Serum Starvation and Full Confluency for Cell Cycle Synchronization of Domestic Cat (Felis catus) Foetal Fibroblasts

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Short-term urea feeding decreases in vitro hatching of bovine blastocysts

Cows fed high-protein diets may have impaired reproductive performance. Although the pathogenesis has not been completely elucidated, it appears that not only the uterus, but also the follicle and oocyte, are affected by excessive plasma urea nitrogen (PUN) concentrations. Thus, the objective was to determine the effects of short-term urea feeding on the competence of bovine oocytes. Forty crossbred heifers (Bos indicus vs Bos taurus) were allocated to two groups, namely CONTROL (maintenance diet) and UREA (maintenance diet supplemented with 75 g of urea/day), following a cross-over design. Heifers received their respective diets for 6 d (without adaptation). On the sixth day, blood samples were harvested both before and 3 h after feeding, and cumulus oocyte complexes (COCs) were collected by ovum pick-up. Although PUN concentrations were higher in UREA than CONTROL heifers (31.31 mg/dL +/- 1.13 vs 22.12 mg/dL +/- 0.86; mean +/- SEM), neither the number of COCs recovered (8.8 +/- 1.0 vs 9.2 +/- 0.8, UREA vs CONTROL, respectively) nor their quality (based on morphology) differed significantly between groups. Next, oocytes were fertilized and cultured in vitro to assess developmental rates. There was an absence of significant differences between groups for rates of cleavage (Day 3) or blastocyst formation (Days 6, 7 and 9), but the hatched blastocyst rate on Day 11 after fertilization was lower (P < 0.05) in the UREA than the CONTROL groups (64.3 vs 83.5%). Therefore, we inferred that the effects of urea were only manifest later in development. In conclusion, high PUN concentrations decreased oocyte competence in heifers, reinforcing the hypothesis that poor reproductive performance in cows with high PUN was due, at least in part, to a deleterious effect on oocytes. (C) 2011 Elsevier Inc. All rights reserved.

Haplotypes of the bovine IgG2 heavy gamma chain in tick-resistant and tick-susceptible breeds of cattle

Bovines present contrasting, heritable phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus. Tick salivary glands produce IgG-binding proteins (IGBPs) as a mechanism for escaping from host antibodies that these ectoparasites ingest during blood meals. Allotypes that occur in the constant region of IgG may differ in their capacity to bind with tick IGBPs; this may be reflected by the distribution of distinct allotypes according to phenotypes of tick infestations. In order to test this hypothesis, we investigated the frequency of haplotypes of bovine IgG2 among tick-resistant and tick-susceptible breeds of bovines. Sequencing of the gene coding for the heavy chain of IgG2 from 114 tick-resistant (Bos taurus indicus, Nelore breed) and tick-susceptible (B. t. taurus, Holstein breed) bovines revealed SNPs that generated 13 different haplotypes, of which 11 were novel and 5 were exclusive of Holstein and 3 of Nelore breeds. Alignment and modeling of coded haplotypes for hinge regions of the bovine IgG2 showed that they differ in the distribution of polar and hydrophobic amino acids and in shape according to the distribution of these amino acids. We also found that there was an association between genotypes of the constant region of the IgG2 heavy chain with phenotypes of tick infestations. These findings open the possibility of investigating if certain IgG allotypes hinder the function of tick IGBPs. If so, they may be markers for breeding for resistance against tick infestations.

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering, that there is limited information about the preovulatory LH surge in Zebu Cattle (Bos indicus). the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n = 12) had their estrus synchronized with all intravaginal device containing progesterone (CIDR-B (R)) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2 alpha (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 11). Starting 38 h after the first PGF2 alpha injection, blood sampling and ovarian ultrasonography took place every 4 h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7 +/- 4.9 and 72.3 +/- 3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin (R) total dose = 200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2 alpha, to minimize stress. only seven blood samples were collected at 4 h interval each. and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6 +/- 1.6 and 59.5 +/- 1.9 h. respectively. after removal of progesterone source. The maximum values for LH in those animals were 19.0 +/- 2.6 ng/ml (mean +/- S.E.M.). It is concluded that, in Nelore COWS submitted to a ovarian superstimulation Protocol, the LH surge occurs approximately 35 It after removal of intravaginal device containing progesterone, and approximately 12h before the LH surge observed after an induced estrus without ovarian superstimulation (C) 2008 Elsevier B.V. All rights reserved.

Bispectral index in dogs at three intravenous infusion rates of propofol

Objective To establish the correlation between the bispectral index (BIS) and different rates of infusion of propofol in dogs. Study design Prospective experimental trial. Animals Eight adult dogs weighing 6-20 kg. Methods Eight animals underwent three treatments at intervals of 20 days. Propofol was used for induction of anesthesia (10 mg kg(-1) IV), followed by a continuous rate infusion (CRI) at 0.2 mg kg(-1) minute(-1) (P2), 0.4 mg kg(-1) minute(-1) (P4) or 0.8 mg kg(-1) minute(-1) (P8) for 55 minutes. The BIS values were measured at 10, 20, 30, 40, and 50 minutes (T10, T20, T30, T40, and T50, respectively) after the CRI of propofol was started. Numeric data were submitted to analysis of variance followed by Tukey test (p < 0.05). Results The BIS differed significantly among groups at T40, when P8 was lower than P2 and P4. At T50, P8 was lower than P2. The electromyographic activity (EMG) in P2 and P4 was higher than P8 at T40 and T50. Conclusion An increase in propofol infusion rates decreases the BIS values and EMG.

PRECLINICAL INVESTIGATION OF THE RETINAL BIOCOMPATIBILITY OF SIX NOVEL VITAL DYES FOR CHROMOVITRECTOMY

Purpose: To investigate the retinal biocompatibility of six novel vital dyes for chromovitrectomy. Methods: An amount of 0.05 mL of 0.5% and 0.05% light green (LG), fast green (FG), Evans blue (EB), brilliant blue (BriB), bromophenol blue (BroB), or indigo carmine (IC) was injected intravitreally in the right eye, whereas in the left eye balanced salt solution was applied for control in rabbits` eyes. Clinical examination, fluorescein angiography, histology with light microscopy, and transmission electron microscopy were performed after 1 and 7 days. Retinal cell layers were evaluated for morphologic alterations and number of cells. The electroretinographic changes were assessed at baseline, 24 hours and 7 days. Results: Fluorescein angiography disclosed hypofluorescent spots only in the 0.5% EB group. Light microscopy and transmission electron microscopy disclosed slight focal morphologic changes in eyes exposed to 0.05% IC, FG, BriB, similar to the control at 1 and 7 days. In the lower dose groups, EB, LG, and BroB caused substantial retinal alterations by light microscopy. At the higher dose, BroB and EB produced diffuse cellular edema and vacuolization within the ganglion cells, bipolar cells, and photoreceptors. FG and IC at 0.5% caused slight retinal alterations similar to balanced salt solution injection. LG at 0.5% caused diffuse vacuolization of bipolar cells after 1 and 7 days. Injection of 0.5% EB caused a significant decrease in neuroretinal cell counts in comparison to control eyes in the 7-day examination (P < 0.05). Electroretinography revealed intermittent prolonged latency and decreased amplitude in eyes injected with 0.5% EB, LG, BriB, and BroB, while at the lower dose, only LG and EB induced few functional changes. Conclusion: The progressive order of retinal biocompatibility, from safest to most toxic, was IC, FG, BriB, BroB, LG, EB.

The influence of erbium:yttrium-aluminum-garnet laser ablation with variable pulse width on morphology and microleakage of composite restorations

The objective of this study was to evaluate the influence of various pulse widths with different energy parameters of erbium:yttrium-aluminum-garnet (Er:YAG) laser (2.94 mu m) on the morphology and microleakage of cavities restored with composite resin. Identically sized class V cavities were prepared on the buccal surfaces of 54 bovine teeth by high-speed drill (n = 6, control, group 1) and prepared by Er:YAG laser (Fidelis 320A, Fotona, Slovenia) with irradiation parameters of 350 mJ/ 4 Hz or 400 mJ/2 Hz and pulse width: group 2, very short pulse (VSP); group 3, short pulse (SP); group 4, long pulse (LP); group 5, very long pulse (VLP). All cavities were filled with composite resin (Z-250-3 M), stored at 37A degrees C in distilled water, polished after 24 h, and thermally stressed (700 cycles/5-55A degrees C). The teeth were impermeabilized, immersed in 50% silver nitrate solution for 8 h, sectioned longitudinally, and exposed to Photoflood light for 10 min to reveal the stain. The leakage was evaluated under stereomicroscope by three different examiners, in a double-blind fashion, and scored (0-3). The results were analyzed by Kruskal-Wallis test (P > 0.05) and showed that there was no significant differences between the groups tested. Under scanning electron microscopy (SEM) the morphology of the cavities prepared by laser showed irregular enamel margins and dentin internal walls, and a more conservative pattern than that of conventional cavities. The different power settings and pulse widths of Er:YAG laser in cavity preparation had no influence on microleakage of composite resin restorations.

Micromorphology of Resin-Dentin Interfaces Using One-Bottle Etch&Rinse and Self-Etching Adhesive Systems on Laser-Treated Dentin Surfaces: A Confocal Laser Scanning Microscope Analysis

Background and Objectives: This study evaluated the hybrid layer (HL) morphology created by three adhesive systems (AS) on dentin surfaces treated with Er:YAG laser using two irradiation parameters. Study Design: Occlusal flat dentin surfaces of 36 human third molars were assigned into nine groups (n = 4) according to the following ASs: one bottle etch&rinse Single Bond Plus (3M ESPE), two-step Clearfil Protect Bond (Kuraray), and all-in-one S3 Bond (Kuraray) self-etching, which were labeled with rhodamine B or fluorescein isothiocyanate dextran and were applied to dentin surfaces that were irradiated with Er:YAG laser at either 120 (38.7 J/cm(2)) or 200 mJ/pulse (64.5 J/cm(2)), or were applied to untreated dentin surfaces (control group). The ASs were light-activated following MI and the bonded surfaces were restored with resin composite Z250 (3M ESPE). After 24 hours of storage in vegetable oil, the restored teeth were vertically, serially sectioned into 1-mm thick slabs, which had the adhesive interfaces analyzed with confocal laser microscope (CLSM-LSM 510 Meta). CLSM images were recorded in the fluorescent mode from three different regions along each bonded interface. Results: Non-uniform HL was created on laser-irradiated dentin surfaces regardless of laser irradiation protocol for all AS, while regular and uniform HL was observed in the control groups. ""Stretch mark""-like red lines were found within the HL as a result of resin infiltration into dentin microfissures, which were predominantly observed in 200 mJ/pulse groups regardless of AS. Poor resin infiltration into peritubular dentin was observed in most regions of adhesive interfaces created by all ASs on laser-irradiated dentin, resulting in thin resin tags with neither funnel-shaped morphology nor lateral resin projections. Conclusion: Laser irradiation of dentin surfaces at 120 or 200 mJ/pulse resulted in morphological changes in HL and resin tags for all ASs evaluated in the study. Lasers Surg. Med. 42:662-670, 2010. (C) 2010 Wiley-Liss, Inc.

Comparative analysis of root surface smear layer removal by different etching modalities or erbium:yttrium-aluminum-garnet laser irradiation. A scanning electron microscopy study

The purpose of this study was to evaluate the effect of erbium:yttrium-aluminum-garnet (Er:YAG) laser (2.94 mu m) irradiation on the removal of root surface smear layer of extracted human teeth and to compare its efficacy with that of citric acid, ethylenediamine tetra-acetic acid (EDTA), or a gel containing a mixture of tetracycline hydrochloride (HCl) and citric acid, using scanning electron microscopy (SEM). Thirty human dentin specimens were randomly divided into six groups: G1 (control group), irrigated with 10 ml of physiologic saline solution; G2, conditioned with 24% citric acid gel; G3, conditioned with 24% EDTA gel; G4, conditioned with a 50% citric acid and tetracycline gel; G5, irradiated with Er:YAG laser (47 mJ/10 Hz/5.8 J/cm(2)/pulse); G6, irradiated with Er:YAG laser (83 mJ/10 Hz/10.3 J/cm(2)/pulse). Electron micrographs were obtained and analyzed according to a rating system. Statistical analysis was conducted with Kruskal-Wallis and Mann-Whitney tests (P < 0.05). G1 was statistically different from all the other groups; no statistically significant differences were observed between the Er:YAG laser groups and those undergoing the other treatment modalities. When the two Er:YAG laser groups were compared, the fluency of G6 was statistically more effective in smear layer removal than the one used in G5 (Mann-Whitney test, P < 0.01). Root surfaces irradiated by Er:YAG laser had more irregular contours than those treated by chemical agents. It can be concluded that all treatment modalities were effective in smear layer removal. The results of our study suggest that the Er:YAG laser can be safely used to condition diseased root surfaces effectively. Furthermore, the effect of Er:YAG laser irradiation on root surfaces should be evaluated in vivo so that its potential to enhance the healing of periodontal tissues can be assessed.

Influence of etching with erbium, chromium:yttrium-scandium-gallium-garnet laser on microleakage of class V restoration

The aim of this in vitro study was to evaluate some parameters of dental etching when irradiated with an erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser. One-hundred sound human third molars were selected and randomly distributed into ten groups (n = 10). The class V cavities of group 1 (control) were prepared with a bur and etched with 37% phosphoric acid, while groups G2 to G10, were prepared with laser (5 W, 88.46 J/cm(2), 90/70% air/water) and etched with the following powers: G3 and G4, 0.25 W; G5 and G6, 0.5 W; G7 and G8, 0.75 W; G9 and G10, 1 W. Group G2 received no laser etching. Prior to restoration, G2, G4, G6, G8 and G10 received acid etching. After restoration, all samples were submitted to a microleakage test. According to statistical analysis (Kruskal-Wallis and Dunn`s tests), G10 presented the lowest microleakage values (P < 0.05). The other groups showed no differences between them. Etching with Er,Cr:YSGG laser (1 W) followed by phosphoric acid was effective in reducing the microleakage of class V restorations.

Calcitonin, sodium alendronate and high intensity laser in the treatment of traumatized teeth: a preliminary study

The aim of this study was to evaluate the influence of erbium:yttrium-aluminum-garnet (Er:YAG) laser compared with traditional treatment on dentin permeability to calcitonin and sodium alendronate. Forty bovine roots were sectioned and divided into eight groups. Groups 1 and 2 (G1/G2) were immersed in saline solution; G1T/G2T were immersed in ethylene diamine tetra-acetic acid plus sodium lauryl ether sulfate (EDTA-T) and sodium hypochlorite (NaOCl); G1I/G2I were irradiated with Er:YAG laser (2.94 mu m, 6 Hz, 40.4 J/cm(2)); G1TI/G2TI were immersed in EDTA-T, NaOCl and subjected to Er:YAG irradiation. After 4 h the radioactivity of the saline solution was measured. Statistical analysis revealed a significant difference (P < 0.05) when the groups treated with EDTA-T and NaOCl followed by Er:YAG laser irradiation were compared with the groups treated with EDTA-T only and with the groups that received no treatment. Er:YAG laser associated with traditional procedures significantly increased the diffusion of calcitonin and sodium alendronate through dentin. All groups showed calcitonin and sodium alendronate diffusion.

Effect of laser phototherapy on recurring herpes labialis prevention: an in vivo study

Alternative treatment for recurrent labial infection by herpes simplex virus (HSV) have been considered. The aim of this study was to evaluate the effectiveness of laser phototherapy in prevention and reduction of severity of labial manifestations of herpes labialis virus. Seventy-one patients, divided into experimental (n = 41) and control (n = 30) groups were followed up for 16 months. Patients in the control group were treated topically with aciclovir and patients in the experimental group were subjected to laser phototherapy (one session per week, 10 weeks): 780 nm, 60 mW, 3.0 J/cm(2) or 4.5 J/cm(2) on healthy (no HSV-1 infection) and affected (with HSV-1 infection) tissues. Patients in the experimental group presented a significant decrease in dimension of herpes labialis lesions (P = 0.013) and inflammatory edema (P = 0.031). The reduction in pain level (P = 0.051) and monthly recurrences (P = 0.076) did not reach statistical significance. This study represents an in vivo indication that this treatment should be further considered as an effective alternative to therapeutic regimens for herpes labialis lesions.

Morphological assessment of dentine and cementum following apicectomy with Zekrya burs and Er:YAG laser associated with direct and indirect Nd:YAG laser irradiation

Objectives. This study aimed to assess the apical surface morphology of maxillary central incisors resected 3.0 mm from the tooth apex using Zekrya burs or Er:YAG laser, with or without subsequent direct Nd:YAG laser irradiation (apical and buccal surfaces) and indirect irradiation (palatal surface). Study design. Forty maxillary central incisors were instrumented and obturated. The roots were divided into 4 groups according to the root resection method (Zekrya bur or Er: YAG laser -1.8 W, 450 mJ, 4 Hz, 113 J/cm(2)) and further surface treatment (none or Nd: YAG laser -2.0 W, 100 mJ, 20 Hz, 124 J/cm(2)). The teeth were prepared for SEM analysis. Scores ranging from 1 to 4 were attributed to cut quality and morphological changes. The data were analyzed by the Kruskal-Wallis test and by Dunn`s test. Results. SEM images showed irregular surfaces on the apical portions resected with Zekrya burs, with smear layer and grooves in the resected dentine and slight gutta-percha displacement and plasticization. On the other hand, apicectomies carried out with Er: YAG laser showed morphological changes compatible with ablated dentine, with rough surfaces and craters. In spite of the presence of plasticized gutta-percha, with the presence of bubbles, an irregular adaptation of the filling material to the root walls was also observed. Direct Nd: YAG laser irradiation of the apical and buccal surfaces of the resected roots resulted in areas of resolidification and fusion in the dentine and cementum, with a vitrified aspect; indirect Nd: YAG laser irradiation of the palatal surfaces yielded a lower number of changes in the cementum, with irregular resolidification areas. Conclusions. There were no differences in terms of cut quality between the use of burs and Er: YAG laser or between the 2 surfaces (apical and buccal) treated with Nd: YAG laser with direct irradiation. However, morphological changes were significantly less frequent on surfaces submitted to indirect irradiation (palatal) when compared with those directly irradiated. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: e77-e82)

Mast cell concentration in the wound healing process of incisions made by different instruments

The aim of this study was to compare the concentration of mast cells (MCs) in the healing process of incisions. Thirty rats were submitted to six linear incisions each, performed in the dorsal skin by carbon dioxide (CO(2)) and diode lasers, electrocautery and conventional scalpel. The animals were euthanized at intervals of 0 h, 24 h, 48 h, 72 h, 7 days and 14 days after the incisions had been made. Histological sections were obtained and stained with toluidine blue for identification of MCs, which were manually counted by conventional microscopy in 20 microscopic fields in the border of the incision, near the granulation tissue, or in the area of new collagen formation, depending on intervals. The concentration of MCs was significantly higher in the wounds made by scalpel than in those made by other techniques at 48 h and 72 h. After 72 h the number of MCs was also significantly higher after electrocautery than after incisions made by 4 W CO(2) laser. On days 7 and 14, there was no significant difference in the MC count among the different types of incisions. In summary, the MC concentration varied after different surgical incisions at early phases of wound healing. At the end of the healing process, however, there were similar MC concentrations around the incisions, suggesting that, in standard incisions in the surgical techniques studied, the wound healing process ultimately occurred in a similar pattern.