Cloxacillin nanostructured formulation for the treatment of bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is a widespread, contagious ocular disease that affects cattle, especially dairy breeds. The disease is caused by Gram-negative bacteria mainly Moraxella bovis, and its treatment consists of parenteral or topic antibiotic therapy. The topic treatment approach is used more commonly in lactating cows, to avoid milk disposal. However, treatment failures are common, because the antibiotic is removed during lacrimation. This study aimed to evaluate the susceptibility of commercial cloxacillin and evaluate the efficacy of nanostructured cloxacillin in clinical cases of IBK by Moraxella. The minimum inhibitory concentration (MIC) of nanoparticle cloxacillin nanocoated, the nanoparticle without the antibiotic and the commercial cloxacillin were determined in vitro with field samples of Moraxella ovis (5) and Moraxella bovis (5). The efficiency of nanoparticles was tested in three cows naturally infected that were treated with 1.0 mL (with 0.32 mg of nanostructured cloxacillin) for the ocular route. Moraxella bovis was isolated and identified by biochemical and molecular methods before the treatment. The animals were treated every 12 h for six days. The cure was considered by the absence of clinical symptoms and bacteria after treatment. The mucoadhesive nanoparticle-based formulation promoted clinical cure with a low number of doses of antibiotics, probably due to the maintenance of the MIC in the ocular mucosa for longer due to the mucoadhesive characteristics of the nanoparticle. The results indicate that the use of nanocoated cloxacillin is possible to control infectious bovine keratoconjunctivitis.

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)

Perfil de progesterona em novilhas com diferente atividade luteal e tratadas com implantes vaginais / Serum progesterone in heifers with different luteal activity treated with vaginal devices

O dispositivo de liberação de progesterona (DLP) é muito importante em protocolos de Inseminação Artificial em Tempo Fixo (IATF). Representa cerca de 43% dos custos e é objeto de estudos sobre a eficiência da sua reutilização. No entanto, perfis de liberação de progesterona (P4) em animais com diferentes concentrações endógenas desse esteroide não são claramente descritos. Este estudo teve como objetivo avaliar a concentração sérica de P4 em fêmeas com diferentes situações de atividade luteal, tratadas com DLP novo (1g de P4) por 8 dias. Trinta novilhas mestiças cíclicas foram divididas em três grupos: em G1 e G2, o DLP foi inserido (D0) sete dias após a ovulação induzida. Adicionalmente, 0,15mg de D-cloprostenol foi administrado três dias depois para promover a luteólise em G2. Para G3, o corpo lúteo foi lisado antes da inserção do DLP para que a P4 exógena fosse a única fonte desse hormônio. O sangue foi coletado no D0, D3, D5 e D8, e a P4 avaliada por RIA. Médias de P4 foram comparadas entre os grupos em cada dia e dentro do grupo, entre os dias, utilizando o teste Tukey. Antes da inserção do implante (D0), os níveis de P4 foram, nos grupos, semelhantes em G1 e G2, e superiores a G3 (5,3±3,1a e 5,3±1,4avs 0,6±0.3bng/mL, respectivamente-P<0,05). No D3, ocorreu o mesmo perfil (5,7±2,6a e 5,4±2,0a e 3,6±0.8bng/mL, respectivamente para G1 e G2 vs G3, P<0,05). Trinta e seis horas (D5) após a PGF, a P4 no G2 caiu para níveis semelhantes aos do grupo G3 e ambos diferiram (P<0,05) de G1 (3,3±1,6b vs 2,4±0,9b e 2,1±0.7bng/mL). Essa diferença se manteve (P<0,05) em D8 (3,1±1,3a, 1,8±0,8b e 1,6±0.6b ng/mL). O aumento da P4 após a inserção (D3 - D0) foi maior (P<0,05) em G3 que em G1 e G2 (2,8±0,9a vs 0,4±1,8b e 0,2±1.4bng/mL). Os animais com maior P4 endógena levam a menor liberação de P4 exógena a partir do DLP. Portanto, os níveis remanescentes de P4 no DLP após o uso dependem da concentração endógena de P4 do animal e possíveis alterações durante a permanência.(AU)

The progesterone (P4) device is a very important step in the ovulation control in Timed Artificial Insemination (TAI) protocols. It represents about 43% of the hormone costs, thus it has been the subject of several studies on efficiency of the reused device as an alternative to reduce TAI costs. However, to our knowledge, profiles for P4 release in animals with different endogenous concentrations of P4 are not clearly described. This study aimed to evaluate serum concentration of P4 in females with different ovarian conditions - related to luteal activity - and treated with a new intravaginal device (1g of P4) for 8 days. Thirty normally cyclic crossbred heifers were divided into three groups: for G1 and G2, P4 device was inserted (D0) seven days after ovulation (7 day old CL). Additional PGF (0.15 mg of D-cloprostenol) was given three days later to promote luteolysis in the G2 group. For G3, CL was killed before P4 insertion and the exogenous progesterone was the only source of this hormone. Blood samples were collected on D0, D3, D5 and D8 and P4 concentration was measured by radioimmunoassay (RIA). Means for P4 concentration were compared among groups in each day and within the group among days using the Tukey test. Before P4 device insertion (D0), P4 levels were higher (P<0.05) in G1 and G2 when compared to G3 (5.3±3.1 and 5.3±1.4 vs. 0.6±0.3ng/mL, respectively). Three days later (D3), the same pattern was observed (5.7±2.6 and 5.4±2.0 and 3.6±0.8ng/mL, respectively for G1 and G2 vs. G3, P<0.05). Thirty-six hours (D5) after PGF injection (G2), P4 in G2 dropped to levels similar to the G3 group and both differed (P<0.05) from G1 (3.3±1.6 vs. 2.4±0.9 and 2.1±0.7ng/mL, G1 vs. G2 and G3, respectively). There were no differences (P>0.05) among groups on D8 (3.1±1.3, 1.8±0.8 and 1.6±0.6ng/mL, respectively, for G1, G2 and G3). Progesterone increase after P4 insertion (D3 - D0) was higher (P<0.05) in G3 compared to G1 and G2 (2.8±0.9 vs. 0.4±1.8 and 0.2±1.4ng/mL, respectively). The interpretation was that animals with higher endogenous P4 promote less release of the exogenous P4 from the device. Therefore, the remaining P4 levels from used progesterone devices depend on the physiological condition of the animal at the time of insertion and possible changes during the treatment.(AU)

Redução da resposta superovulatória de fêmeas bovinas superestimuladas com FSH em doses split / Reduced superovulatory response of bovine females overstimulated with FSH in split doses

Objetivou-se avaliar a superovulação (SOV) de vacas zebuínas, utilizando protocolo convencional ou protocolo com número menor de aplicações e similar dosagem (dose split). Utilizaram-se 16 fêmeas (total 32 SOV), com idade entre 17-42 meses e escore de condição corporal 2,5-4 (escala de 1-5), em delineamento tipo cross-over. No início do tratamento (D0), os animais receberam um dispositivo de progesterona e 2mg de benzoato de estradiol. As fêmeas do grupo convencional receberam 250UI de FSH/LH divididas em oito doses decrescentes administradas em intervalos de 12h (FSH/LH no D4, D5, D6 e D7 no período da manhã e tarde, nas respectivas dosagens: 50,0 UI; 37,5 UI; 25,0 UI; 12,5 UI). No D7 pela manhã, as fêmeas foram tratadas com 150μg de D+cloprostenol, e a remoção da progesterona foi realizada no D7 à tarde. As fêmeas do grupo split também receberam 250 UI de FSH/LH. No D4 de manhã, administraram-se 62,5 UI de FSH/LH via IM e 125 UI por via SC. Quarenta e oito horas após (D6) administraram-se 62,5 UI via SC e na manhã do D7 foi removida a progesterona e aplicaram-se 150μg de D+cloprostenol. As fêmeas de ambos os grupos receberam 50μg de análogo de GnRH no D8 pela manhã e foram inseminadas 12 e 24 horas após. No D15 realizou-se a colheita dos embriões em ambos os tratamentos. Avaliou-se a resposta superovulatória pela contagem do número de folículos e CLs de cada ovário, com auxílio de ultrassom. Todas as variáveis foram submetidas ao teste T de Student para amostras pareadas. Houve diferença (P<0,05) na quantidade de folículos acima de 8mm no D8 (9,06±4,54 e 5,50±4,59); número de CLs no dia da colheita (8,12± 3,26 e 4,69±3,46), número de embriões totais (6,69±3,05 e 3,37±2,50) e de embriões viáveis (5,25±2,29 e 2,37±1,78) nas vacas do grupo convencional em relação às do split, respectivamente. Conclui-se que o protocolo split tem pior resposta superovulatória e de produção in vivo de embriões, em vacas zebuínas, quando comparado ao protocolo convencional...

The aim of this study was to evaluate the superovulation (SOV) response of zebu cows, using conventional protocol or other protocol with similar dose but smaller number of applications (split dose). 16 females (32 SOV), aged 17-42 months, and body condition score of 2.5-4 (1-5 scale) were used in randomized cross-over. At the start of treatment (D0), the animals received progesterone device and 2mg of estradiol benzoate. The females from the conventional group received 250 IU of FSH/LH divided into eight decreasing doses administered at intervals of 12 hours (FSH/LH in D4, D5, D6 and D7, with their respective strengths: 50.0 IU, 37.5 IU, 25.0 IU, 12.5 IU). In D7, the females were treated with 150μg of D+cloprostenol, and the removal of progesterone device was held in the afternoon. The females from the split group also received 250 IU of FSH/LH. In the morning D4 was administered in 62.5 IU FSH/LH IM and 125 IU subcutaneously. Forty eight hours later (D6) 62.5 IU was administered subcutaneously in the morning and on D7, the progesterone device was removed and 150 μg of D+cloprostenol was applied. The females in both groups were given 50 μg of GnRH in the morning and on D8 were inseminated after 12 and 24 hours. On D15 the embryo collection was performed in both treatments. The evaluation of superovulatory response was done by counting the number of follicles and corpus luteum (CL) in each ovary, with the aid of ultrasound. The variables were assessed by Student's t test for paired samples...

Comparison of conventional freezing and vitrification with dimethylformamide and ethylene glycol for cryopreservation of ovine embryos.

The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re-expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re-expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p < 0.05). The number of dead cells in vitrified embryos of Gr. 2 and Gr. 3 was higher (42.6 ± 26.2 and 63.2 ± 34.65, respectively) in comparison with Gr. 1 (conventional freezing, 10.1 ± 8.5, p < 0.05). Embryos vitrified with dimethylformamide included the same quality of apoptotic cells that Gr. 1 (conventional freezing) and fresh embryos. In conclusion, the dimethylformamide and ethylene glycol used as cryoprotectant to vitrify ovine embryos, in the concentrations and exposition time tested in this work, were not as efficient as the conventional freezing for cryopreservation of ovine embryos Thus, the conventional freezing with ethylene glycol was the most efficient method to cryopreserve ovine embryos in comparison with vitrification.

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.

Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm.

Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl-beta-cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl-beta-cyclodextrin/50 x 10(6) sperm; and (T4) T3 with 0.156 mg methyl-beta-cyclodextrin/50 x 10(6) sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen-thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3+/-5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5+/-11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3+/-7.1% and T4: 11.8+/-9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm.