Mini-percoll gradient may be used for frozen-thawed sperm selection in sheep / Gradiente de mini-Percoll pode ser utilizado na seleção espermática de sêmen congelado de ovinos

This study evaluated the effect of increasing centrifugal force and reducing centrifugation time and volume in Percoll protocols on ram sperm parameters. Commercial semen of Santa Inês rams were used and five treatments were performed: traditional Percoll and mini-Percoll (MP) techniques (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). At post-thawing (PT) and post-selection protocols (0h), samples were assessed for spermatozoa recovery rate, motility, plasma membrane (PM) integrity, sperm capacitation and morphology and incubated at 37 C for 1, 2 and 3h. The sperm recovery rate averaged 9.1±1.4%, and most motility parameters were similar (P> 0.05) among protocols. VCL (µm/s) was higher (P< 0.05) after MP-II, III and IV (66.1±4.5) than traditional Percoll (46.3±4.9). Capacitation status and PM integrity were similar (P> 0.05) among treatments. For the first time, we have demonstrated the reduction of the gradient volume and centrifugation time associated with an increase on centrifugation force at Percoll can be successfully used for frozen-thawed ram sperm selection. MP may be used instead of traditional Percoll, decreasing costs and semen handling time.(AU)

O presente estudo avaliou o efeito do aumento da força de centrifugação, bem como da redução do tempo de centrifugação e do volume do gradiente de Percoll em diferentes protocolos nos parâmetros espermáticos de ovinos. Foi utilizado sêmen comercial de carneiros da raça Santa Inês, e cinco tratamentos foram realizados: Percoll tradicional e quatro técnicas de mini-Percoll (I- 5000 x g, 5min; II- 2500 x g, 5min; III- 1250 x g, 5min; IV- 700 x g, 10min). Após o descongelamento e a seleção espermática em cada técnica utilizada (0h), amostras foram avaliadas quanto à taxa de recuperação espermática, motilidade, integridade de membrana plasmática, capacitação e morfologia. Ao final, foram incubadas a 37 ºC por uma, duas e três horas. A taxa de recuperação média (9,1±1,4%) e a maioria dos parâmetros de motilidade foram similares (P>0,05) entre os tratamentos. VCL foi maior (P<0,05) após MP-II, III e IV (66,1±4,5) quando comparados ao Percoll tradicional (46,3±4,9). O status da capacitação e a integridade de membrana foram similares (P>0,05) entre os tratamentos. Pela primeira vez, foi demonstrado que a redução do volume do gradiente utilizado e do tempo de centrifugação, associada com o aumento da força de centrifugação nos protocolos de Percoll, pode ser usada com sucesso na seleção espermática de sêmen congelado de ovinos. O mini-Percoll pode ser utilizado em alternativa à técnica de Percoll tradicional, diminuindo custos e tempo de manipulação do sêmen durante a técnica.(AU)

Major seminal plasma proteome of rabbits and associations with sperm quality.

The present study was conducted to describe the major seminal plasma proteome of rabbits and potential associations between seminal proteins and semen criteria. Semen samples were collected from 18 New Zealand adult rabbits, and seminal plasma proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Sperm motility, vigor, concentration, morphology and membrane sperm viability were evaluated. Rabbits ejaculated 364 ±â€¯70 million sperm/ml, with 81 ±â€¯6.1% motile cells, 3.8 ±â€¯0.2 vigor and 66.7 ±â€¯2.5% sperm with normal morphology. Based on the viability and acrosome integrity assay, there were 65.8 ±â€¯2.5% live sperm with intact acrosome and most spermatozoa had both intact acrosome and functional membrane. On average, 2-D gels of rabbit seminal plasma had 232 ±â€¯69.5 spots, as determined by PDQuest software (Bio Rad, USA). Mass spectrometry allowed the identification of 137 different proteins. The most abundant proteins in rabbit seminal plasma were hemoglobin subunit zeta-like, annexins, lipocalin, FAM115 protein and albumin. The intensity of the spots associated with these five proteins represented 71.5% of the intensity of all spots detected in the master gel. Multiple regression models were estimated using sperm traits as dependent variables and seminal plasma proteins as independent ones. Also, sperm motility had positive association with beta-nerve growth factor and cysteine-rich secretory protein 1-like and a negative one with galectin-1. The percentage of rabbit sperm with intact membrane was related to seminal plasma protein FAM115 complex and tropomyosin. Then, the population of morphologically normal sperm in rabbit semen was positively linked to carcinoembryonic antigen-related cell adhesion molecule 6-like and down regulated by seminal plasma isocitrate dehydrogenase. Based on another regression model, the variation in the percentage of live sperm with intact acrosome was partially explained by the amount of leukocyte elastase inhibitor and the peptidyl-prolyl cis-trans isomerase A in the rabbit seminal fluid. The current study reports the identification of 137 proteins of rabbit seminal plasma. Major proteins of seminal secretion relate primarily to prevention of damages caused by lipid peroxide radicals and oxidative stress, membrane functionality, transport of lipids to the sperm membrane and temperature regulation. Moreover, finding seminal plasma proteins as indicators of semen parameters will improve assisted reproductive technologies.

Desempenho produtivo e econômico do confinamento de bovinos zebuínos alimentados com três dietas de alto concentrado / Economic performance of zebu cattle under feedlot fed with three high concentrate diets

Estudaram-se o desempenho produtivo e a viabilidade econômica no confinamento de 24 bovinos, inteiros, com idade média de 23 meses, sendo 12 Nelore (NEL) e 12 F1 Nelore x Brahman (NBR), divididos em três dietas: SIL - silagem de milho e ração concentrada; PEL - exclusiva de pellets; GRN - milho grão inteiro e pellets. O experimento foi realizado em um delineamento inteiramente ao acaso, em esquema fatorial 2x3, com quatro repetições. Os animais NBR apresentaram resultados semelhantes aos da raça NEL quanto aos desempenhos alimentar e econômico, para todas as variáveis analisadas durante o confinamento. Entre as três dietas, a dieta PEL, apesar de não ser a de maior custo em R$/kg, obteve o menor desempenho, com maior COT/kg, menor RT e menores margens líquidas. A dieta SIL apresentou os menores COT/kg e custo/kg de carcaça, com maiores receitas e margens líquidas. A dieta GRN obteve resultados econômicos semelhantes à SIL, porém obteve menor GMD em relação à SIL, além de ter o custo de dieta (R$/kg) mais oneroso em relação aos três tratamentos. Todas as dietas foram economicamente viáveis ao final do experimento, com ML/Kg de carcaça médios de 2,12, 0,35 e 1,09 para SIL, PEL e GRN, respectivamente, no entanto a dieta SIL foi a de melhor desempenho produtivo e econômico.

The production performance and economic viability of a feedlot of 24 cattle, with an average age of 23 months, 12 Nellore (NEL) and 12 F1 Nellore x Brahman (NBR), divided into three diets: SIL - corn silage and concentrate diet; PEL - exclusive of pellets; GRN - corn and whole grain pellets were studied. The experiment was conducted in a completely randomized scheme in a 2x3 factorial with four replications. The NBR animals showed similar results to the NEL in performance and economic viability for all variables analyzed. Among the diets, the PEL diet, although not the highest diet cost in R$/kg, had the lowest performance with higher TOC/kg, lower TR and lower net margins. The SIL diet had the lowest TOC/kg and cost/kg of carcass, with higher revenues and net margins. The economic results obtained for the GRN diet were similar to SIL, but had lower ADG compared to SIL and highest diet cost (R$/kg) among all. The diets were economically viable at the end of the trial, with average net margin/kg carcass of 2.12, 0.35 and 1.09 for SIL, FEP and GRN, respectively; however the SIL diet presented better productive and economical performance.

Avaliação de espermatozoides caprinos congelados em meio à base de água de coco em pó (ACP-101®) ou TRIS / Evaluation of goat spermatozoa frozen in media based on powder coconut water media based (ACP-101®) or TRIS

Compararam-se as características cinéticas e morfológicas de espermatozoides caprinos congelados nos meios à base de ACP-101® e TRIS. Os diluentes utilizados foram: ACP-101® (+ 2,5 por cento gema ovo + 7 por cento glicerol) e TRIS (+ 20 por cento gema ovo + 6,8 por cento glicerol). Quarenta e oito ejaculados de quatro bodes foram coletados, avaliados, divididos em duas alíquotas e diluídos nos meios ACP-101® e TRIS, respectivamente, posteriormente congelados e, após 30 dias, descongelados. A avaliação da motilidade espermática por computador foi realizada aos 5, 60 e 120 minutos pós-descongelação. As características de motilidade espermática analisadas foram: motilidade total (MT) ( por cento) e progressiva (MP) ( por cento), velocidades média do trajeto do espermatozoide (VAP) (µm/s) e linear (VSL) (µm/s) e população de espermatozoides rápidos (ER) ( por cento). As avaliações de morfologia espermática quantificaram a porcentagem de espermatozoides normais (N) e as alterações da cabeça (AC), da peça intermediária (API) e do flagelo (AF), aos cinco e 120 minutos pós-descongelação. O diluente TRIS apresentou resultados cinéticos mais elevados que o ACP-101® aos 60 e 120 minutos pós-descongelação. As AC aos 120 minutos pós-descongelação foram mais altas nos espermatozoides congelados em ACP-101®. Conclui-se que o diluente TRIS promoveu maior viabilidade in vitro dos espermatozoides caprinos pós-descongelação.

The aim of the work was to compare kinetic and morphologic characteristics of goat sperm frozen in diluent media based on ACP-101® and TRIS. The employed diluents were: ACP-101® (+ 2.5 percent egg yolk + 7 percent glycerol) and TRIS (+ 20 percent egg yolk + 6.8 percent glycerol). Forty eight ejaculates from four bucks were collected, assessed and divided into two aliquots and diluted into the experimental treatments ACP-101® and TRIS. The samples were frozen and after 30 days, thawed. The computer assisted spermatic motility evaluations were placed into 5, 60 and 120 minutes post-thawing. The motion parameters assessed were: total motility (MT) ( percent), progressive motility (MP) ( percent), average path velocity (VAP) (µm/s), straight linear velocity (VSL) (µm/s), and population of rapid spermatozoa (ER) ( percent). The morphologic parameters: normal spermatozoa (N), head alteration (AC), intermediary piece alteration (API), tail alteration (AF) were evaluated at 5 and 120 min post-thawing. The media based on TRIS showed kinetic results significantly superior to ACP-101® at 60 and 120min. After 120min post-thawing the AC was higher in frozen sperm in media based on ACP-101®. The TRIS media promoted better goat spermatozoa in vitro viability post-thawing.

An MD-PhD program in Brazil: students’ concepts of science and of common sense

In 1995, a pioneering MD-PhD program was initiated in Brazil for the training of medical scientists in experimental sciences at the Federal University of Rio de Janeiro. The program’s aim was achieved with respect to publication of theses in the form of papers with international visibility and also in terms of fostering the scientific careers of the graduates. The expansion of this type of program is one of the strategies for improving the preparation of biomedical researchers in Brazil. A noteworthy absence of interest in carrying out clinical research limits the ability of young Brazilian physicians to solve biomedical problems. To understand the students’ views of science, we used qualitative and quantitative triangulation methods, as well as participant observation to evaluate the students’ concepts of science and common sense. Subjective aspects were clearly less evident in their concepts of science. There was a strong concern about "methodology", "truth" and "usefulness". "Intuition", "creativity" and "curiosity" were the least mentioned thematic categories. Students recognized the value of intuition when it appeared as an explicit option but they did not refer to it spontaneously. Common sense was associated with "consensus", "opinion" and ideas that "require scientific validation". Such observations indicate that MD-PhD students share with their senior academic colleagues the same reluctance to consider common sense as a valid adjunct for the solution of scientific problems. Overcoming this difficulty may be an important step toward stimulating the interest of physicians in pursuing experimental research.

Comparative histopathological study of sporotrichosis and American tegumentary leishmaniasis in dogs from Rio de Janeiro.

Pyogranulomatous lesions from 80 dogs with sporotrichosis and 26 dogs with American tegumentary leishmaniasis (ATL) were compared microscopically in order to identify features that would support the diagnostic suspicion and direct the subsequent search for the aetiological agent of either infection. Odds ratios and their respective 95% confidence intervals were calculated in order to evaluate the impact of the microscopical findings on the diagnosis of either disease. Lesions with well-formed granulomata were 14 times more likely to be due to sporotrichosis than ATL. Marked neutrophil infiltration into granulomata was 12.26 times more likely to be associated with sporotrichosis when compared with lesions having mild neutrophilic infiltration. Absence of lymphocytes and macrophages in the peripheral infiltrate was associated with a 9.71 and 4.93 higher chance, respectively, of being sporotrichosis rather than ATL compared with lesions where these cells were present. Lesions with a perivascular, perifollicular and interstitial peripheral inflammatory infiltrate were 5.48 times more likely to be due to sporotrichosis than ATL when compared with lesions with a diffuse peripheral infiltrate. Histopathological analysis may therefore contribute to the diagnosis of sporotrichosis or ATL skin lesions in dogs since this method permits the identification of features that direct the diagnostic suspicion, thus facilitating the search for the aetiological agent in histological sections, permitting the precise request of subsequent tests and thereby reducing costs and time taken to achieve a definitive diagnosis and the initiation of appropriate therapy.

Histopathology of canine sporotrichosis: a morphological study of 86 cases from Rio de Janeiro (2001-2007).

The present study reports the histopathological findings of 86 skin lesions of dogs with sporotrichosis from Rio de Janeiro. Suppurative granulomatous inflammation was the predominant finding and was observed in 76 (88.37%) cases. Plasma cells surrounding the suppurative granulomas were detected in 68 (89.5%) cases and an inflammatory infiltrate at the periphery of these granulomatous lesions was observed in 63 (82.9%). Fungus-specific staining revealed yeast cells compatible with Sporothrix schenckii in 36 cases. These fungal elements were only detected in lesions characterized by suppurative granulomatous inflammation. Thus, specific staining of serial sections is recommended in the case of dogs with skin lesions whose histopathological presentation is consistent with sporotrichosis. However, due to the generally small number of yeast cells in lesions, the hypothesis of sporotrichosis should not be ruled out even if the result is negative, especially in epidemic areas where correlation with epidemiological data is particularly useful.

Dosimetric audits of photon beams in radiation therapy centres in Rio de Janeiro, Brazil.

Data related to 11 y of high-energy photon radiotherapy beam dosimetry are presented and analysed. Dosimetric evaluations were carried out using water phantoms and thimble ionisation chambers and are part of the radiation protection regulatory licensing process for medicine facilities of Brazilian government. Measurements were done at reference conditions for a standard absorbed dose of 100 cGy [cGy (=1 rad)]. The absolute per cent deviation between the measured and presumed delivered doses should not exceed the tolerance level of +/-3%. The first dosimetry survey from 1996 to 1998 showed a situation that was an object of concern. Deviations of 22 and 18.7% could be measured, although small deviations were also obtained. After 1998, the improvement in dosimetry quality control by the radiotherapy centres became clear, with most of the deviations situated within the +/-3% range. The decrease in the measured deviations presents the effective success of the Institute of Radiation Protection and Dosimetry audit programme for the improvement in the control of radiotherapy photon beams in Rio de Janeiro. Also, it is possible to recommend to Brazilian regulatory organisation a decrease in the tolerance level for dosimetric deviations in order to achieve a more precise dose delivered to patients in radiotherapy centres.

Effect of subchronic caffeine treatment on MK-801-induced changes in locomotion, cognition and ataxia in mice.

N-Methyl-D-aspartate (NMDA) receptor antagonists cause hyperlocomotion and cognitive deficits in rodents, and caffeine-tolerant mice show diminished locomotor response to NMDA receptor antagonists. The aim of this study was to evaluate the effect of subchronic caffeine treatment on MK-801-induced hyperlocomotion, ataxia and cognitive deficits, as well as amphetamine-induced hyperlocomotion in mice. Mice were treated subchronically with caffeine (0, 0.1, 0.3 and 1 mg/ml and 1, 3 and 7 days) and evaluated for locomotor activity, working memory (delayed alternation test), long-term memory (inhibitory avoidance task) and ataxia. Hyperlocomotion induced by MK-801 (0.25 mg/kg i.p.) was diminished after 3 days and almost abolished after 7 days of caffeine treatment at the 1 mg/ml dose, and this effect was also dose-dependent. Ataxia induced by 0.5 mg/kg MK-801 was not affected by caffeine treatment, but a short-lived hyperlocomotor effect was observed. Performance deficit in the inhibitory avoidance task induced by MK-801 (0.01 mg/kg) was prevented in mice treated with caffeine for 7 days at 1 mg/ml, and perseverative errors in the T-maze by MK-801 (0.4 mg/kg) were attenuated. The locomotor effect of amphetamine (5 mg/kg) was unaffected by subchronic caffeine treatment. The findings that hyperlocomotion and cognitive effects induced by MK-801 can be specifically influenced by reduced adenosinergic activity agree with a model of adenosine hypofunction in schizophrenia, since NMDA receptor antagonists are pharmacological models for this disorder.