Teofilina como agente capacitante do sêmen bovino / Theophylline as a capacitating agent of bovine semen

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 (P<0,05) no tempo zero hora, enquanto para espermatozoides mortos, as maiores taxas (P<0,05) foram nos tempos de 12h (84,46±5,82) e 18h (86,75±4,19). A taxa de embriões produzidos (37,97±13) e a taxa de eclosão (33,50±14) foram maiores (P<0,05) para o tratamento HEP. Não foi observada diferença (P>0,05) 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.(AU)

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 (P<0.05) in time 0h, while sperm dead rates were highest (P<0.05) at 12h (84.46±5, 82), and 18h (86.75±4.19). The produced embryos rate (37.97±13) and hatching rate (33.50±14) were larger (P<0.05) for HEP treatment. There was no difference (P>0.05) 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.(AU)

Real-time PCR for detection of Brucella ovis and Histophilus somni in ovine urine and semen / PCR em tempo real para detecção de Brucella ovis e Histophilus somni em urina e sêmen ovino

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.

Atividade folicular ovariana em avestruz (Struthio camelus) avaliada por ultrassonografia e sua relação com fotoperíodo e postura / Follicular activity in ostriches (Struthio camelus) evaluated by ultrasonography, and its relation with the photoperiod and egg laying

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<0.01) with photoperiod. However, there was no correlation between the egg laying and the follicular activity with the amount of rain. Considering the technique adopted, the right antimere offered better ultrasonographic access. The method was efficient for ovary follicular evaluation in the ostrich, offering adequate subsidies for the evaluation of the reproductive activity of the female.

Use of nonlinear models for describing scrotal circumference growth in Guzerat bulls raised under grazing conditions.

The objective was to use various nonlinear models to describe scrotal circumference (SC) growth in Guzerat bulls on three farms in the state of Minas Gerais, Brazil. The nonlinear models were: Brody, Logistic, Gompertz, Richards, Von Bertalanffy, and Tanaka, where parameter A is the estimated testis size at maturity, B is the integration constant, k is a maturating index and, for the Richards and Tanaka models, m determines the inflection point. In Tanaka, A is an indefinite size of the testis, and B and k adjust the shape and inclination of the curve. A total of 7410 SC records were obtained every 3 months from 1034 bulls with ages varying between 2 and 69 months (<240 days of age = 159; 241-365 days = 451; 366-550 days = 1443; 551-730 days = 1705; and >731 days = 3652 SC measurements). Goodness of fit was evaluated by coefficients of determination (R(2)), error sum of squares, average prediction error (APE), and mean absolute deviation. The Richards model did not reach the convergence criterion. The R(2) were similar for all models (0.68-0.69). The error sum of squares was lowest for the Tanaka model. All models fit the SC data poorly in the early and late periods. Logistic was the model which best estimated SC in the early phase (based on APE and mean absolute deviation). The Tanaka and Logistic models had the lowest APE between 300 and 1600 days of age. The Logistic model was chosen for analysis of the environmental influence on parameters A and k. Based on absolute growth rate, SC increased from 0.019 cm/d, peaking at 0.025 cm/d between 318 and 435 days of age. Farm, year, and season of birth significantly affected size of adult SC and SC growth rate. An increase in SC adult size (parameter A) was accompanied by decreased SC growth rate (parameter k). In conclusion, SC growth in Guzerat bulls was characterized by an accelerated growth phase, followed by decreased growth; this was best represented by the Logistic model. The inflection point occurred at approximately 376 days of age (mean SC of 17.9 cm). We inferred that early selection of testicular size might result in smaller testes at maturity.

Diagnosis of Brucella ovis infection by serology and PCR in urine samples from naturally infected rams in the state of Piauí / Diagnóstico da infecção por Brucella ovis por sorologia e reação em cadeia pela polimerase (PCR) em amostras de urina de carneiros naturalmente infectados no estado do Piauí

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 <0,05), sugerindo que a associação de métodos de diagnóstico como a técnica de PCR em amostras de urina e sorologia por IDGA e a avaliação clínica dos animais é necessária para um diagnóstico eficiente na infecção por B. ovis.

A comparison of two agar gel immunodiffusion methods and a complement fixation test for serologic diagnosis of Brucella ovis infection in experimentally infected rams / Comparação entre dois métodos de imunodifusão em gel de Agar e um método de fixação de complemento para o diagnóstico sorológico da infecção por Brucella ovis em carneiros experimentalmente infectados

A infecção por Brucella ovis é considerada uma das principais causas de epididimite e infertilidade em carneiros, resultando em falhas reprodutivas e perdas econômicas significativas em rebanhos ovinos ao redor do mundo. O estudo teve o objetivo de avaliar três testes sorológicos disponíveis para o diagnóstico da brucelose ovina por B. ovis, utilizando 181 soros ovinos. Amostras de soro provenientes de carneiros experimentalmente infectados foram coletadas ao longo de 192 dias pós-infecção (n=117) e durante o período pré-infecção (n=9). Adicionalmente, amostras de soro foram obtidas de ovinos provenientes de um rebanho livre para B. ovis (n=55). As técnicas de imunodifusão em gel de agar (IDGA), utilizando dois antígenos disponíveis comercialmente, e de fixação de complemento foram comparadas (FC). Foram obtidos resultados de sensibilidade especificidade semelhantes para ambos os métodos de IDGA e ainda, a técnica de IDGA foi mais eficiente do que a da FC para o diagnóstico sorológico da infecção por B. ovis.

Extenders containing dimethylformamide associated or not with glycerol are ineffective for ovine sperm cryopreservation.

The study aimed at testing the effectiveness of dimethylformamide, alone or combined with glycerol, as cryoprotectant for freezing ram semen. Ejaculates from nine rams were cryopreserved in Tris-based extenders, containing 5% of glycerol, association of dimethylformamide with glycerol, in four proportions achieving 5% of cryoprotectors in the media and pure dimethylformamide (2, 3, 4 and 5%) in replacement to glycerol. The samples were diluted to 100 × 10(6) sptz/ml and stored in 0.25-ml straws in liquid nitrogen. After thawing (37 °C for 30 s), motility was preserved better by the extender containing 5% of glycerol (p < 0.05). The extenders containing pure dimethylformamide, or more than 2% in combination with glycerol, provided sperm motilities close to zero. Plasma and acrosomal membrane integrity were preserved better (p < 0.05) in the extender containing 5% glycerol. It can be concluded that dimethylformamide, alone or combined with glycerol, has no beneficial effects on ovine semen cryopreservation.

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen.

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 Inês 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.

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen.

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 (P < 0.05). 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.