Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Cardioprotective effects of alternagin-C (ALT-C), a disintegrin-like protein from Rhinocerophis alternatus snake venom, on hypoxia-reoxygenation-induced injury in fish.

Alternagin-C (ALT-C) is a disintegrin-like peptide purified from Rhinocerophis alternatus snake venom with the property of inducing vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, and angiogenesis. Therefore, this protein could be interesting as a new approach for ischemic heart diseases, an imbalance between myocardial oxygen supply and demand, leading to cardiac dysfunction. We investigated the effects of a single dose of alternagin-C (0.5 mg kg-1, via intra-arterial), after 7 days, on hypoxia/reoxygenation challenge in isolated ventricle strips and on morphological changes and density of blood vessels of the heart, using fish as an alternative experimental model. ALT-C treatment provided protection of cardiomyocytes against hypoxia/reoxygenation-induced negative inotropism. ALT-C also stimulated angiogenesis and improved excitation-contraction coupling during hypoxic conditions. Our results provide a new insight into a functional role of ALT-C against hypoxia/reoxygenation-induced cardiomyocyte injury pointing out to a potential therapeutic strategy for ischemia-related diseases.

Ultrastructure of bovine placenta during all gestational period / Ultraestrutura de placenta bovina durante todo o período gestacional

Placentas from pregnant cows with different gestation periods were used. Placental fragments of all groups were processed and evaluated by transmission electron microscopy. After fragment analysis, bovine placenta was observed to be epitheliochorial type in early pregnancy, becoming progressively sinepiteliocorial at the beginning of the second trimester. There are no ultrastructural evidences of inflammation in the region of caruncles throughout gestation, despite the invasion of caruncle proper lamina by trophoblast cells. However, throughout pregnancy and especially at the end, there were evident signs of cell degeneration in both trophoblast and the uterine epithelium. The active trophoblast cells intensely phagocytize cellular debris. There are complex interdigitations between the surface of the trophoblast and the uterine epithelium, which is related to the increase of the exchange surface between mother and fetus. At the end of pregnancy, interdigitations disappear, favoring the detachment and expulsion of the placenta after birth.(AU)

Foram utilizadas placentas de vacas abatidas em frigorífico com diversos tempos gestacionais. Fragmentos de placentomo de todos os grupos foram processados e avaliados em microscopia eletrônica de transmissão. Após análise dos fragmentos, observou-se que a placenta bovina é do tipo epiteliocorial no início da gestação, tornando-se sinepiteliocorial progressivamente a partir do início do segundo mês de gestação. Não existem evidências ultraestruturais de inflamação na região das carúnculas durante toda a gestação, apesar da invasão da lâmina própria caruncular por células trofoblásticas. No entanto, durante toda a gestação e em especial ao seu final, foram observados sinais evidentes de degeneração celular, tanto do trofoblasto como do epitélio uterino. As células trofoblásticas ativas fagocitam intensamente os debris celulares originados dessas degenerações. Existem complexas interdigitações entre a superfície do trofoblasto e do epitélio uterino, o que estaria relacionado com o aumento da superfície de troca entre mãe e feto. Ao final da gestação, praticamente desaparecem essas interdigitações, favorecendo o descolamento e a expulsão da placenta após o parto.(AU)

Sperm morphometry and chromatin condensation in Nelore bulls of different ages and their effects on IVF.

The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8-2 years), adult (3.5-7 years), and senile (8-14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%-16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy.

Number and density of equine preantral follicles in different ovarian histological section thicknesses.

Regardless of species, advances in preantral follicle culture and cryopreservation and transplant of ovarian tissue techniques are dependent on the number and density of preantral follicles in the ovary. This study tested the effect of different histological section thicknesses on number, classification, and density of equine preantral follicles. An ovarian fragment was obtained from 5- to 10-year-old mares (n = 14) after slaughter, and each fragment was submitted to three histological section thickness treatments: 3, 5, and 7 µm. The area (cm(2)) of each ovarian fragment was measured, and the sections were evaluated by light microscopy. The percentage of morphologically normal follicles (89%) was similar (P > 0.05) among primordial, transitional, and primary follicles and also among histological section thicknesses. A greater (P < 0.05) number of preantral follicles per histological section were seen in the 7-µm (8.0 ± 2.2) than that in the 3-µm (3.4 ± 0.7) treatment. Furthermore, a linear regression analysis reported that the number of preantral follicles increased (P < 0.05) when a thicker section treatment was used. However, no association (P > 0.05) between follicular density and treatment was observed. The mean number of preantral follicles per fragment (45.3 ± 18.8) and the follicular density (3.0 ± 0.5 follicles per cm(2)) were different (P < 0.05) among mares. In conclusion, this study on equine preantral follicles reported that (1) a 7-µm histological section thickness might be recommended because it allowed identification of a greater number of preantral follicles per sample, (2) a large individual variation in follicle population and density was detected regardless of histological section thickness, and (3) mares have a low number and density of preantral follicles when compared with those reported for other species.

Characterization of the virulence, growth temperature and antibiotic resistance of the Campylobacter jejuni IAL 2383 strain isolated from humans.

The objective of this study was to characterize the C. jejuni IAL2383 strain isolated from humans in Brazil. Transcripts for the racR, dnaJ and ciaB genes were found and flaA, plda and cadF genes were present in the genome and bacteria was sensitive to most of the important antimicrobials used to treat humans. C. jejuni IAL2383 is a good experimental model to analyze the interactions with cells.

The spreading process of Ehrlichia canis in macrophages is dependent on actin cytoskeleton, calcium and iron influx and lysosomal evasion.

Ehrlichia canis is an obligate intracellular microorganism and the etiologic agent of canine monocytic ehrlichiosis. The invasion process has already been described for some bacteria in this genus, such as E. muris and E. chaffeensis, and consists of four stages: adhesion, internalisation, intracellular proliferation and intercellular spreading. However, little is known about the spreading process of E. canis. The aim of this study was to analyse the role of the actin cytoskeleton, calcium, iron and lysosomes from the host cell in the spreading of E. canis in dog macrophages in vitro. Different inhibitory drugs were used: cytochalasin D (actin polymerisation inhibitor), verapamil (calcium channel blocker) and deferoxamine (iron chelator). Our results showed a decrease in the number of bacteria in infected cells treated with all drugs when compared to controls. Lysosomes in infected cells were cytochemically labelled with acid phosphatase to allow the visualisation of phagosome-lysosome fusion and were further analysed by transmission electron microscopy. Phagosome-lysosome fusion was rarely observed in vacuoles containing viable E. canis. These data suggest that the spreading process of E. canis in vitro is dependent on cellular components analysed and lysosomal evasion.

Characterization of the virulence, growth temperature and antibiotic resistance of the Campylobacter jejuni IAL 2383 strain isolated from humans

The objective of this study was to characterize the C. jejuni IAL2383 strain isolated from humans in Brazil. Transcripts for the racR, dnaJ and ciaB genes were found and flaA, plda and cadF genes were present in the genome and bacteria was sensitive to most of the important antimicrobials used to treat humans. C. jejuni IAL2383 is a good experimental model to analyze the interactions with cells.

Low-power laser therapy for repairing acute and chronic-phase bone lesions.

To evaluate the therapeutic activity of low-power laser (InGaAlP: 670 nm/30 mW), at doses of 90 J/cm(2), on the process of acute and chronic-phase repair of bone lesions of Wistar rats. Sixty-three adult males were divided into nine groups subjected to bone injury, in order to form the following treatments: T1 (control); T2 (acute-phase); T3 (chronic-phase) which were subdivided into three subgroups (n=7), analyzed on the 9th, 17th and 28th days post-surgery, after a period of daily treatment with laser. The animals with acute-phase treatment presented a more extensive endochondral ossification process. Laser-treated animals showed significant increases in serum alkaline phosphatase levels and had an effect on biomechanical property, resulting in a gradual increase in bone stiffness. Laser therapy aided the bone consolidation process and favored the physiopathologic mechanisms involved in bone tissue repair, and its effects were more prominent when treatment started during the acute phase of the injury.

Participation of the Cytoskeletal and Lysosomal Compartments in Campylobacter jejuni Invasion of Caco-2 cells, the Cellular Response by Morphometric Analysis and the Presence of Cytokine and Chemokine Transcripts.

This study aimed to evaluate the participation of actin and tubulin in the process of internalisation, the interaction of bacterial phagosomes with lysosomes, the morphometric changes and the expression of inflammatory cytokines in Caco-2 cells infected with Campylobacter jejuni. Both actin and tubulin participated in the process of internalisation. Inside the cells, lysosomes fuse with phagosomes, which may lead to bacterial death because after 2 h, the bacteria were not detected by Transmission electron microscopy (TEM). There is increased expression of TGF-ß3 during the early stages, and IL-8 was expressed after 60 min p.i. This work showed that C. jejuni invades and causes major morphometric changes in epithelial cells. In response, the cells increase their expression of cytokines that can lead to inflammation. The mechanisms of invasion are dependent on actin and tubulin, and once internalised, lysosomes fuse with phagosomes.

Effect of sequence of insemination after simultaneous thawing of multiple semen straws on conception rate to timed AI in suckled multiparous Nelore cows.

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 °C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated.

Transfer, viability and colonisation of Campylobacter jejuni in the chicken vitellus and in embryos.

1. The objective of this study was to evaluate the ability of Campylobacter jejuni to penetrate and colonise eggs from specific-pathogen-free (SPF) and heavy breeder hens, and to determine its effects on the viability of SPF embryos. 2. We detected C. jejuni in 10% of breeder hens and 20% of SPF eggs, which demonstrates the ability of the bacteria to go through the pores of eggs and contaminate the vitellus after 3 h of contact. These results indicate that there is a risk of contamination under commercial production conditions, where, after oviposition, there is contact between the egg and organic material such as faeces and blood. 3. We observed that in 80% of SPF eggs analysed, C. jejuni survived the 21-d incubation period. This positive result suggests that this microorganism was also responsible for early embryonic mortality. 4. The ability of C. jejuni to penetrate the eggs in this study suggests that serious problems may occur under natural field conditions, which may cause significant problems for producers.

Avaliação computacional da compactação da cromatina e de características morfométricas da cabeça de espermatozoides de coelho (Oryctolagus cuniculus) / Computational evaluation of chromatin condensation and morphometric characteristics of rabbit (Oryctolagus cuniculus) sperm head

Investigou-se a correlação entre a morfometria da cabeça e a intensidade da condensação e heterogeneidade da cromatina em espermatozoides de coelho (Oryctolagus cuniculus). Para tal, utilizaram-se 35 esfregaços de sêmen de coelhos da raça Nova Zelândia, corados com azul de toluidina e avaliados por análise de imagem computacional. As imagens foram obtidas digitalmente em tons de cinza e avaliadas por algoritmos desenvolvidos em ambiente de programação Scilab. As mensurações obtidas da cabeça dos espermatozoides foram área, perímetro, comprimento, largura, relação comprimento largura, elipsidade, fator de forma, descritores Fourier e simetria lateral e anteroposterior. Também foram avaliadas a intensidade da compactação e a heterogeneidade da cromatina espermática. Os espermatozoides de coelho apresentaram compactação e heterogeneidade cromatínica mais intensas do que os de touro e observou-se correlação significativa entre características morfométricas da cabeça e compactação e heterogeneidade cromatínica. Conclui-se que a cromatina é importante para a constituição morfológica da cabeça de espermatozoides de coelho e que a cromatina espermática de coelho é naturalmente mais heterogênea e menos compactada que a de touro.

The correlation between the head morphometry and the intensity of condensation and heterogeneity of sperm chromatin were investigated in rabbit (Oryctolagus cuniculus). To this, 35 semen smears from New Zealand rabbits were stained with toluidine blue and evaluated by computer image analysis. The images were obtained in digital grayscale and analyzed by algorithms developed in the Scilab programming environment. The measurements obtained from the sperm heads were area, perimeter, length, width, length:width ratio, ellipticity, shape factor, lateral and anterior-posterior symmetries, and Fourier descriptors. The intensity and heterogeneity of the compaction of sperm chromatin was also evaluated. The rabbit spermatozoa showed chromatin heterogeneity and condensation more intense than the bull spermatozoa, and it was observed correlation between morphometric characteristics of the head and chromatin compaction and heterogeneity. The results suggest that chromatin is important for the morphological constitution of the head morphology spermatozoa head, as well as, rabbit sperm chromatin is inherently more heterogeneous and less condensed than the bull sperm chromatin.

Biological characterization of a myotoxin phosphoplipase A2 homologue purified from the venom of the snake Bothrops moojeni

A myotoxin phospholipase A2 homologue, BmooMtx, was isolated from the venom of Bothrops moojeni by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. SDS-PAGE showed the enzyme to be a monomer with a molecular weight of 16,500. BmooMtx induced release of creatine kinase and morphological analyses indicated that it provoked an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 hours after injection. Anti-BmooMTx antibodies partially neutralized the myotoxic activity of BmooMtx and crude B. moojeni venom, as judged by determination of plasma creatine kinase levels and histological evaluation of skeletal muscle in mice. Anti-BmooMTx antibodies were effective in reducing the plasma creatine kinase levels of crude B. alternatus and B. leucurus venoms, evidencing immunological cross-reactivity between BmooMTx and other bothropic venoms. Intraplantar (i.pl.) injection of BmooMtx (1 to 15 ìg/animal) caused a dose- and time-dependent hyperalgesia and edematogenic responses. Dexamethasone (0.4 mg/kg), meloxicam (2 mg/kg) and promethazine (5 mg/kg) markedly reduced the hyperalgesia. Our data suggest that these drugs may likely serve as complementary therapies in cases of accidents with Bothrops moojeni, provided that such pharmacological treatments are administered immediately after the incident.

Eletroacupuntura no reparo do tendão calcanear comum em coelhos após enxertia com peritônio bovino / Eletro-acupuncture in the rabbit acchiles tendon repair using bovine peritoneum graft

Avaliou-se o efeito da eletroacupuntura (EA) sobre os aspectos macroscópicos e microscópicos em 36 coelhos que tiveram o tendão calcanear comum seccionado e que, após 30 dias, receberam enxerto de peritônio bovino conservado em solução saturada de sal. Após a cirurgia, os animais foram distribuídos em três grupos de igual número: os do grupo E10 foram estimulados pela EA do segundo ao 11º dia de pós-operatório, os do grupo E20 do 11º ao 31º dia de pós-operatório e os do grupo C não foram estimulados. Posteriormente, os animais foram redistribuídos em três subgrupos de igual número, os quais foram observados no pós-operatório por 11, 31 e 91 dias. Nos coelhos do E10 e E20, ocorreu diminuição de aderência da área enxertada aos tecidos circunvizinhos. Aos 31 dias de pós-operatório, observou-se nos do E10 tecido conjuntivo. Verificou-se intensa neovascularização no local enxertado em todos os animais que receberam estímulo pela EA. Nos coelhos do E10, aos 91 dias, verificou-se intensa quantidade de tecido conjuntivo, com fibras colágenas organizadas e paralelas ao eixo do tendão. O estímulo pela EA na fase precoce do pós-operatório estimulou a maturação cicatricial com mínima reação inflamatória.

The effects of eletro-acupuncture (EA) were evaluated on clinical and histological aspects in 36 rabbits that had the acchiles tendon sectioned and grafted after 30 days using bovine peritoneum graft preserved in saturated salt solution. The animals were randomly distributed into three groups. They were stimulated by EA from the 2nd to the 11th post-operative day (S10 group), from the 11th to the 31st post-operative day (S20 group), and were not stimulated (C group). Then, the rabbits were distributed into three sub-groups and were observed for 11, 31, and 91 days. The rabbits from S10 and S20 groups presented decreased adhesions between graft and adjacent tissues. The rabbits from S10 group showed connective tissue at 31 days of the post-operative period. Intense graft neovascularization was observed in all animals stimulated by EA. Large amounts of connective tissue with organized and parallel to the tendon axis collagen fibers were detected on the day 91st in the S10 group animals. The early EA stimulation during the post-operative period stimulated the tendon healing with minimal inflammatory reaction.

Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex.

A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatographic steps using DEAE-Sephacel and gel-filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS-PAGE under reducing conditions and gave one main peak at 29,814 KDa in MALDI-TOF/TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the Aalpha-chain of fibrinogen and, more slowly, the Bbeta-chain. Its fibrinogenolytic activity is inhibited by beta-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at >or=80 degrees C. Intraplantar injection of Eumiliin (1-25 microg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1-5h after enzyme injection. Intraplantar injection of Eumiliin (1-25 microg/paw) also caused an oedematogenic response that was maximal after 1h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24h after injection.

Histomorphometry and macroscopic intestinal lesions in broilers infected with Eimeria acervulina.

This study aimed at measuring intestinal villi and assessing the intestinal absorptive area in broilers infected with Eimeria acervulina under different treatments to control coccidiosis. The experiment was divided into two stages, carried out in successive housings, raised in the same environment (or aviary). In the first stage, on 25 May 2008, fifty 12-day-old birds were orally inoculated with 3 x 10(3) oocysts of E. acervulina. In the second stage, on July 2008, other 50 birds were allocated on litter contaminated by the feces of birds on the first housing (natural infection by oocysts present in the reused litter). The experiment was arranged in a complete randomized design with five treatments and three replicates of 10 chicks per treatment. Broiler chicks were housed at 1 day of age and autopsies were performed at 21 days of age. Three 2-cm-long segments of the duodenum were excised from each bird and fixed in 10% buffered formalin. A total of 30 slides were prepared for each treatment, totaling 150 evaluated histological sections using H&E staining. Villus morphology was carried out by the HL Image 97 software. The intestinal absorptive area was calculated and macroscopic lesions were classified according to standard lesion scores. Results showed that intestinal villus measurements and absorptive area are directly affected by E. acervulina and that there is direct and positive correlation between the macro and microscopic findings observed in intestinal coccidiosis. E. acervulina causes shortening of villi and reduction in the intestinal absorptive area, affecting broiler growth. The prevention method of litter fermentation during the interval between housings and oral administration of Diclazuril can reduce the severity of intestinal lesions by E. acervulina in broilers impairing oocyst virulence or viability.

Análise computacional da compactação da cromatina de espermatozoides de galo / Computational analysis of chromatin condensation of rooster spermatozoa

Testaram-se variantes metodológicas utilizando azul de toluidina (AT), até se estabelecer um protocolo confiável para a avaliação computacional da compactação da cromatina em espermatozoides de galo. Para tal, foram utilizados sêmen de 10 galos com 35 semanas de idade e sêmen de 10 galos com 60 semanas de idade. O melhor método foi o de hidrólise com ácido clorídrico 1N por 10 minutos, coloração em cubeta com AT 0,025 por cento, pH 4,0, por 20 minutos, desidratação em álcool, diafanização em xilol e montagem com bálsamo do Canadá. Todas as amostras de sêmen foram submetidas a este protocolo e posteriormente avaliadas por análise de imagem computacional, em que foram feitas mensurações da área, comprimento, largura, perímetro, homogeneidade da compactação da cromatina dentro de cada cabeça e intensidade de compactação da cromatina. Os espermatozoides de galos velhos apresentaram mais alterações na cromatina que os de galos jovens. Os galos jovens apresentaram cabeça dos espermatozoides maior que os galos mais velhos. A análise computacional da compactação da cromatina mostrou-se um método menos subjetivo e mais preciso que a avaliação visual das cabeças dos espermatozoides.

The methodological variants using toluidina blue (AT) to establish a trustworthy protocol for the computational analysis of chromatin condensation of rooster spermatozoa were studied. Twenty semen samples were used: ten from 35-week-old roosters and ten from 60-week-old roosters. Different methods of denaturation and staining were tested. The best method was hydrolysis with 1N HCl for 10 minutes, staining in bucket with 0.025 percent AT, pH 4.0, for 20 minutes, dehydration in alcohol, clearing in xylol, and mounted with Canada balsam. All the semen samples were submitted to this protocol and later evaluated by computational image analysis. Area, length, width, perimeter, and chromatin compaction homogeneity of head spermatozoa were measured. The sperm of older roosters presented more chromatin changes than the ones of younger ones. The spermatozoa of younger roosters presented bigger heads than the ones of older roosters. The computational analysis of chromatin compaction showed to be less subjective and more precise than the visual evaluation for identification of chromatin alterations of rooster spermatozoa.

Morfometria da mucosa duodenal em frangos de corte submetidos à temperatura ambiente cíclica elevada / Intestinal morphometry of the duodenal mucosa in broiler chickens underwent to high cyclic environment temperature

Os efeitos da temperatura ambiente cíclica elevada sobre a morfometria da mucosa duodenal e o peso corporal em frangos de corte foram avaliados. Setenta pintos de corte, machos, foram alojados em gaiolas e distribuídos em dois grupos. Um grupo foi submetido diariamente, durante uma hora, à temperatura ambiente cíclica elevada do primeiro até o 42º dia de idade (ambiente ST); e outro foi mantido em conforto térmico (ambiente TN). Cinco frangos de cada grupo foram sacrificados, semanalmente, por deslocamento cervical para mensuração da altura de vilosidades (VI), profundidade das criptas (CR) e relação vilo/cripta (VI/CR) duodenal. Dez aves de cada grupo foram pesadas semanalmente em balança digital. Utilizou-se delineamento inteiramente ao acaso em esquema fatorial 7x2 (sete idades: um, sete, 14, 21, 28, 35 e 42 dias, e dois ambientes: ST e TN). Os ambientes foram comparados pelo teste de Fisher (P<0,05), e, para avaliar o efeito da idade, foi realizada análise de regressão polinomial. As aves do ambiente ST apresentaram menores VI aos 14 e 21 dias, menor CR aos 28 dias e menor VI/CR aos 21 dias de idade do que as aves do ambiente TN. A temperatura ambiente cíclica elevada teve efeito danoso sobre a estrutura da mucosa duodenal de frangos de corte até a quarta semana de idade e sobre o peso corporal ao final do ciclo produtivo.

The effects of high cyclic environment temperature on body weight and morphometry of the duodenal mucosa in broiler chicken were evaluated. Seventy one-day-old male broiler chicks were sheltered in cages and distributed in two groups. One group was daily exposed to high cyclic environment temperature for an hour, from hatching to 42 days of age (group ST), the other one was kept under thermoneutral conditions (group TN). Five chickens of each group were weekly slaughtered by cervical delocation to mesure the villosities height (VI), crypts depth (CR), and villo/crypt ratio (VI/ CR) in the duodenum. Ten chickens of each group were weighted weekly on a digital balance. A completely randomized experimental design in a 7x2 factorial arrangement (hatching, seven, 14, 21, 28, 35, and 42 days of age and two environments: ST and TN). The environments were compared by Fisher test (P<0.05) and the effects of days of life by polynomial regression. The ST group had reduction in VI at 14 and 21 days of age (P<0.01), CR at 28 days of age (P<0.05), and in VI/CR at 21 days of age (P<0.01). Cyclic high environment temperature had harmful effect on intestinal structure of broiler from hatching to four weeks of age and on body weigh at the end of the productive cycle.

Immunolocalization and pathological alterations following Strongyloides venezuelensis infection in the lungs and the intestine of MHC class I or II deficient mice.

The present study, investigated the mechanisms involved in the immune responses of Major Histocompatibility Complex class I or class II knockout mice, following Strongyloides venezuelensis infection. Wild-type C57BL/6 (WT), MHC II(-/-) and MHC I(-/-) mice were individually inoculated with 3000 larvae (L3) of S. venezuelensis and sacrificed on days 1, 3, 5, 8, 13 and 21 post-infection (p.i.). Samples of blood, lungs and small intestines were collected. The tissue samples were stained with hematoxylin-eosin for the pathological analysis. The presence of the parasite was demonstrated by immunoperoxidase analysis. MHC II(-/-) mice presented a significantly higher number of adult worms recovered from the small intestine on day 5p.i. and presented elevated numbers of eggs in the feces. The infection by S. venezuelensis was completely eliminated 13 days after infection in WT as well as in MHC I(-/-) mice. In MHC II(-/-) mice, eggs and adult worms were still found on day 21 p.i., however, there was a significant reduction in their numbers. In the lung, the parasite was observed in MHC I(-/-) on day 1 p.i. and in MHC II(-/-) mice on days 1 and 5 p.i. In the small intestine of WT mice, a larger number of parasites were observed on day 8 p.i. and their absence was observed after day 13 p.i. Through immunohistochemistry analysis, the parasite was detected in the duodenum of WT on days 5 and 8 p.i., and in knockout mice on days 5, 8 and 13 p.i.; as well as in posterior portions of the small intestine in MHC I(-/-) and MHC II(-/-) on day 13 p.i., a finding which was not observed in WT mice. We concluded that immunohistochemistry analysis contributed to a more adequate understanding of the parasite localization in immunodeficient hosts and that the findings aid in the interpretation of immunopathogenesis in Strongyloides infection.