chemokines CCL3, CXCL8, and CXCL12 as well as nitric oxide (NO), we evaluated the production of these mediators in supernatants of cultured human dental pulp fibroblasts (HDPF) stimulated by heat-killed Enterococcus faecalis (HKEF). Methods: Primary cultures of HDPF were stimulated with medium alone or HKEF (1:1, 10:1, or 100:1 bacteria:fibroblast) for 1, 6, and 24 hours. Chemokines and NO were assessed through enzyme-linked immunosorbent assay and Griess reaction, respectively. Statistical analysis was performed by using analysis of variance and Tukey post test. Results: CCL3 was not detected, whereas constitutive CXCL8 was not affected. Production of CXCL12 was increased at 1 and 6 hours, and NO was increased at the concentration of 1:1 bacteria:fibroblast at 24 hours. Viability and proliferation assays did not reveal cell number differences. Conclusions: These findings demonstrate that heat-killed E. faecalis is able to increase production of CXCL12 and NO by HDPF. (J Endod 2010;36:91-94)

Study of the effectiveness of propolis extract as a storage medium for avulsed teeth

The purpose of the present study was to evaluate the efficacy of propolis extract in maintaining the viability of human periodontal ligament (PDL) cells, and to radiographically analyze tooth replantation and the adjacent periodontium in dogs after storage in this extract. Human PDL cells were incubated with the experimental media propolis, milk, saliva, Hank`s balanced salt solution (HBSS), and Dulbecco`s modified Eagles medium (DMEM, positive controls), and distilled water (negative control). Cell viability was determined 0, 1, 3, 6, 12, and 24 h later by colorimetric MTT assay. Thirty incisors from dogs were divided into two storage time blocks (1 and 3 h) and were maintained in the experimental media. HBSS served as a positive control, and dry teeth (on gauze) as a negative control. The replanted teeth were radiographed once per month for 6 months. The radiographic images were standardized by the shortening/lengthening factor, and were both qualitatively and quantitatively analyzed. The in vitro results showed that the efficacy of propolis in maintaining functional viability of PDL cells was similar to that of milk. Propolis and milk were significantly better than controls from the 6-h time period. The in vivo results showed that teeth maintained in propolis medium exhibited replacement resorption with significant reduction in tooth length, similar to teeth maintained in saliva and dried teeth. This resorption was less intense with the 3-h storage time than the 1-h storage time. Conditions close to normal were found in teeth maintained in milk, similar to the HBSS control. Therefore, although propolis was effective in maintaining the viability of human PDL cells, resorption of the tooth replantation in dogs occurred under these experimental conditions.

Transdentinal protective role of sodium ascorbate against the cytopathic effects of H(2)O(2) released from bleaching agents

Objectives. The objectives of this study were to evaluate the transdentinal cytotoxicity of 10% and 16% carbamide peroxide gel (CP), as well as the ability of the antioxidant, 10% sodium ascorbate (SA), to protect the odontoblasts in culture. Study design. Human dentin discs of 0.5-mm thickness were obtained and were placed into artificial pulp chambers. MDPC-23 odontoblastlike cells were seeded on pulp surface of the discs and the following groups were established: G1-No Treatment (control), G2-10% SA/6hs, G3-10%/CP6hs, G4-10%SA/6hs+10%CP/6hs, G5-16%CP/6hs, and G6-10%SA/6hs+16%CP/6hs. The cell viability was measured by the MTT assay. Results. In groups where 16% CP was used, decreased cell viability was observed. Conversely, the application of 10% SA on the dentin discs, before the use of the CP, reduced the cytotoxic effects of these products on cells. Conclusions. The 16% CP cause a significant decrease in MDPC-23 cell viability and 10% SA was able to partially prevent the toxic effects of CP. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: e70-e76)

Chronic ethanol intake inhibits in vitro osteogenesis induced by osteoblasts differentiated from stem cells

The study investigated whether chronic ethanol (ETH) intake and subsequent ETH exposure of cell cultures affects osteoblast differentiation by evaluating key parameters of in vitro osteogenesis. Rats were treated with 5-20% (0.85-3.43 mM) ETH, increasing by 5% per week for a period of 4 weeks (habituation), after which the 20% level was maintained for 15 days (chronic intake). Bone-marrow stem cells from control (CONT) or ETH-treated rats were cultured in osteogenic medium which was either supplemented (ETH) or not supplemented (CONT) with 1.3 mm ethanol. Thus, four groups relating to rat treatment/culture supplementation were evaluated: (1) CONT/CONT, (2) ETH/CONT, (3) CONT/ETH and (4) ETH/ETH Cell morphology, proliferation and viability, total protein content, alkaline phosphatase (ALP) activity and bone-like nodule formation were evaluated. Chronic ethanol intake significantly reduced both food and liquid consumption and body weight gain. No difference was seen in cell morphology among treatments. Cell number was affected at 7 and 10 days as follows: CONT/CONT = CONT/ETH < ETH/CONT = ETH/ETH. Doubling time between 3 and 10 days was greater in groups of CONT animals: ETH/ETH = ETH/CONT < CONT/ETH = CONT/CONT. Cell viability and ALP activity were not affected by either animal treatment or culture exposure to ethanol. At day 21, the total protein content was affected as follows: ETH/ETH = CONT/ETH < ETH/CONT = CONT/CONT. Bone-like nodule formation was affected as follows: ETH/ETH < CONT/ETH < ETH/CONT < CONT/CONT. These results show that chronic ethanol intake, followed by the exposure of osteoblasts to ethanol, inhibited the differentiation of osteoblasts, as indicated by an increased proliferation rate and reduced bone-like nodule formation. Copyright (C) 2007 John Wiley & Sons, Ltd.

Bone cell responses to the composite of Ricinus communis polyurethane and alkaline phosphatase

The aim of this study was to evaluate the response of osteoblastic cells to the composite of Ricinus cominunis polyurethane (RCP) and alkaline phosphatase (ALP) incubated in synthetic body fluid (SBF). RCP pure (RCPp) and RCP blended with ALP 6 mg/mL polymer (RCP+ALP) were incubated in SBF for 17 days. Four groups of RCP were tested: RCPp, RCP+ALP, and RCPp and RCP+ALP incubated in SBF (RCPp/SBF and RCP+ALP/SBF). Stem cells from rat bone marrow were cultured in conditions that allowed osteoblastic differentiation on RCP discs and were evaluated: cell adhesion, culture growth, cell viability, total protein content, ALP activity, and bone-like nodule formation. Data were compared by ANOVA or Kruskal-Wallis test. The group RCP-A P was highly cytotoxic and, therefore, was not considered here. Cell adhesion (p = 0.14), culture growth (p = 0.39), viability (p = 0.46) and total protein content (p = 0.12) were not affected by either RCP composition or incubation in SBE ALP activity was affected (p = 0.0001) as follows: RCPp < RCPp/SBF < RCP+ALP/SBF. Bone-like nodule formation was not observed on all evaluated groups. The composite RCP+ALP prior to SBF incubation is cytotoxic and must not be considered as biomaterial, but the incorporation of ALP to the RCP followed by SBF incubation could be a useful alternative to improve the biological properties of the RCP. (c) 2007 Wiley Periodicals, Inc.