Effects of equine chorionic gonadotropin and type of ovulatory stimulus in a timed-AI protocol on reproductive responses in dairy cows

The objectives were to evaluate the effects of equine chorionic gonadotropin (eCG) supplementation (with or without eCG) and type of ovulatory stimulus (GnRH or ECP) on ovarian follicular dynamics, luteal function, and pregnancies per AI (P/AI) in Holstein cows receiving timed artificial insemination (TAI). On Day 0, 742 cows in a total of 782 breedings, received 2 mg of estradiol benzoate (EB) and one intravaginal progesterone (P4) insert (CIDR). On Day 8, the CIDR was removed, and all cows were given PGF2 alpha and assigned to one of four treatments in a 2 x 2 factorial arrangement: (1) CG: GnRH 48 h later; (2) CE: ECP; (3) EG: eCG + GnRH 48 It later; (4) EE: eCG + ECP. There were significant interactions for eCG x ovulatory stimulus and eCG x BCS. Cows in the CG group were less likely (28.9% vs. 33.8%; P < 0.05) to become pregnant compared with those in the EG group (odds ratio [OR] = 0.28). There were no differences in P/AI between CE and EE cows (30.9% vs. 29.1%; OR = 0.85; P = 0.56), respectively. Thinner cows not receiving eCG had lower P/AI than thinner cows receiving eCG (15.2% vs. 38.0%; OR = 0.20; P < 0.01). Treatment with eCG tended to increase serum progestesterone concentrations during the diestrus following synchronized ovulation (P < 0.10). However, the treatment used to induce ovulation did not affect CL volume or serum progesterone concentrations. In conclusion, both ECP and GnRH yielded comparable P/AI. However, eCG treatment at CIDR removal increased pregnancy rate in cows induced to ovulate with GnRH and in cows with lower BCS. (C) 2009 Elsevier Inc. All rights reserved.

Effects of gonadotropin-releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers

Two experiments evaluated the effects of the first GnRH injection of the 5-d timed artificial insemination (AI) program on ovarian responses and pregnancy per AT (P/AI), and the effect of timing of the final GnRH to induce ovulation relative to AT on P/AI. In experiment 1, 605 Holstein heifers were synchronized for their second insemination and assigned randomly to receive GnRH on study d 0 (n = 298) or to remain as untreated controls (n = 307). Ovaries were scanned on study d 0 and 5. All heifers received a controlled internal drug-release (CIDR) insert containing progesterone on d 0, a single injection of PGF(2 alpha),, and removal of the CIDR on d 5, and GnRH concurrent with timed AT on d 8. Blood was analyzed for progesterone at AI. Pregnancy was diagnosed on d 32 and 60 after AI. Ovulation on study d 0 was greater for GnRH than control (35.4 vs. 10.6%). Presence of a new corpus luteum (CL) at PGF(2 alpha),, injection was greater for GnRH than for control (43.1 vs. 20.8%), although the proportion of heifers with a CL at PGF(2 alpha) did not differ between treatments and averaged 87.1%. Progesterone on the day of AT was greater for GaRH than control (0.50 +/- 0.07 vs. 0.28 +/- 0.07 ng/mL). The proportion of heifers at AI with progesterone <0.5 ng/mL was less for GURH than for control (73.8 vs. 88.2%). The proportion of heifers in estrus at AI did not differ between treatments and averaged 66.8%. Pregnancy per AI was not affected by treatment at d 32 or 60 (GnRH = 52.5 and 49.8% vs. control = 54.1 and 50.0%), and pregnancy loss averaged 6.0%. Responses to GnRH were not influenced by ovarian status on study d 0. In experiment 2, 1,295 heifers were synchronized for their first insemination and assigned randomly to receive a CIDR on d 0, PGF(2 alpha) and removal of the CIDR on d 5, and either GnRH 56 h after PGF(2 alpha) and AI 16 h later (OVS56, n = 644) or GnRH concurrent with AI 72 h after PGF(2 alpha) (COS72; n = 651). Estrus at AI was greater for COS72 than for OVS56 (61.4 vs. 47.5). Treatment did not affect P/AI on d 32 in heifers displaying signs of estrus at AI, but COS72 improved P/AI compared with OVS56 (55.0 vs. 47.6%) in those not in estrus at AI. Similarly, P/AI on d 60 did not differ between treatments for heifers displaying estrus, but COS72 improved P/AI compared with OVS56 (53.0 vs. 44.7%) in those not in estrus at AI. Administration of GnRH on the first day of the 5-d timed AI program resulted in low ovulation rate and no improvement in P/AI when heifers received a single PGF(2 alpha) injection 5 d later. Moreover, extending the proestrus by delaying the finAI GnRH from 56 to 72 h concurrent with AI benefited fertility of dairy heifers that did not display signs of estrus at insemination following the 5-d timed AI protocol.

A new presynchronization system (Double-Ovsynch) increases fertility at first postpartum timed AI in lactating dairy cows

This study evaluated a novel presynchronization method, using Ovsynch prior to the Ovsynch-timed AI protocol (Double-Ovsynch) compared to Presynch-Ovsynch. Lactating Holstein (n = 337) cows, were assigned to two treatment groups: (1) Presynch (n = 180), two injections of PGF 14 d apart, followed by the Ovsynch-timed AI protocol 12 d later; (2) Double-Ovsynch (n = 157), received GnRH, PGF 7 d later, and GnRH 3 d later, followed by the Ovsynch-timed AI protocol 7 d later. All cows received the same Ovsynch-timed AI protocol: GnRH (G1) at 68 +/- 3 DIM (mean +/- SEM), PGF 7 d later, GnRH (G2) 56 h after PGF, and AI 16 to 20 h later. Pregnancy was diagnosed 39-45 d after timed AI. Double-Ovsynch increased the pregnancies per AI (P/AI) compared to Presynch-Ovsynch (49.7% vs 41.7%, P = 0.03). Surprisingly, Double-Ovsynch increased P/AI only in primiparous (65.2% vs 45.2%; P = 0.02) and not multiparous (37.5% vs 39.3%) cows. In a subset of 87 cows, ovarian ultrasonography and progesterone (P4) measurements were performed at G1 and 7 d later. Double-Ovsynch decreased the percentage of cows with low P4 (<1 ng/mL) at G1 (9.4% vs 33.3%) and increased the percentage of cows with high P4 (>= 3 ng/mL) at PGF (78.1% vs 52.3%). Thus, presynchronization of cows with Double-Ovsynch increased fertility in primiparous cows compared to a standard Presynch protocol, perhaps due to induction of ovulation in non-cycling cows and improved synchronization of cycling cows. Future studies are needed, with a larger number of cows, to further test the hypothesis of higher fertility with Double-Ovsynch, and to elucidate the physiological mechanisms that underlie apparent changes in fertility with this protocol. (C) 2008 Elsevier Inc. All rights reserved.

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TA!) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2 x 2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5 +/- 2.3 d postpartum, received a 3 mg norgestomet ear implant se, plus 3 mg norgestomet and 5 mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 mu g gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means +/- SEM, 1.53 +/- 0.1 vs. 0.48 +/- 0.1 mm/d; P < 0.0001), its diameter on Day 11(11.4 +/- 0.6 vs. 9.3 +/- 0.7 mm; P = 0.03), as well as ovulation rate (80.8% vs. 50.0%, P = 0.02), whereas GnRH improved the synchrony of ovulation (72.0 +/- 1.1 VS. 71.1 +/- 2.0 h). In Experiment 2 (n = 599 cows, 40 to 120 d postpartum), pregnancy rates differed (P = 0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control. GnRH, eCG, and eCG + GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 318%, P = 0.002; and 48.0% vs 37.6%, P = 0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol. (C) 2010 Elsevier Inc. All rights reserved.

Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox

Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 A, beta = 96.03 degrees. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V (M) of 2.12 A3 Da-1, corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques.

Severity of Oral Mucositis in Patients Undergoing Hematopoietic Cell Transplantation and an Oral Laser Phototherapy Protocol: A Survey of 30 Patients

Background Data and Objective: Oral mucositis (OM) is one of the worst cytotoxic effects of chemotherapy and radiotherapy in patients undergoing hematopoietic cell transplantation (HCT), and it causes severe morbidity. Laser phototherapy has been considered as an alternative therapy for prevention and treatment of OM. The aim of this study was to describe the incidence and severity of OM in HCT patients subjected to laser phototherapy, and to discuss its effect on the oral mucosa. Patients and Methods: Information concerning patient age and gender, type of basic disease, conditioning regimen, type of transplant, absence or presence of pain related to the oral cavity, OM grade, and adverse reactions or unusual events were collected from 30 patients undergoing HCT (allogeneic or autologous). These patients were given oral laser phototherapy with a InGaAIP laser (660 nm and 40 mW) daily. The data were tabulated and their frequency expressed as percentages. Results: In the analysis of those with OM, it was observed that 33.4% exhibited grade I, 40% grade II, 23.3% grade III, and 3.3% grade IV disease. On the most critical post-HCT days (D+5 and D+8), it was observed that 63.3% of patients had grade I and 33.3% had grade II disease; no patients had grade III or IV disease in this period. This severity of OM was similar to that seen in other studies of laser phototherapy and OM. Conclusion: The low grades of OM observed in this survey show the beneficial effects of laser phototherapy, but randomized clinical trials are necessary to confirm these findings.

Bone Marrow Concentrate and Bovine Bone Mineral for Sinus Floor Augmentation: A Controlled, Randomized, Single-Blinded Clinical and Histological Trial-Per-Protocol Analysis

Purpose: The purpose of this work was to evaluate the potential of substituting autogenous bone (AB) by bone marrow aspirate concentrate (BMAC). Both AB and BMAC were tested in combination with a bovine bone mineral (BBM) for their ability of new bone formation (NBF) in a multicentric, randomized, controlled, clinical and histological noninferiority trial. Materials and Methods: Forty-five severely atrophied maxillary sinus from 26 patients were evaluated in a partial cross-over design. As test arm, 34 sinus of 25 patients were augmented with BBM and BMAC containing mesenchymal stem cells. Eleven control sinus from 11 patients were augmented with a mixture of 70% BBM and 30% AB. Biopsies were obtained after a 3-4-month healing period at time of implant placement and histomorphometrically analyzed for NBF. Results: NBF was 14.3%+/- 1.8% for the control and nonsignificantly lower (12.6%+/- 1.7%) for the test (90% confidence interval: -4.6 to 1.2). Values for BBM (31.3%+/- 2.7%) were significantly higher for the test compared with control (19.3%+/- 2.5%) (p < 0.0001). Nonmineralized tissue was lower by 3.3% in the test compared with control (57.6%; p = 0.137). Conclusions: NBF after 3-4 months is equivalent in sinus, augmented with BMAC and BBM or a mixture of AB and BBM. This technique could be an alternative for using autografts to stimulate bone formation.

Comparison of the Salmonella/Microsome Microsuspension Assay with the new Microplate Fluctuation Protocol for Testing the Mutagenicity of Environmental Samples

The objective of this study was to compare the responses of the Salmonella/microsome microsuspension assay with the new microplate fluctuation protocol (MPF) for the evaluation of the mutagenic activity of environmental samples. Organic extracts of total particulate atmospheric air samples, surface waters, and effluents were tested in dose-response experiments. The assays were performed with strain TA98 in the absence and presence of S9 mix. Both protocols produced similar results, despite the fact that the maximum score of the MPF is limited to 48 wells, whereas in the regular plate assay it is possible to count up to 1,500 colonies using an automatic counter. Similar sensitivities based on the lowest dose that resulted in a positive response were obtained for both assays. The MPF procedure is less laborious (e.g., all-liquid format, use of multi-channel pipettors) and allows for automation of the pipetting and dispensing steps, thus, reducing time of the analysis which is particularly important in environmental quality monitoring programs or in effect-directed analysis. The results show that the MPF procedure is a promising tool to test environmental samples for mutagenic activity. Environ. Mol. Mutagen. 51:31-38, 2010. (C) 2009 Wiley-Liss, Inc.

Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial

Background: Blood pressure (BP) within pre-hypertensive levels confers higher cardiovascular risk and is an intermediate stage for full hypertension, which develops in an annual rate of 7 out of 100 individuals with 40 to 50 years of age. Non-drug interventions to prevent hypertension have had low effectiveness. In individuals with previous cardiovascular disease or diabetes, the use of BP-lowering agents reduces the incidence of major cardiovascular events. In the absence of higher baseline risk, the use of BP agents reduces the incidence of hypertension. The PREVER-prevention trial aims to investigate the efficacy, safety and feasibility of a population-based intervention to prevent the incidence of hypertension and the development of target-organ damage. Methods: This is a randomized, double-blind, placebo-controlled clinical trial, with participants aged 30 to 70 years, with pre-hypertension. The trial arms will be chlorthalidone 12.5 mg plus amiloride 2.5 mg or identical placebo. The primary outcomes will be the incidence of hypertension, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new sub-clinical atherosclerosis, and sudden death. The study will last 18 months. The sample size was calculated on the basis of an incidence of hypertension of 14% in the control group, a size effect of 40%, power of 85% and P alpha of 5%, resulting in 625 participants per group. The project was approved by the Ethics committee of each participating institution. Discussion: The early use of blood pressure-lowering drugs, particularly diuretics, which act on the main mechanism of blood pressure rising with age, may prevent cardiovascular events and the incidence of hypertension in individuals with hypertension. If this intervention shows to be effective and safe in a population-based perspective, it could be the basis for an innovative public health program to prevent hypertension in Brazil.

Otolaryngology and Speech Therapy evaluation in the assessment of oropharyngeal dysphagia: a combined protocol proposal

Dysphagia is a symptom associated with an array of anatomical and functional changes which must be assessed by a multidisciplinary team to guarantee optimal evaluation and treatment, preventing potential complications. Aim: The aim of the present study is to present the combined protocol of clinical and swallowing videoendoscopy carried by ENT doctors and speech therapists in the Dysphagia Group of the ENT Department - University Hospital. Materials and Methods: Retrospective study concerning the use of a protocol made up of patient interview and clinical examination, followed by an objective evaluation with swallowing videoendoscopy. The exam was performed in 1,332 patients from May 2001 to December 2008. There were 726 (54.50%) males and 606 (45.50%) females, between 22 days and 99 years old. Results: We found: 427 (32.08%) cases of normal swallowing, 273 (20.48%) mild dysphagia, 224 (16.81%) moderate dysphagia, 373 (27.99%) severe dysphagia and 35 (2.64%) inconclusive exams. Conclusion: The combined protocol (Otolaryngology and Speech Therapy), is a good way to approach the dysphagic patient, helping to achieve early and safe deglutition diagnosis as far as disorder severity and treatment are concerned.

Tadalafil and fluoxetine in premature ejaculation: Prospective, randomized, double-blind, placebo-controlled study

Introduction: Premature ejaculation ( PE) is a common male sexual disorder. An ideal, reliable and effective treatment is desired by many men and couples affected by this condition. Aim: Evaluate if the association of a phosphodiesterase- 5 inhibitor, tadalafil, and a selective serotonin reuptake inhibitor, fluoxetine, can prolong the intravaginal ejaculatory latency time ( IELT) in men with lifelong premature ejaculation. Methods: Sixty patients with lifelong premature ejaculation and without erectile dysfunction ( ED) with IELT less than 90 s were enrolled in the protocol and randomized into 4 groups to use a combination of medications: ( 1) tadalafil 20 mg plus fluoxetine 90 mg, ( 2) fluoxetine 90 mg plus placebo, ( 3) tadalafil 20 mg plus placebo, and ( 4) two different placebo capsules ( control). Before starting the medications, each man timed his IELT with a stopwatch, and likewise during the treatment period. Fluoxetine 90 mg or placebo was taken once a week plus tadalafil 20 mg or placebo within a 36- hour frame of intended sexual intercourse with a steady partner. Patients were prospectively followed for 12 weeks. One- way ANOVA was used for statistical comparisons of IELT results in each group. Results: Mean IELT before starting treatment was 51.3 +/- 23 s. With one- way ANOVA, a statistically significant difference in post- treatment IELT was seen with combination treatment compared to placebo ( p < 0.001). There were increases in IELT from baseline in patients using fluoxetine plus tadalafil ( 49.57 +/- 25.87 to 336.13 +/- 224.77) (p < 0.001), fluoxetine (56.55 +/- 18.55 to 233.62 +/- 105.08) ( p < 0.001) and tadalafil (49.26 +/- 19.43 to 186.53 +/- 159.05) (p = 0.001). The increases in each group were statistically significant compared to the placebo (49.86 +/- 19.43 to 67.82 +/- 46.18) ( p = 0.042). Conclusion: Fluoxetine plus tadalafil significantly increased the IELT from baseline in men with lifelong premature ejaculation when compared to placebo, tadalafil or fluoxetine. Copyright (C) 2008 S. Karger AG, Basel.

PROWESS-SHOCK TRIAL: A PROTOCOL OVERVIEW AND PERSPECTIVES

Sepsis remains a challenge for intensive care physicians, as it keeps up with high mortality rate in spite of the high costs associated with its treatment. Several studies indicate that the infusion of Drotrecogin-alpha activated (DrotAA) reduce mortality in patients at high risk of death when administered early and secured the appropriate initial treatment of sepsis as recommended by Surviving Sepsis Campaign. Europe and United States of America differ regarding the criteria of high risk of death in sepsis, two or more organ dysfunctions and Acute Physiology and Chronic Health Evaluation 25 or more, respectively. In addition to varied definitions of high risk of death for inclusion of patients in sepsis studies, the possibility of bleeding related to drug use and intrinsic limitations related to study design led the Company to develop a new randomized, multinational, placebo-controlled, double-blind study to assess the effectiveness of drug in patients with septic shock in adults.

The remodeling of collagen fibers in rats ankles submitted to immobilization and muscle stretch protocol

To evaluate the remodeling of collagen fibers in the articular cartilage of rat ankles, with and without immobilization, after application of muscle stretching protocol. Twenty three Wistar rats were divided into four groups: immobilized (I), n = 6; immobilized and stretched (IS), n = 6; stretched (S), n = 6 and control (C), n = 5. The animals in groups I and IS were submitted to immobilization. After the period of immobilization, the animals in groups IS and S were submitted to a muscle stretching protocol. At the end of the experiment, the animals were euthanized and the joints removed, processed and stained with Picrosirius red. The analysis was carried out using a polarized light microscope. The density of collagen fibers were quantified according to the intensity of birefringence displayed. By way of statistical analyses, the right and left hind limbs of the different groups were compared based on the total density of collagen fibers, the density of thick collagen fibers and the density of thin collagen fibers. Immobilization promoted a reduction in density of the thin fibers and of total collagen. The muscle stretching protocol after immobilization promoted a reduction in density of the total collagen and of the thick fibers, but the density of the thin fibers showed the same values as control. The collagen fibers were remodeled by the different stimuli. Immobilization was harmful to the collagen fibers and the muscle stretching protocol only recovered the thin collagen fibers.