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RESUMO - Objetivou-se avaliar a teofilina como agente capacitante substituto ou associado à heparina sobre a reação acrossômica dos espermatozoides e o desenvolvimento de embriões produzidos in vitro. O experimento foi realizado com quatro touros e três tratamentos, totalizando 12 grupos experimentais. O sêmen dos touros foi avaliado nos tratamentos descritos a seguir: tratamento 1 (HEP): heparina ? 10µg/mL; tratamento 2 (TEO): teofilina ? 5mM; tratamento 3 (HEP + TEO): heparina (10µg/mL) + teofilina (5mM), por zero, seis, 12 e 18 horas, corados com trypan blue/Giemsa para avaliação da reação acrossômica. Para a produção dos embriões, os agentes capacitantes foram adicionados aos meios de fertilização. Na análise espermática, a taxa de reação acrossômica verdadeira foi maior (P0,05) entre touros na análise de reação acrossômica nem na PIVE. A utilização da teofilina foi tão eficiente quanto a da heparina na indução da reação acrossômica, no entanto resultou em menores taxas de produção embrionária. ABSTRACT - The sperm capacitating process should take special attention during in vitro embryo production (IVEP) once that affects the success of embryo production. The study aimed to evaluate theophylline as substitute capacitating agent or in combination with heparin on the sperm acrosome reaction and development of embryos produced in vitro. The experiment was carried out using 4 bulls and 3 treatments, establishing 12 experimental groups. Each bull was evaluated in the following treatments: Treatment 1 (HEP): Heparin - 10mg/mL; Treatment 2 (THEO): Theophylline - 5mM; Treatment 3 (HEP + THEO), Heparin (10mg/mL) + Theophylline (5mM). The semen of bulls was incubated in each treatment for 0, 6, 12 and 18h, stained with Trypan blue / Giemsa and analyzed by electron microscopy for assessment of acrosome reaction. Using sperm of same bulls, capacitating agents were added to the fertilization media, for IVEP. In sperm analysis, the true acrosome reaction rate was higher (P0.05) between bulls in acrosome reaction analysis neither for IVEP. The use of theophylline was as effective as heparin in the induction of the acrosome reaction, although it resulted in lower embryo production rates. Made available in DSpace on 2018-01-23T16:05:24Z (GMT). No. of bitstreams: 1 Cnpgl2017ArqBrasMVZVaragoTeofilina.pdf: 618605 bytes, checksum: bfa66dc554da02ac69db9f6d25dc0dcd (MD5) Previous issue date: 2018-01-17

The objective of the experiment was to verify, through ultrasonography, the follicular activity of ostriches in different seasons of the year, correlating them with photoperiod, number of rainy days in each month and egg laying. Eight females were evaluated monthly, during 12 consecutive months in an ostrich farm located in the Center-West of Minas Gerais, Brazil. It was found that the time of the year for egg laying lasts for eight months, from June to January. The egg laying was positively correlated (r = 0.48; P

Brucella ovis is considered the most important infectious cause of reproductive disorders in sheep. The disease is characterized by epididymitis, subfertility and infertility in rams. B. ovis occasionally results in abortion in ewes, as well. The aim of this study was to evaluate kinetic changes in the reproductive organs of rams experimentally infected with B. ovis. Nine rams were experimentally inoculated intrapreputially with 2mL of a suspension containing 1.2×109CFU (colony-forming units)/mL of B. ovis (strain ATCC25840). In addition, 50μL of a suspension containing 1.2×1010CFU/mL of the same B. ovis strain was inoculated into each conjunctival sac, resulting in 3.6×109CFU total per ram. Six of nine infected rams had developed clinical changes in the tail of the epididymis at 30 days post-infection (dpi), but these changes regressed in 50% of these rams. Ultrasound demonstrated an increase in the area of the tail of the epididymis (P

RESUMO A epididimite infecciosa ovina é uma das principais enfermidades reprodutivas de carneiros. O presente estudo teve por objetivo desenvolver protocolos de PCR em tempo real para B. ovis e H. somni e avaliar sua aplicabilidade em amostras de sêmen e urina de carneiros. Delinearam-se primers e sondas espécie-específicos para cada agente. As sondas foram delineadas com o sistema TaqMan incorporando um marcador FAM para B. ovis e Cy5 para H. somni na extremidade 5' e um quencher na extremidade 3'. A PCR em tempo real para B. ovis e H. somni foi altamente sensível, uma vez que a amplificação de DNA ocorreu com até 0,2ng de DNA/reação. A especificidade dos iniciadores e sondas foi avaliada com amostras de DNA de outros agentes causadores de epididimite ovina e nenhuma amplificação inespecífica foi observada. A aplicabilidade da técnica em amostras biológicas também foi confirmada, pois não houve perda de eficácia (P>0,05) quando comparada à PCR convencional com amostras de sêmen e urina de carneiros experimentalmente infectados.

Infectious epididymitis is considered a major cause of economic losses for the sheep industry worldwide. This study aimed to investigate clinical and pathological changes associated with experimental infections withA. seminisandH. somniin rams. Twenty rams of age 18 to 24 months were infected by intraepididymal inoculation ofA. seminis(n=10) andH. somni(n=10). Rams were weekly examined and biological samples were collected during six weeks. All rams inoculated withA. seminisand 80% inoculated withH. somnibecame infected. The recovery of bacteria was possible in semen and urine samples and tissues in both experimental groups. Clinically, there were a decrease in testicular consistency and an increase in measures of the left epididymis tails in both experimental groups. The main gross changes were observed in the reproductive tract. Microscopically, the main lesions were inflammatory changes in the genitourinary tract and testicular degeneration.A. seminisandH. somniwere able to colonize several organs of the genitourinary tract in rams, being indistinguishable by clinical exam, necropsy or histopathology. For differential diagnosis, it is important to use diagnostic techniques for direct confirmation of the etiologic agent.

Background Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. Results The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. Conclusions The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

A brucelose ovina causada pela Brucella ovis é uma doença reprodutiva de carneiros caracterizada por epididimite, orquite, com consequente diminuição da fertilidade e prejuízos econômicos significativos. O presente trabalho teve por objetivo avaliar a aplicabilidade da técnica de PCR como um método de diagnóstico em campo, comparado-a com a técnica de IDGA. Foram coletadas amostras de urina e soro de 90 carneiros oriundos de 31 rebanhos localizados no Estado do Piauí. Quatro das 31 (12,9%) propriedades avaliadas apresentaram animais positivos. Dezoito (20%) amostras de urina foram positivas pela PCR, enquanto o método de IDGA identificou 16 (17,8%) carneiros soropositivos. Embora os métodos tenham apresentado concordância baixa na estatística Kappa (k=0,04), não foi observada diferença estatística entre as técnicas (P>0,05) pelo teste exato de Fisher. A combinação dos dois testes aumentou significativamente a detecção de animais positivos para 34,4% (P

Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis -induced genital disease. Two mutant strains were generated in this study (the Δ abcAB and Δ hmg strains). The B. ovis Δ abcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain ( P < 0.001). Conversely, the Δ hmg strain infected mice at the same level as WT B. ovis , suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the Δ abcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the Δ abcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo .

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Ines cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10(6) sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST(+) (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI(-)/PSA(-) (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10(6) spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.

Background: Leptospirosis has a worldwide distribution with high social and economic impact. It is caused by a variety of Leptospira spp. serovars, particularly in tropical regions. Various species of animals can be affected by Leptospira spp. including cattle, horses, sheep, goats and swine, and can act as a reservoir for human infection. In Brazil, sheep leptospirosis has been diagnosed in a variety of States, however, little is known about sheep flocks of Mato Grosso. Considering the marked growth of sheep industry in Mato Grosso State in past recent years, this study aimed to evaluate the frequency of serologically positive sheep for Leptospira spp. serovars in the State of Mato Grosso (Brazil).Materials, Methods & Results: Blood samples of 346 sheep, which belonged to 16 sheep flocks of the medium northern region of Mato Grosso was obtained by jugular vein puncture. Serum was obtained by blood centrifugation and stored at -20°C until the test to be performed. The diagnosis was performed using the microscopic agglutination test. Serovars Australis, Autumnalis, Bataviae, Bolívia, Castelonis, Celledoni, Grippotyphosa, Hardjo, Hardjobovis, Hebdomadis, Icterohaemorrhagiae, Javanica, Lagoa, Norma, Panama, Pomona, Sejroe, Shermani, Szwajizak, and Wolffi were tested. Reactions were considered positive with minimum titration of 1:100. Considering all serovars, 54.9% of sheep were positive, and the serovar more frequently detected was Celledoni (22.8%), followed by Javanica (19.4%), Castellonis (16.5%), and Norma (15.3%), with predominance of titration of 100 for these serovars, while only one sheep had a titration of 1:800 detected for serovar Hardjo. All flocks (100%) were positive (at least one positive animal), with the frequency of animals positive to Leptospira spp. varying of 31.3% to 79.2% in flocks.Discussion: Infection for Leptospira spp. in humans has a large social and sanitary impact. Under an animal health perspective, beyond sanitary impact, leptospirosis have a large economic impact and can act as important reservoirs for Leptospira spp. for human infection, especially in tropical conditions, which favor the persistence of agent in the environment. The occurrence of sheep leptospirosis has been diagnosed in several States of Brazil as Rio Grande do Sul, Paraíba, Rio de Janeiro, São Paulo and Maranhão, with frequency of herds positive for Leptospira spp. varying of 5.4% to 47.4%, however, the frequency of positive sheep in this study was higher than these previous reports in other Brazilian States. The most frequent serovar in sheep flocks have a great importance for prophylactic control. However, there were identified as main frequent serovar Celledoni and Javanica, which are considered unusual in sheep, and indicates a peculiar profile of ovine serovars for the region. Considering de high frequency of positive farms (100%) in Mato Grosso and the lack of studies about the occurrence of leptospirosis in this region, our results suggest the occurrence of risk factors that can favor the dispersion and survival of agent. Contact of the sheep evaluated in this study with other animal species such as cattle, dogs, and horses that can serve as a source for sheep infection or may become contaminated from sheep. Then, the profile of sheep infection by Leptospira spp. in the State of Mato Grosso has a peculiar profile of serovars, with high frequency of infection of animals and herds.