Histological Evidence of Wound Healing Improvement in Rats Treated with Oral Administration of Hydroalcoholic Extract of Vitis labrusca.

Plant extracts rich in phenolic compounds have been demonstrated to accelerate wound healing, but their use by oral route has been poorly studied. The leaves of Vitis labrusca are rich in phenolic acids and flavonoids. The goal of this study was to assess the healing properties of the oral administration of hydroalcoholic extract of V. labrusca leaves (HEVL) in a murine model. HEVL was obtained by Soxhlet and dynamic maceration, and their yield and phenolic acids and flavonoid contents were determined. For the wound healing assay, 8 mm wounds were performed on the back of 48 Wistar rats, assigned into four groups (n = 12): CTR (distilled water), HEVL100, HEVL200, and HEVL300 (HEVL at 100, 200, and 300 mg/kg, respectively). On days 7 and 14, wound closure rates were assessed, and the healing wounds were subjected to histological analysis. Soxhlet-obtained extract was selected for the wound healing assay because it provided a higher yield and phenolic acid and flavonoid contents. HEVL significantly reduced leukocytosis in the peripheral blood (p < 0.05), accelerated wound closure (p < 0.05), and improved collagenization (p < 0.05) on day 7, as well as enhanced the epidermal tissue thickness (p < 0.001) and elastic fiber deposition on day 14 (p < 0.01). Furthermore, HEVL promoted an increase in the histological grading of wound healing on both days 7 and 14 (p < 0.01). The doses of 200 and 300 mg/kg provided better results than 100 mg/Kg. Our data provide histological evidence that the oral administration of HEVL improves wound healing in rodents. Therefore, the extract can be a potential oral medicine for healing purposes.

Season does not have a deleterious effect on proportions of stallion seminal plasma proteins.

The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding proteins as well as of heparin-binding proteins was found. Analysis of semen characteristics revealed a somewhat contrasting picture. While only minor variations in sperm kinematics and sperm morphology were found between seasons, the flow-cytometric measurements of mitochondrial membrane potential and also, to some extent, reactive oxygen species production indicated lower sperm quality in the breeding season. Chromatin integrity and testosterone levels were unchanged between seasons. The results suggest that stallion ejaculates could be used year-round for freezing, since only minor differences in protein composition exist between the breeding and non-breeding seasons, as well as between good and bad freezers. In addition, sperm quality is not impaired during the non-breeding season.

Effect of red propolis extract isolated or encapsulated in nanoparticles on the in vitro culture of sheep preantral follicle: Impacts on antrum formation, mitochondrial activity and glutathione levels.

This study aimed to characterize the hydroethanolic extract of red propolis (HERP) and nanoparticles containing HERP for using as an additive in the culture medium of isolated ovine preantral follicles. HERP was characterized by high-performance liquid chromatography (HPLC) and determination of flavonoid content, and the nanoparticles by the mean particle diameter, polydispersity index (PI) and encapsulation efficiency (EE). The effect of HERP (10 and 20 Î·g/ml-HERP10 and HERP20 groups) and nanoparticles (NP10 and NP20 groups) on isolated secondary follicles cultured in vitro for 12 days was observed by morphological evaluation, oxidative stress markers (reactive oxygen species-ROS and glutathione-GSH) and active mitochondria. HPLC showed formononetin as the major compound in the HERP (63.92 ± 0.21 µg/mL). The content of flavonoids ranged from 2.14% to 11.00%. The nanoparticles showed mean diameter of 287.5 ± 3.9 and 479 ± 18.1 Î·m; PI of 0.117 ± 0.018 and 0.316 ± 0.039; and EE of 67.15% and 41%, respectively, for the NP10 and NP20 groups. After 12 days of culture, HERP20 and NP20 increased (p < 0.05) the percentage of normal follicles compared to NP10. HERP20 showed significantly higher percentages of antrum formation than control medium (MEM) and NP10 (p < 0.05). HERP20 also showed an increase (p < 0.05) in mitochondrial activity compared to the other treatments, except NP20 (p > 0.05), and increased GSH levels (p < 0.05) compared to MEM and HERP10. In conclusion, use of HERP (20 Î·g/ml) on in vitro culture of isolated ovine preantral follicles can increase antrum formation, mitochondrial activity and GSH levels.

Decreased inflammatory response in rat bladder after intravesical administration of capsaicin-loaded liposomes.

The objective of this work was to study the reduction in the capsaicin toxicity by encapsulation in liposomes. Capsaicin was extracted from peppers and characterized with high performance liquid chromatography (HPLC). We determined the zeta potential, polydispersivity index (PdI) and vesicle size of liposomes. Wistar rats were submitted to intravesical instillation of liposomes (LIP), capsaicin (CAP) or liposomes with capsaicin (CAPLIP). After 24 hours, bladders were removed for histological analysis. Vesicle size ranged from 68 to 105 nm with PdI smaller than 0.2 and zeta potential around -30 mV. The vesicles maintained stability over the 14-day study. The histological analysis of the CAP group showed intense inflammation in almost all bladder layers, as well as ulcer formation. Conversely, the CAPLIP group showed a smooth inflammatory reaction and hyperemia. In conclusion, the liposomes effectively protected the bladder against the irritative action of capsaicin.

Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis.

BACKGROUND: Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge. RESULTS: The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5). CONCLUSIONS: rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro.

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.

Sperm Quality in Young Bull Semen Can Be Improved by Single Layer Centrifugation.

Interest in using semen from young bulls is increasing due to identifying promising animals by genomic selection. However, sperm quality in these ejaculates may not reach currently accepted standards for the cattle breeding industry. The purpose of this study was to determine if centrifugation of semen from young bulls through the Bovicoll colloid could improve sperm quality sufficiently for the frozen semen to be acceptable for artificial insemination. Ejaculates from 19 young bulls were split and either processed by Single-Layer Centrifugation (SLC) or not (CON) before freezing. After thawing, sperm quality was evaluated by determination of membrane integrity, mitochondrial membrane potential, DNA integrity, production of reactive oxygen species, sperm morphology and motility. Approximately half of the CON samples reached acceptable post-thaw quality (membrane integrity ≥ 40%) despite being below the breeding company´s desired sperm concentration threshold pre-freezing. In the remaining samples, sperm quality was improved by SLC such that 45% of them reached acceptable quality post-thaw. Almost 75% of the young bull sperm samples could have produced usable frozen semen doses by adjusting the breeding company´s current processing protocols. Since lowering the generation interval has a direct effect on the genetic gain per year, SLC could aid genetic progress in cattle breeding.

Post-thaw semen quality in young bull ejaculates before being accepted for commercial semen doses.

BACKGROUND: Genomic selection enables bulls with desirable characteristics to be identified at a young age, but sperm quality can be poor in the ejaculates of young bulls. Few studies have been done on post-thaw sperm quality in bulls less than 10 months old. The objective of this study was to determine the age at which post-thaw sperm quality was acceptable for artificial insemination. METHODS: Semen was collected by artificial vagina; samples containing 100-500 million spermatozoa/ml were frozen for this study. Post-thaw analyses of membrane integrity (MI), mitochondrial membrane potential (MMP), chromatin integrity, morphology, production of reactive oxygen species and sperm kinematics were made. RESULTS: The age at which ejaculates exceeded the breeding company's thresholds of acceptance varied considerably among individuals, with 285 days being the earliest. Morphology (p < 0.003), MI (p = 0.0096), high MMP (p = 0.043) and superoxide production (p = 0.0084) increased between the first and last ejaculates but reached acceptable levels at different ages for individual bulls. CONCLUSIONS: It was possible to obtain acceptable post-thaw sperm quality from samples even though sperm concentration was lower than the breeding company's threshold. Therefore, it might be feasible to use ejaculates earlier than is currently considered possible, by modifying semen handling protocols.

Hydrophilic Scaffolds Containing Extracts of Stryphnodendron adstringens and Abarema cochliacarpa for Wound Healing: In Vivo Proofs of Concept.

The present work aimed to evaluate the healing effect of hydrophilic polymeric resorbable biomembrane scaffolds containing plant extracts obtained from two different species, both popularly known as Stryphnodendron adstringens or Barbatimão. The hydrogel-based scaffolds were characterized and submitted to biological tests using Wistar rats to evaluate their healing capacity. The wound retraction index and the evaluation of the inflammatory process and tissue collagenization were recorded. The extracts showed antioxidant activity with IC50 between 10 and 20 µg/mL (DPPH assay) and 4-6 mmol Trolox/g (FRAP assay). The extract of Stryphnodendron adstringens (SA) presented gallocatechin, epigallocatechin, and O-methylpigalocatechin, while the extract of Abarema cochliacarpa (AC) presented catechin, dimers of procyanidins, di-O-hydroxide, O-deoxyhexosi-hexoside, and epicatechin. The membranes containing SA extract (GELSA) were more rigid, with a more intense color, but less thick, with a more compact structure and few pores. The membranes containing AC extract (GELAC) presented a mechanical profile like the gelatin membrane (GEL), with greater permeability to water vapor. The GELAC and GELSA membranes showed similar thermal degradation profiles. The wounds treated with the membranes containing the extracts obtained high levels of retraction of the wounds with values around 60% and 80% in three and seven days, respectively. These data indicate that the compounds of both species have promising biological activities in the repair process, showing that the extracts accelerated the healing process due to the lower intensity of the inflammatory reaction and the presence of compounds such as catechin and epigallocatechin.

In Silico Screening of Putative Corynebacterium pseudotuberculosis Antigens and Serological Diagnosis for Caseous Lymphadenitis in Sheep by Enzyme-Linked Immunosorbent Assay.

Corynebacterium pseudotuberculosis is the etiologic agent of Caseous Lymphadenitis (CLA), a disease leading to severe damage in sheep and goats farming due to the lack of serological diagnosis, treatment, and effective prophylaxis. In this context, several strategies in an attempt to discover new antigens to compose diagnosis assays or vaccines are fundamental. Therefore, this study aimed to use bioinformatics software to evaluate the critical chemical characteristics of unknown proteins of C. pseudotuberculosis by selecting them for heterologous expression in Escherichia coli. For this purpose, six protein sequences of ascorbate transporter subunit, UPF protein, MMPL family transporter, Ribonuclease, Iron ABC transporter domain-containing permease, and fimbrial subunit were obtained. In silico analyses were performed using amino acid sequences to access immunodominant epitopes and their antigenic and allergenic potential and physicochemical characterization. The expressed proteins were used as an antigen for serological diagnosis by ELISA. All proteins showed distinct immunodominant epitopes and potential antigenic characteristics. The only proteins expressed were PTS and Ribonuclease. In parallel, we expressed CP40 and all were used with ELISA antigen in 49 CLA positive sera and 26 CLA negative sera. The proteins alone showed 100% sensitivity and 96.2%, 92.3%, and 88.5% specificity for rPTS, rRibonuclease, and rCP40, respectively. When proteins were combined, they showed 100% sensitivity and 84.6%, 92.3%, 88.5%, and 92.3% specificity for rPTS/rCp40, rRibonuclease/rCP40, rPTS/rRibonuclease, and rPTS/rRibonuclease/rCP40, respectively. The results of this study show an excellent correlation of sensitivity and specificity with all proteins. None of the specificity values preclude the potential of rPTS, rRibonuclease, or rCP40 for use in ELISA diagnostic assays since the results of this work are superior to those of other studies on CLA diagnosis described in the literature.

Characterization of a new multidrug-resistant Brazilian K. pneumoniae isolate and 172 Klebsiella spp. sequenced strains: Genomic island, multilocus sequence typing and capsule locus dataset.

The genus Klebsiella comprises species that cause nosocomial and community-acquired infections. A dataset was created to compile the sequence type (ST) and capsule type (K-locus) information predicted for 172 worldwide isolates of Klebsiella spp. whose complete genomes could be retrieved from the GenBank (NCBI) repository. The dataset also includes information related to one multidrug-resistant strain (B31) isolated from a patient who was admitted to an intensive care unit in the Northeast region of Brazil. This strain was phenotypically characterized and submitted to whole-genome sequencing and comparative genomics analysis as we recently reported [1]. The dataset also compiles information on Pathogenicity Islands (PIs), Resistance Islands (RIs) and Miscellaneous Islands (MIS) present in the genome of strain B31. The information provided here may support outbreak prevention policies and future epidemiological studies involving Klebsiella spp.

Comparative genomics with a multidrug-resistant Klebsiella pneumoniae isolate reveals the panorama of unexplored diversity in Northeast Brazil.

The emergence of community acquired infections increases the public health concern on K. pneumoniae and closely related bacteria among which antimicrobial resistance spreads. We report a multidrug-resistant K. pneumoniae isolate, B31, of a patient infected in the community and admitted to an intensive care unit in Northeast Brazil. Antimicrobial susceptibility and genome information were thoroughly investigated to characterize B31 in front of 172 sequenced strains of different countries. Assigned to the Sequence Type 15, which is globally spread, B31 presented extended spectrum beta-lactamase, tigecycline and ciprofloxacin resistance. Genome sequencing revealed most resistance genes being carried by plasmids with high dissemination potential. The absence of main virulence factors, like yersiniabactin and colibactin, apparently suggests a mild pathogenic strain which, on the contrary, persisted and caused severe infection in a previously healthy patient. The present study contributes to unveil the unclear genomic scenario of virulent and multidrug-resistant K. pneumoniae in Brazil.

Steady-state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro.

The aims of this study were to investigate steady-state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT-PCR was used to analyze caprine steady-state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1-3 mm) and large (3-6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM(+)) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT-PCR demonstrated an increase in steady-state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady-state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM(+) after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth.

Effects of cashew gum and nanoparticles on cooled stallion semen.

BACKGROUND: Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes at 4 °C. RESULTS: Aliquots were analysed by computer-assisted sperm motility analysis on the day of collection, and after 24 h and 48 h of cold storage. After 48 h, the total motility with NP1 (78.53 + 6.31%) was similar to control 85.79 + 6.31% at 0 h. The same pattern was observed for progressive motility. Membrane integrity assessed by flow cytometer was similar between control, TOC and G1 at all storage times. The DNA fragmentation in the control group increased at all time points, whereas chromatin integrity was maintained after 24 h in TOC and NP0.5 compared to 0 h. There was no increase in the proportion of live spermatozoa producing hydrogen peroxide, but there was a tendency for an increased proportion of spermatozoa in the live superoxide category in CG1 after 24 h cooled storage. CONCLUSIONS: The addition of CG or CG-derived NP to extender for stallion semen was not harmful to the sperm cells.

Sperm quality variables as indicators of bull fertility may be breed dependent.

A means of discriminating among bulls of high fertility based on sperm quality is needed by breeding centers. The objective of the study was to examine parameters of sperm quality in bulls of known fertility to identify useful indicators of fertility. Frozen semen was available from bulls of known fertility (Viking Genetics, Skara, Sweden): Swedish Red (n=31), Holstein (n=25) and Others (one each of Charolais, Limousin, Blonde, SKB). After thawing, the sperm samples were analyzed for motility (computer assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity and reactive oxygen species. A fertility index score based on the adjusted 56-day non-return rate for >1000 inseminations was available for each bull. Multivariate data analysis (Partial Least Squares Regression and Orthogonal Partial Least Squares Regression) was performed to identify variables related to fertility; Pearson univariate correlations were made on the parameters of interest. Breed of bull affected the relationship of sperm quality variables and fertility index score, as follows: Swedish Red: %DNA Fragmentation Index, r=-0.56, P<0.01; intact plasma membrane, r=0.40, P<0.05; membrane damaged, not acrosome reacted, r=-0.6, P<0.01; Linearity, r=0.37, P<0.05; there was a trend towards significance for Wobble, r=0.34, P=0.08. Holstein: Linearity was significant r=0.46, P<0.05; there was a trend towards significance for Wobble, r=0.45, P=0.08. In conclusion, breed has a greater effect on sperm quality than previously realized; different parameters of sperm quality are needed to indicate potential fertility in different breeds.

Recombinant CP40 from Corynebacterium pseudotuberculosis confers protection in mice after challenge with a virulent strain.

BACKGROUND: Caseous Lymphadenitis (CLA) is a contagious, infectious, chronic disease caused by Corynebacterium pseudotuberculosis, which affects mainly sheep and goats. The clinical prevalence of CLA in Brazil is 30%, resulting in decreased milk production, weight loss, and unusable meat and leather. Prophylaxis is based on vaccination; however, current vaccinations do not offer effective protection against the infection, which makes the development of a new vaccine essential to control this disease. EXPERIMENTAL APPROACH: Here, we developed a recombinant vaccine based on CP40 protein (rCP40) combined with an adjuvant (Freund's complete adjuvant or saponin) and evaluated its efficacy in a murine model of CLA. Female BALB/c mice were used in an immunization assay. KEY RESULTS: rCP40 induced high levels of IgG2a and IgG2b antibodies. After challenge with a virulent strain of C. pseudotuberculosis C57 (10(4)CFU/mL), the levels of IgG2a and IgG2b were sustained, indicating a Th1 response. The groups immunized with rCP40 protein (GES and GEF groups) showed 100% protection and was statistically significant in the GES and GEF groups (p<0.037 and p<0.0952, respectively). CONCLUSIONS: The results indicated the recombinant protein CP40 induced an specific immune response in mice that was able to afford protection after challenge, regardless the adjuvant used in the formulation.

In vivo antitumor effect, induction of apoptosis and safety of Remirea maritima Aubl. (Cyperaceae) extracts.

BACKGROUND: Remirea maritima has been widely used in the treatment of diarrhea, kidney disease, and high fever and for therapeutic purposes, such as an analgesic and anti-inflammatory. However, few scientific research studies on its medicinal properties have been reported. PURPOSE: The present study aimed to investigate the anticancer potential of aqueous extract (AE), 40% hydroalcoholic extracts (40HA) and 70% (70HA) from R. maritima in experimental models and to identify its phytochemical compounds. METHODS: The chemical composition of AE, 40HA and 70HA was assessed by HPLC-DAD and ESI-IT-MS/MS. In vitro activity was determined on cultured tumor cell, NCI-H385N (Broncho-alveolar carcinoma), OVCAR-8 (Ovarian carcinoma) and PC-3M (prostate carcinoma) by the MTT assay, and the in vivo antitumor activity was assessed in Sarcoma 180-bearing mice. Toxicological parameters were also evaluated as well as the humoral immune response. RESULTS: Among the aqueous and hydroalcoholic extracts of R. maritima, only 40HA showed in vitro biological effect potential, presenting IC50 values of 27.08, 46.62 and >50µg/ml for OVCAR-8, NCI-H385M and PC-3M cells lines, respectively. Regarding chemical composition, a mixture of isovitexin-2''-O-ß-D-glucopyranoside, vitexin-2''-O-ß-D-glucopyranoside, luteolin-7-O-glucuronide and 1-O-(E)-caffeoyl-ß-D-glucose were identified as the major phytochemical compounds of the extracts. In the in vivo study, the tumor inhibition rates were 57.16-62.57% at doses of 25mg/kg and 50mg/kg, respectively, and the tumor morphology presented increasing numbers of apoptotic cells. Additionally, 40HA also demonstrated significantly increased of OVA-specific total Ig. CONCLUSIONS: 40HA exhibited in vitro and in vivo anticancer properties without substantial toxicity that could be associated with its immunostimulating properties.

Histological analysis of the periodontal ligament and alveolar bone during dental movement in diabetic rats subjected to low-level laser therapy.

OBJECTIVE: The purpose of this research was to evaluate the histological changes of the periodontal ligament and alveolar bone during dental movement in diabetic rats subjected to low level laser therapy (LLLT). METHODS: The movement of the upper molar was performed in 60 male Wistar rats divided into four groups (n=15): CTR (control), DBT (diabetic), CTR/LT (irradiated control) and DBT/LT (irradiated diabetic). Diabetes was induced with alloxan (150 mg/kg, i.p.). LLLT was applied with GaAlAs laser at 780 nm (35 J/cm(2)). After 7, 13 and 19 days, the periodontal ligament and alveolar bone were histologically analyzed. RESULTS: The mean of osteoblasts (p<0.01) and blood vessels (p<0.05) were significantly decreased in DBT compared with CTR at 7 days, whereas the mean of osteoclasts was lower at 7 (p<0.001) and 13 days (p<0.05). In DBT/LT, only the mean of osteoclasts was lower than in CTR (p<0.05) at 7 days, but no difference was observed at 13 and 19 days (p>0.05). The collagenization of the periodontal ligament was impaired in DBT, whereas DBT/LLT showed density/disposition of the collagen fibers similar to those observed in CTR. CONCLUSIONS: LLLT improved the periodontal ligament and alveolar bone remodeling activity in diabetic rats during dental movement.

Progesterone and Follicle Stimulating Hormone interact and promote goat preantral follicles survival and development in vitro / Progesterona e Hormônio Folículo-Estimulante interagem e promovem a sobrevivência e o desenvolvimento in vitro de folículos pré-antrais caprinos

We investigated the effects of progesterone and follicle stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) alone or containing progesterone (1, 2.5, 5, 10 or 20ng/mL), FSH (50ng/mL) or the interaction between progesterone and FSH. Fresh (non-cultured control) and cultured ovarian tissues were processed for histological and ultrastructural studies. After 7 days the addition of FSH to all progesterone concentrations maintained the percentage of normal follicles similar to fresh control. At day 7 of culture, a higher percentage of developing follicles was observed only in 2.5ng/ml of progesterone associated with FSH or 10ng/ml of progesterone alone when compared with control. From day 1 to day 7 of culture, a significant increase in the percentage of developing follicles was observed in MEM and 2.5ng/ml of progesterone + FSH. In addition, after 7 days, in all treatments, there was a significant increase in follicular diameter when compared with control, except for MEM alone and in 5ng/ml of progesterone + FSH or 10ng/ml of progesterone alone. Ultrastructural studies confirmed follicular integrity after 7 days of culture in 2.5ng/ml of progesterone with FSH. In conclusion, this study demonstrated that the interaction between progesterone and FSH maintains ultrastructural integrity, stimulates primordial follicles activation and further growth of cultured caprine preantral follicles.

Este trabalho verificou os efeitos da progesterona e do hormônio folículo-estimulante (FSH) na sobrevivência e no crescimento de folículos pré-antrais caprinos. Fragmentos de tecido ovariano foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (MEM) sozinho ou contendo progesterona (1, 2.5, 5, 10 ou 20ng/mL), FSH (50ng/mL) ou a combinação entre esses dois hormônios. O tecido fresco (controle não-cultivado) e o cultivado foram processados para análise histológica e ultra-estrutural. Após 7 dias a adição de FSH a todas as concentrações de progesterone manteve o percentual de folículos normais similar ao controle fresco. No dia 7 de cultivo, um alto percentual de folículos em desenvolvimento foi observado somente no tratamento com 2,5ng/ml de progesterona associada ao FSH ou com 10ng/ml de progesterona sozinha, em relação ao controle fresco. Do dia 1 para o dia 7 de cultivo, um aumento significativo no percentual de folículos em desenvolvimento foi observado no MEM sozinho e adicionado de 2,5ng/ml de progesterona + FSH. Além disso, após 7 dias, em todos os tratamentos, houve um aumento significativo no diâmetro folicular em relação ao controle, exceto nos tratamentos com MEM sozinho, 5ng/ml de progesterona + FSH ou 10ng/ml de progesterona sozinha. A análise ultra-estrutural confirmou a integridade follicular após 7 dias de cultivo no tratamento com 2,5ng/ml de progesterona + FSH. Em conclusão, este estudo demonstrou que a interação entre progesterona e FSH mantém a integridade ultra-estrutural, estimula a ativação de folículos primordiais e o posterior crescimento de folículos pré-antrais caprinos cultivados in vitro.

Bone morphogenetic protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro / A proteína morfogenética óssea-6 (BMP-6) induz a atresia em folículos primordiais caprinos cultivados in vitro

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

O presente estudo investigou os efeitos da proteína morfogenética óssea-6 (BMP-6) no desenvolvimento in vitro de folículos primordiais caprinos. Amostras de córtex ovariano de cabras foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (meio controle) suplementado com diferentes concentrações de BMP-6. As taxas de sobrevivência, ativação e crescimento foram avaliadas por histologia clássica e microscopia eletrônica de transmissão (MET). Após 7 dias de cultivo, a análise histológica demonstrou que a BMP-6 aumentou o percentual de folículos primordiais degenerados no dia 7 quando comparados ao controle fresco (D0). Além disso, houve um aumento significativo do diâmetro folicular e oocitário em ambos os períodos de cultivo em todos os tratamentos na presença de BMP-6. Com a progressão do cultivo do dia 1 para o dia 7, nos tratamentos com 1 ou 50ng/ml de BMP-6, foi observado um aumento significativo no diâmetro folicular. Entretanto, contrário ao observado no meio controle, a MET revelou que os folículos cultivados nesses tratamentos apresentavam sinais evidentes de atresia. Em conclusão, esse estudo demonstrou que a BMP-6 afeta negativamente a sobrevivência e a ultra-estrutura de folículos primordiais caprinos.