Evaluation of fertilization capability of frozen-thawed completely immotile spermatozoa collected from a white bengal tiger after interspecific ICSI with bovine oocytes

The objective of this study was to evaluate the fertilization capability of White Bengal Tiger frozen-thawed completely immotile spermatozoa after interspecific intracytoplasmic sperm injection (ICSI) with bovine oocytes. The fertilization status of presumptive zygotes was assessed 18 h after ICSI by immunofluorescence staining and confocal microscopy. The fertilization rate was 34.8% (8/23), as confirmed by the extrusion of two polar bodies, or male and female pronuclei formation. For unfertilized oocytes (65.2%, 15/23), one activated oocyte had an activated spermatozoon but most were unactivated oocytes with unactivated spermatozoa (1/15, 6.7% vs 10/15, 66.7%, respectively, p < 0.05). These results showed that White Bengal Tiger frozen-thawed completely immotile spermatozoa retained the capacity to fertilize bovine oocytes after interspecific ICSI. This is the first report of in vitro produced zygotes using tiger immotile sperm with bovine oocytes by interspecific ICSI technique, which provides an efficient and feasible method for preservation and utilization of endangered feline animals.(AU)

Melittin-induced metabolic changes on the Madin-Darby Bovine Kidney cell line / [Mudanças metabólicas induzidas por melitina em células da linhagem Madin-Darby Bovine Kidney]

In this study, the toxic effects of melittin on Madin-Darby Bovine Kidney cells (MDBK) were analyzed with respect to mitochondrial functionality by reduction of MTT and flow cytometry, apoptosis potential, necrosis, oxygen reactive species (ROS) production, lipid peroxidation, and DNA fragmentation using flow cytometry and cell membrane destabilization by confocal microscopy. The toxicity presented dose-dependent characteristics and mitochondrial activity was inhibited by up to 78.24 ±3.59% (P<0.01, n = 6) in MDBK cells exposed to melittin (10µg/mL). Flow cytometry analysis revealed that melittin at 2µg/mL had the highest necrosis rate (P<0.05) for the cells. The lipoperoxidation of the membranes was also higher at 2µg/mL of melittin (P<0.05), which was further confirmed by the microphotographs obtained by confocal microscopy. The highest ROS production occurred when the cells were exposed to 2.5µg/mL melittin (P<0.05), and this concentration also increased DNA fragmentation (P<0.05). There was a significative and positive correlation between the lipoperoxidation of membranes with ROS (R=0.4158), mitochondrial functionality (R=0.4149), and apoptosis (R=0.4978). Thus, the oxidative stress generated by melittin culminates in the elevation of intracellular ROS that initiates a cascade of toxic events in MDBK cells.(AU)

Neste estudo, os efeitos tóxicos da melitina em células Madin-Darby Bovine Kidney (MDBK) foram analisados quanto à funcionalidade mitocondrial, por redução de MTT e citometria de fluxo, potencial de apoptose, necrose, produção de espécies reativas de oxigênio (ROS), peroxidação lipídica e fragmentação de DNA, utilizando-se citometria de fluxo e desestabilização da membrana celular, por microscopia confocal. A toxicidade apresentou características dose-dependentes e a atividade mitocondrial foi inibida até 78,24±3,59% (P<0,01, n = 6) em células MDBK expostas à melitina (10µg/mL). Análises por citometria de fluxo revelaram que a melitina a 2µg/mL apresentou o maior índice necrótico celular (P<0,05). A maior lipoperoxidação de membranas também foi na concentração de 2µg/mL de melitina (P<0,05), o que foi posteriormente confirmado por microscopia confocal. A maior produção de ROS aconteceu quando as células foram expostas a 2,5µg/mL de melitina (P<0,05), e essa concentração também aumentou a fragmentação de DNA (P<0,05). Houve uma significativa correlação positiva entre a lipoperoxidação de membranas e a produção de ROS (R=0,4158), funcionalidade mitocondrial (R=0,4149) e apoptose (R=0,4978). Portanto, o estresse oxidativo gerado pela melitina culminou na elevação de ROS intracelular, que inicia uma cascata de eventos tóxicos nas células MDBK.(AU)

Suppression of Xanthomonas oryzae pv. oryzae biofilm formation by Acacia mangium methanol leaf extract / Supressão de Xanthomonas oryzae pv. oryzae: formação do biofilme oryzae pelo extrato de folhas de metanol de Acacia mangium

Xanthomonas oryzae pv. oryzae (Xoo), a pathogen responsible for rice bacterial leaf blight, produces biofilm to protect viable Xoo cells from antimicrobial agents. A study was conducted to determine the potency of Acacia mangium methanol (AMMH) leaf extract as a Xoo biofilm inhibitor. Four concentrations (3.13, 6.25, 9.38, and 12.5 mg/mL) of AMMH leaf extract were tested for their ability to inhibit Xoo biofilm formation on a 96-well microtiter plate. The results showed that the negative controls had the highest O.D. values from other treatments, indicating the intense formation of biofilm. This was followed by the positive control (Streptomycin sulfate, 0.2 mg/mL) and AMMH leaf extract at concentration 3.13 mg/mL, which showed no significant differences in their O.D. values (1.96 and 1.57, respectively). All other treatments at concentrations of 6.25, 9.38, and 12.5 mg/mL showed no significant differences in their O.D. values (0.91, 0.79, and 0.53, respectively). For inhibition percentages, treatment with concentration 12.5 mg/mL gave the highest result (81.25%) followed by treatment at concentrations 6.25 and 9.38 mg/mL that showed no significant differences in their inhibition percentage (67.75% and 72.23%, respectively). Concentration 3.13 mg/mL resulted in 44.49% of biofilm inhibition and the positive control resulted in 30.75% of biofilm inhibition. Confocal laser scanning microscopy (CLSM) analysis of Xoo biofilm inhibition and breakdown showed the presence of non-viable Xoo cells and changes in aggregation size due to increase in AMMH leaf extract concentration. Control slides showed the absence of Xoo dead cells.(AU)

Xanthomonas oryzae pv. oryzae (Xoo), um patogênico responsável pela influência bacteriana na folha do arroz, produz biofilme para proteger células Xoo viáveis de agentes antimicrobianos. Foi conduzido um estudo para determinar a potência do extrato de folha de Acacia mangium methanol (AMMH) como um inibidor de biofilme Xoo. Quatro concentrações (3,13, 6,25, 9,38 e 12,5 mg/mL) de extrato de folha de AMMH foram testadas quanto à sua capacidade de inibir a formação de biofilme Xoo em uma placa de microtitulação de 96 poços. Os resultados mostraram que os controles negativos tiveram o maior valor de OD do que os outros tratamentos, indicando a intensa formação de biofilme. Isso foi seguido do controle positivo (sulfato de estreptomicina, com concentração de 0,2 mg/mL, e extrato de folha de AMMH, com concentração de 3,13 mg/mL), que não apresentou diferenças significativas nos seus valores OD (1,96 e 1,57, respectivamente). Todos os outros tratamentos com concentrações de 6,25, 9,38, e 12,5 mg/mL não tiveram diferenças significativas nos seus valores OD (0,91, 0,79, e 0,53, respectivamente). Para percentagens de inibição, o tratamento com concentração 12,5 mg/mL apresentou o maior resultado (81,25%), seguido do tratamento em concentrações de 6,25 e 9,38 mg/mL, que não mostraram diferenças significativas na sua percentagem de inibição (67,75 e 72,23%, respectivamente). Concentração 3,13 mg/mL resultou em 44,49% de inibição do biofilme, e o controle positivo resultou em 30,75% de inibição do biofilme. Análise por microscopia confocal de leitura a laser de inibição e separação de biofilme Xoo revelou a presença de células Xoo não viáveis e alterações no tamanho da agregação por causa do aumento na concentração de extrato de folha de AMMH. Slides de controle mostraram a ausência de células Xoo mortas.(AU)

Suppression of Xanthomonas oryzae pv. oryzae biofilm formation by Acacia mangium methanol leaf extract

Abstract Xanthomonas oryzae pv. oryzae (Xoo), a pathogen responsible for rice bacterial leaf blight, produces biofilm to protect viable Xoo cells from antimicrobial agents. A study was conducted to determine the potency of Acacia mangium methanol (AMMH) leaf extract as a Xoo biofilm inhibitor. Four concentrations (3.13, 6.25, 9.38, and 12.5 mg/mL) of AMMH leaf extract were tested for their ability to inhibit Xoo biofilm formation on a 96-well microtiter plate. The results showed that the negative controls had the highest O.D. values from other treatments, indicating the intense formation of biofilm. This was followed by the positive control (Streptomycin sulfate, 0.2 mg/mL) and AMMH leaf extract at concentration 3.13 mg/mL, which showed no significant differences in their O.D. values (1.96 and 1.57, respectively). All other treatments at concentrations of 6.25, 9.38, and 12.5 mg/mL showed no significant differences in their O.D. values (0.91, 0.79, and 0.53, respectively). For inhibition percentages, treatment with concentration 12.5 mg/mL gave the highest result (81.25%) followed by treatment at concentrations 6.25 and 9.38 mg/mL that showed no significant differences in their inhibition percentage (67.75% and 72.23%, respectively). Concentration 3.13 mg/mL resulted in 44.49% of biofilm inhibition and the positive control resulted in 30.75% of biofilm inhibition. Confocal laser scanning microscopy (CLSM) analysis of Xoo biofilm inhibition and breakdown showed the presence of non-viable Xoo cells and changes in aggregation size due to increase in AMMH leaf extract concentration. Control slides showed the absence of Xoo dead cells.

Resumo Xanthomonas oryzae pv. oryzae (Xoo), um patogênico responsável pela influência bacteriana na folha do arroz, produz biofilme para proteger células Xoo viáveis de agentes antimicrobianos. Foi conduzido um estudo para determinar a potência do extrato de folha de Acacia mangium methanol (AMMH) como um inibidor de biofilme Xoo. Quatro concentrações (3,13, 6,25, 9,38 e 12,5 mg/mL) de extrato de folha de AMMH foram testadas quanto à sua capacidade de inibir a formação de biofilme Xoo em uma placa de microtitulação de 96 poços. Os resultados mostraram que os controles negativos tiveram o maior valor de OD do que os outros tratamentos, indicando a intensa formação de biofilme. Isso foi seguido do controle positivo (sulfato de estreptomicina, com concentração de 0,2 mg/mL, e extrato de folha de AMMH, com concentração de 3,13 mg/mL), que não apresentou diferenças significativas nos seus valores OD (1,96 e 1,57, respectivamente). Todos os outros tratamentos com concentrações de 6,25, 9,38, e 12,5 mg/mL não tiveram diferenças significativas nos seus valores OD (0,91, 0,79, e 0,53, respectivamente). Para percentagens de inibição, o tratamento com concentração 12,5 mg/mL apresentou o maior resultado (81,25%), seguido do tratamento em concentrações de 6,25 e 9,38 mg/mL, que não mostraram diferenças significativas na sua percentagem de inibição (67,75 e 72,23%, respectivamente). Concentração 3,13 mg/mL resultou em 44,49% de inibição do biofilme, e o controle positivo resultou em 30,75% de inibição do biofilme. Análise por microscopia confocal de leitura a laser de inibição e separação de biofilme Xoo revelou a presença de células Xoo não viáveis e alterações no tamanho da agregação por causa do aumento na concentração de extrato de folha de AMMH. Slides de controle mostraram a ausência de células Xoo mortas.

Post-seminal structure and development of the hemiparasitic plant Escobedia grandiflora (Orobanchaceae)

Escobedia grandiflora(L.f.) Kuntze is a wild hemiparasitic plant with orange roots. Little is known about the development of initial parasitism with the host, despite the significant value of roots for Central and South American communities. Therefore, this study aimed to characterize post-seminal structure and development of E. grandiflorain Pennisetum purpureumhost. To analyze the structure and development of E. grandiflora, seedlings, stems and roots samples were processed and examined underlight, confocal and scanning electron microscopy. Escobedia grandifloraseeds are composed of seed coat, perisperm, and embryo. Emergence of the radicle began eleven days after imbibition. Seedlings showed a root hair collar encircling the axis at the root-hypocotyl junction with elongation of internal cortical cells. Seedlings formed haustoria and successfully reached of the host roots 22 days following root emergence. In the root many starch grains were observed, albeit more scarce in the hypocotyl. After 43 days of root emergence, the seedling stage was finished with the formation of the definitive leaves, and star of the plant stage. After 64 days, root ramification, amount of starch, and orange pigmentation increased with formation of haustoria. The developmental pattern of E. grandiflora plants was slow, but the roots grew faster than the stem. Escobedia grandifloraseeds were not endospermic and have limited nutritional value. After root emergence, the young seedling must develop roots and starch storage towards to haustorium formation and attachment to host roots.

Biofilm production of Leptospira spp. strains

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods & Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader.[...]

Biofilm production of Leptospira spp. strains

Background: Leptospirosis is a zoonosis that affects many species of mammals and occurs endemically in Brazil. The biofilm matrix provides structure and protection to the biofilm cells working as a physical barrier to antibiotic agents, which are attached or consumed by the matrix components. However, this attribute varies according to the matrix, antimicrobial agent and biofilm age. Leptospira may change morphologically according to environmental conditions, including cell aggregation and biofilm formation. Leptospira can colonize the ducts of kidney from hosts for a long time, forming a biofilm, which is believed to be an important factor for their maintenance in animals and in the environment. Thus, the objective of this research was to determine the biofilm formation capacity of four strains of Leptospira interrogans.Materials, Methods & Results: The strains were typified by WHO/FAO/OIE and National Collaborating Center for Reference and Research on Leptospirosis (Kit Biomedical Research, Amsterdam, Netherlands). Leptospira interrogans strains, two isolated from cattle and two isolated from dogs were biofilms tested for adhesion on polystyrene plates, extracellular matrix composition and confocal microscopy. In the plating adhesion test, the suspension was inoculated into 96-well sterile polystyrene microplates with flat bottom at a ratio of 1:200 in EMJH medium, followed by 24 h incubation at 28°C, with medium renewal after 12 h. After this period the wells were washed three times with sterile PBS and following incubation; the plates were dried in the oven at 60°C for 30 min and added 200 μL of 1% violet crystal for five min. Subsequently, the plates were washed with distilled water, after complete removal, 200 μL of acetic acid 33% was added and the readings were performed at 570 nm in the ELISA reader.[...](AU)

Morphological and morphometric study of pre-ovigerous and post-ovigerous adults of Tanaisia (Paratanaisia) bragai (Santos, 1934) (Digenea, Eucotylidae)

The aim of this study was to obtain data on the morphology and morphometry of pre-ovigerous and post-ovigerous adults of the species Tanaisia (Paratanaisia) bragai, using confocal laser scanning microscopy to obtain tomographic images of the suckers and tegument. For morphometric analysis, 45 specimens (30 pre-ovigerous adults and 15 post-ovigerous adults) were measured with the aid of an ocular micrometer coupled to the objective of a photonic microscope. Pre-ovigerous and post-ovigerous adult individuals, stained with Mair carmalumen and mounted in permanent preparations, were analyzed by means of confocal laser scanning microscopy. Positive correlation was detected between the body length and ovary length of postovigerous adults (rs: 0.774; p< 0.05), the body length and the length of uterus (rs: 0.839; p< 0,01) and between the ovary width and egg length (rs: 0.777; p p<0.01). Morphological study of the pre-ovigerous adults demonstrated that the ovary and testes develop simultaneously before the development of the uterus and vitelline glands. The acetabulum was detected in pre-ovigerous adults stained with hematoxilin and observed using light microscopy. In these specimens, the acetabulum measured 36.7 ± 6.9 µm (25-50 µm) in width and 39.91 ± 6.8 µm (25-55 µm) in length. The acetabulum was not detected in post-ovigerous adults observed with light microscopy. However, this structure was detected using confocal miscrocopy. In the post-ovigerous specimens, the acetabulum presented a reduced size compared to the pre-ovigerous adults. This may imply that this structure has more functional significance in the larval and pre-ovigerous stages.(AU)

Morphological and morphometric study of pre-ovigerous and post-ovigerous adults of Tanaisia (Paratanaisia) bragai (Santos, 1934) (Digenea, Eucotylidae)

The aim of this study was to obtain data on the morphology and morphometry of pre-ovigerous and post-ovigerous adults of the species Tanaisia (Paratanaisia) bragai, using confocal laser scanning microscopy to obtain tomographic images of the suckers and tegument. For morphometric analysis, 45 specimens (30 pre-ovigerous adults and 15 post-ovigerous adults) were measured with the aid of an ocular micrometer coupled to the objective of a photonic microscope. Pre-ovigerous and post-ovigerous adult individuals, stained with Mair carmalumen and mounted in permanent preparations, were analyzed by means of confocal laser scanning microscopy. Positive correlation was detected between the body length and ovary length of postovigerous adults (rs: 0.774; p< 0.05), the body length and the length of uterus (rs: 0.839; p< 0,01) and between the ovary width and egg length (rs: 0.777; p p<0.01). Morphological study of the pre-ovigerous adults demonstrated that the ovary and testes develop simultaneously before the development of the uterus and vitelline glands. The acetabulum was detected in pre-ovigerous adults stained with hematoxilin and observed using light microscopy. In these specimens, the acetabulum measured 36.7 ± 6.9 µm (25-50 µm) in width and 39.91 ± 6.8 µm (25-55 µm) in length. The acetabulum was not detected in post-ovigerous adults observed with light microscopy. However, this structure was detected using confocal miscrocopy. In the post-ovigerous specimens, the acetabulum presented a reduced size compared to the pre-ovigerous adults. This may imply that this structure has more functional significance in the larval and pre-ovigerous stages.

Suppression of Xanthomonas oryzae pv. oryzae biofilm formation by Acacia mangium methanol leaf extract

Abstract Xanthomonas oryzae pv. oryzae (Xoo), a pathogen responsible for rice bacterial leaf blight, produces biofilm to protect viable Xoo cells from antimicrobial agents. A study was conducted to determine the potency of Acacia mangium methanol (AMMH) leaf extract as a Xoo biofilm inhibitor. Four concentrations (3.13, 6.25, 9.38, and 12.5 mg/mL) of AMMH leaf extract were tested for their ability to inhibit Xoo biofilm formation on a 96-well microtiter plate. The results showed that the negative controls had the highest O.D. values from other treatments, indicating the intense formation of biofilm. This was followed by the positive control (Streptomycin sulfate, 0.2 mg/mL) and AMMH leaf extract at concentration 3.13 mg/mL, which showed no significant differences in their O.D. values (1.96 and 1.57, respectively). All other treatments at concentrations of 6.25, 9.38, and 12.5 mg/mL showed no significant differences in their O.D. values (0.91, 0.79, and 0.53, respectively). For inhibition percentages, treatment with concentration 12.5 mg/mL gave the highest result (81.25%) followed by treatment at concentrations 6.25 and 9.38 mg/mL that showed no significant differences in their inhibition percentage (67.75% and 72.23%, respectively). Concentration 3.13 mg/mL resulted in 44.49% of biofilm inhibition and the positive control resulted in 30.75% of biofilm inhibition. Confocal laser scanning microscopy (CLSM) analysis of Xoo biofilm inhibition and breakdown showed the presence of non-viable Xoo cells and changes in aggregation size due to increase in AMMH leaf extract concentration. Control slides showed the absence of Xoo dead cells.

Resumo Xanthomonas oryzae pv. oryzae (Xoo), um patogênico responsável pela influência bacteriana na folha do arroz, produz biofilme para proteger células Xoo viáveis de agentes antimicrobianos. Foi conduzido um estudo para determinar a potência do extrato de folha de Acacia mangium methanol (AMMH) como um inibidor de biofilme Xoo. Quatro concentrações (3,13, 6,25, 9,38 e 12,5 mg/mL) de extrato de folha de AMMH foram testadas quanto à sua capacidade de inibir a formação de biofilme Xoo em uma placa de microtitulação de 96 poços. Os resultados mostraram que os controles negativos tiveram o maior valor de OD do que os outros tratamentos, indicando a intensa formação de biofilme. Isso foi seguido do controle positivo (sulfato de estreptomicina, com concentração de 0,2 mg/mL, e extrato de folha de AMMH, com concentração de 3,13 mg/mL), que não apresentou diferenças significativas nos seus valores OD (1,96 e 1,57, respectivamente). Todos os outros tratamentos com concentrações de 6,25, 9,38, e 12,5 mg/mL não tiveram diferenças significativas nos seus valores OD (0,91, 0,79, e 0,53, respectivamente). Para percentagens de inibição, o tratamento com concentração 12,5 mg/mL apresentou o maior resultado (81,25%), seguido do tratamento em concentrações de 6,25 e 9,38 mg/mL, que não mostraram diferenças significativas na sua percentagem de inibição (67,75 e 72,23%, respectivamente). Concentração 3,13 mg/mL resultou em 44,49% de inibição do biofilme, e o controle positivo resultou em 30,75% de inibição do biofilme. Análise por microscopia confocal de leitura a laser de inibição e separação de biofilme Xoo revelou a presença de células Xoo não viáveis e alterações no tamanho da agregação por causa do aumento na concentração de extrato de folha de AMMH. Slides de controle mostraram a ausência de células Xoo mortas.

Growth, viability and architecture of biofilms of Listeria monocytogenes formed on abiotic surfaces

The pathogenic bacterium Listeria monocytogenes can persist in food processing plants for many years, even when appropriate hygienic measures are in place, with potential for contaminating ready-to-eat products and, its ability to form biofilms on abiotic surfaces certainly contributes for the environmental persistence. In this research, L. monocytogenes was grown in biofilms up 8 days attached to stainless steel and glass surfaces, contributing for advancing the knowledge on architecture of mature biofilms, since many literature studies carried out on this topic considered only early stages of cell adhesion. In this study, biofilm populations of two strains of L. monocytogenes (serotypes 1/2a and 4b) on stainless steel coupons and glass were examined using regular fluorescence microscopy, confocal laser scanning microscopy and classic culture method. The biofilms formed were not very dense and microscopic observations revealed uneven biofilm structures, with presence of exopolymeric matrix surrounding single cells, small aggregates and microcolonies, in a honeycomb-like arrangement. Moreover, planktonic population of L. monocytogenes (present in broth media covering the abiotic surface) remained stable throughout the incubation time, which indicates an efficient dispersal mechanism, since the culture medium was replaced daily. In conclusion, even if these strains of L. monocytogenes were not able to form thick multilayer biofilms, it was noticeable their high persistence on abiotic surfaces, reinforcing the need to focus on measures to avoid biofilm formation, instead of trying to eradicate mature biofilms.(AU)

Morphology, ontogeny and structure of the stipular nectaries in Caamembeca spectabilis (Polygalaceae) / Morfologia, ontogenia e estrutura dos nectários estipulares em Caamembeca spectabilis (Polygalaceae)

Nodal glands are found in one third of the Polygalaceae genera and have valuable taxonomic, ecological and evolutionary significance. In Brazil, they occur in five of the eleven genera already registered. However, there is still a controversy regarding the origin of these structures. The objective of this study was to characterize the morphology and the origin of nodal glands inCaamembeca spectabilis, in order to increase the structural and functional knowledge of these glands in the genera. Samples of nodal regions were collected, fixed and processed according to the methods of light microscopy and electron scanning. Ants were observed and identified along the stem axis. The glucose in exudate allows us to classify these glands as extrafloral nectaries. They are located in pairs on the nodal region. However, its origin is in the leaf trace. In the longitudinal section, the nectaries were present in the apex of cells with anticlinal walls impregnated with suberin, which represents the first record for the family. In this region there is also the formation of a hole by lysis. The secretory tissue is surrounded by phloem. Xylem vessels were observed only on the basis of the nectary, where there are also idioblasts with crystals in druse type. We have studied the ontogeny of the glands nodal in Caamembeca spectabilis and unveiled that these glands are linked to the leaves as stipular nectaries. In addition, the new findings presented here may add support for the understanding of morphology and anatomy of nodal glands in Caamembeca.

Glândulas nodais são encontradas em um terço dos gêneros de Polygalaceae e possuem grande importância taxonômica, ecológica e evolutiva. No Brasil, ocorrem em cinco, dos onze gêneros já registrados. Contudo, ainda há controvérsias quanto à origem dessas estruturas. O objetivo deste trabalho foi caracterizar a morfologia e a origem das glândulas nodais em Caamembeca spectabilis, visando ampliar o conhecimento estrutural e funcional dessas glândulas no gênero. Amostras das regiões nodais foram coletadas, fixadas e processadas segundo os métodos em microscopia de luz e eletrônica de varredura. Formigas foram observadas e identificadas ao longo do eixo caulinar. A glicose no exsudato permite tratar as glândulas como nectários extraflorais. Esses estão localizados aos pares na região nodal. Porém, sua origem ocorre no traço foliar. Os nectários, em secção longitudinal, apresentam as células do ápice com paredes anticlinais impregnadas com suberina, sendo este o primeiro registro para a família. Nessa região também ocorre a formação de um orifício por lise. O tecido secretor do nectário é circundado pelo floema. Vasos de xilema foram observados apenas na base do nectário, onde também ocorrem idioblastos com cristais do tipo drusas. Dessa forma, este estudo confirma a trajetória ontogenética das glândulas nodais em C. spectabilis, as quais, de fato, estão ligadas às folhas, como nectários estipulares. Além de apresentar dados inéditos que auxiliam a compreensão morfológica e anatômica das mesmas em Caamembeca.

Morphology, ontogeny and structure of the stipular nectaries in Caamembeca spectabilis (Polygalaceae) / Morfologia, ontogenia e estrutura dos nectários estipulares em Caamembeca spectabilis (Polygalaceae)

Nodal glands are found in one third of the Polygalaceae genera and have valuable taxonomic, ecological and evolutionary significance. In Brazil, they occur in five of the eleven genera already registered. However, there is still a controversy regarding the origin of these structures. The objective of this study was to characterize the morphology and the origin of nodal glands inCaamembeca spectabilis, in order to increase the structural and functional knowledge of these glands in the genera. Samples of nodal regions were collected, fixed and processed according to the methods of light microscopy and electron scanning. Ants were observed and identified along the stem axis. The glucose in exudate allows us to classify these glands as extrafloral nectaries. They are located in pairs on the nodal region. However, its origin is in the leaf trace. In the longitudinal section, the nectaries were present in the apex of cells with anticlinal walls impregnated with suberin, which represents the first record for the family. In this region there is also the formation of a hole by lysis. The secretory tissue is surrounded by phloem. Xylem vessels were observed only on the basis of the nectary, where there are also idioblasts with crystals in druse type. We have studied the ontogeny of the glands nodal in Caamembeca spectabilis and unveiled that these glands are linked to the leaves as stipular nectaries. In addition, the new findings presented here may add support for the understanding of morphology and anatomy of nodal glands in Caamembeca.(AU)

Glândulas nodais são encontradas em um terço dos gêneros de Polygalaceae e possuem grande importância taxonômica, ecológica e evolutiva. No Brasil, ocorrem em cinco, dos onze gêneros já registrados. Contudo, ainda há controvérsias quanto à origem dessas estruturas. O objetivo deste trabalho foi caracterizar a morfologia e a origem das glândulas nodais em Caamembeca spectabilis, visando ampliar o conhecimento estrutural e funcional dessas glândulas no gênero. Amostras das regiões nodais foram coletadas, fixadas e processadas segundo os métodos em microscopia de luz e eletrônica de varredura. Formigas foram observadas e identificadas ao longo do eixo caulinar. A glicose no exsudato permite tratar as glândulas como nectários extraflorais. Esses estão localizados aos pares na região nodal. Porém, sua origem ocorre no traço foliar. Os nectários, em secção longitudinal, apresentam as células do ápice com paredes anticlinais impregnadas com suberina, sendo este o primeiro registro para a família. Nessa região também ocorre a formação de um orifício por lise. O tecido secretor do nectário é circundado pelo floema. Vasos de xilema foram observados apenas na base do nectário, onde também ocorrem idioblastos com cristais do tipo drusas. Dessa forma, este estudo confirma a trajetória ontogenética das glândulas nodais em C. spectabilis, as quais, de fato, estão ligadas às folhas, como nectários estipulares. Além de apresentar dados inéditos que auxiliam a compreensão morfológica e anatômica das mesmas em Caamembeca.(AU)

ATIVIDADE ANTI-HELMÍNTICA in vitro DE QUIMIOTIPOS DO ÓLEO ESSENCIAL DE Lippia alba SOBRE Haemonchus contortus

O controle de nematoides gastrintestinais de pequenos ruminantes é realizado convencionalmente com a administração de anti-helmínticos sintéticos e semissintéticos. Entretanto, o uso inadequado de determinado anti-helmíntico, seleciona indivíduos que possuem a capacidade de sobreviver a esse quimioterápico tornando essencial a busca por métodos alternativos. Os produtos derivados de plantas têm sido amplamente investigados quanto ao seu potencial anti-helmíntico sobre Haemonchus contortus. O objetivo do presente estudo foi avaliar os efeitos dos óleos essenciais de quimiotipos carvona (CV) e citral (CT) de Lippia alba sobre H. contortus. A caracterização química foi feita por meio de cromatografia gasosa acoplada a espectrometria de massa (CG/MS). O efeito anti-helmíntico dos óleos essenciais foi avaliado no teste de eclosão de ovos (TEO) e teste de motilidade de nematoides adultos (TMA), utilizando um isolado de H. contortus multirresistente, Kokstad. Foi realizada microscopia confocal de varredura a laser (MCVL) dos ovos e adultos de H. contortus e microscopia eletrônica de varredura (MEV) dos adultos após tratamento com os óleos para observações qualitativas de seus efeitos. Os resultados foram submetidos à análise de variância (ANOVA) e comparados pelo teste Tukey (P>0,05). A concentração inibitória de 50% (CE50) para o TEO foi calculada a partir do programa SPSS 17.0 program for Windows. Os resultados demonstraram a existência de dois quimiotipos, CV e CT do óleo essencial de L. alba (La). O quimiotipo CV do óleo essencial de L. alba (LaCV) apresentou 70,2% de CV e o quimiotipo CT do óleo essencial de L. alba (LaCT) estava constituído por 20,40% e 29,49% de neral e geranial, respectivamente. No TEO, a CE50 de LaCV e LaCT foi de 0,2 e 0,3mg/mL, respectivamente. No TMA, após 12h de exposição ao LaCV e LaCT, 100% dos nematoides adultos estavam imóveis. A MCVL evidenciou alterações na motilidade da larva dentro do ovo provocada por LaCV, enquanto que LaCT impediu a formação da larva. Nos adultos expostos aos dois óleos foram observadas aparentes alterações na porção anterior, em nível de esôfago. Na MEV foram observadas alterações morfológicas na porção anterior, cápsula bocal e na porção medial. Conclui-se que os dois óleos essenciais de L. alba causaram aparentes alterações morfológicas e inibiram a eclosão de ovos, assim como a motilidade de nematoides adultos de H. contortus. Porém é importante avaliar o efeito in vivo, para identificar níveis seguros de eficácia e toxicidade antes de confirmar sua utilização como antihelmíntico.

The control of gastrointestinal nematodes in small ruminants is carried out conventionally with the administration of synthetic and semi-synthetic anthelmintics. However, the inappropriate use of a certain anthelmintic, selects individuals who have the capacity to survive this chemotherapy, making the search for alternative methods essential. Plant derived products have been extensively investigated for their anthelmintic potential on H. contortus. The aim of the present study was to evaluate the effects of the essential oils of Lippia alba chemotypes carvone (CV) and citral (CT) on H. contortus. Chemical characterization was done by means of gas chromatography coupled to mass spectrometry (CG/MS). The anthelmintic effect of essential oils was assessed in the egg hatch test (EHT) and adult nematode motility test (ANMT), using a multidrug-resistant H. contortus isolate, Kokstad. Confocal laser scanning microscopy (CLSM) of eggs and adults of H. contortus and scanning electron microscopy (SEM) of adults was performed after treatment with oils for qualitative observations of their effects. The results were submitted to analysis of variance (ANOVA) and compared by the Tukey test (P> 0.05). The 50% inhibitory concentration (EC50) for EHT was calculated using the SPSS 17.0 program for Windows. The results demonstrated the existence of two chemotypes, CV and CT of the essential oil of L. alba (La). The chemotype CV of the essential oil of L. alba (LaCV) presented 70.2% of CV and the chemotype CT of the essential oil of L. alba (LaCT) was constituted by 20.40% and 29.49% of neral and geranial, respectively. In the EHT, the EC50 of LaCV and LaCT was 0.2 and 0.3 mg/mL, respectively. In ANMT, after 12h of exposure to LaCV and LaCT, 100% of adult nematodes were immobile. CLSM showed changes in the larval motility inside the egg caused by LaCV, while LaCT promoted changes in the larva formation. In adults exposed to both oils, apparent changes were observed in the anterior portion, at the level of the esophagus. In SEM, morphological changes were observed in the anterior portion, mouthpiece and medial portion. It is concluded that the two oils of L. alba, chemotypes CV and CT, caused apparent morphological changes and inhibited the hatching of eggs, as well as the motility of adult H. contortus nematodes. However, it is important to evaluate the effect in vivo to identify safe levels of efficacy and toxicity before confirming its use as an anthelmintic.

DESENVOLVIMENTO DE SORVETE FUNCIONAL SIMBIÓTICO COM POLPA DE CAGAITA (Eugenia dysenterica)

A inovação tecnológica bioinspirada na fabricação de sorvetes tem impulsionado o mercado de derivados lácteos. Desta forma, aumentar a densidade nutricional e funcionais dos sorvetes, com o uso de soro lácteo, leitelho, inulina, probiótico e fruto do Cerrado constituem alternativa viável para o setor. Objetivou-se a produção e caracterização físico-química, microbiológica e sensorial de diferentes sorvetes variando as concentrações da relação leite/creme, soro lácteo e leitelho definidas pelo Delineamento Simplex Centroide, sendo posteriormente adicionado cepa probiótica de Lactobacillus acidophilus, inulina e polpa de cagaita (Eugenia dysenterica). Foram produzidas, inicialmente, nove amostras de sorvetes funcionais com teores de soro e de leitelho, variando de 5% a 15%, e relação leite/creme de 70% a 90%, conforme estipulado por Delineamento Simplex Centroide. Todas as formulações apresentaram resultados dentro dos padrões estabelecidos pela legislação vigente. Os sorvetes apresentaram maior teor dos ácidos graxos palmítico, oleico e esteárico. Destacaram-se as formulações com maior concentração dos coprodutos por apresentarem melhor composição nutricional, considerando-se os altos teores de proteína, frações proteicas, lipídeos e lactose. A formulação com maior concentração de soro e leitelho, bem como as formulaçõescontrole, totalizando seis amostras, foram incrementadas com 0,5% de Lactobacillus acidophilus, sendo três amostras com adição de 20% de polpa de cagaita e três amostras sem adição de polpa. Posteriormente, as formulações foram incrementadas com 5% de inulina. Realizou-se as análises físico-químicas, microbiológicas, levantamento de perfil de ácidos graxos, análise descritiva quantitativa, reologia, análise de perfil de textura e microscopia confocal. Todas as formulações apresentaram resultados dentro dos padrões estabelecidos pela legislação vigente. Os sorvetes funcionais probióticos e simbióticos foram categorizados em sorvetes padrão e premium em relação ao teor de gordura. As formulações com polpa de cagaita em sua composição, apresentaram maiores valores de acidez, umidade e menor velocidade de derretimento e maior dureza instrumental. Os sorvetes exibiram equivalência em relação à semi-quantificação dos pixels das imagens, obtidas pela análise de microscopia confocal, com a quantificação de lipídeos e proteínas. Sorvetes com adição de polpa apresentaram maior intensidade da cor amarela e aroma cítrico. Todos os sorvetes são categorizados como alimentos probióticos e apresentaram contagem de 8 Log10 UFC/g, sendo ótimos veículos para fornecer cultura probiótica e todos os seus benefícios ao ser consumido.

Bio-inspired technological innovation in ice cream manufacturing has driven the market for dairy products. In this way, increasing the nutritional density and approving ice cream, with the use of whey, buttermilk, inulin, probiotic and fruit from the Cerrado are viable alternative for the sector. The objective was the production and physicochemical, microbiological and sensory characterization of different ice creams, varying those due to the milk/cream ratio, whey and buttermilk defined by the Simplex Centroide Design, and subsequently added a probiotic strain of Lactobacillus acidophilus, inulin and cagaita pulp (Eugenia dysenterica). Nine ice creams were produced with whey and buttermilk contents, ranging from 5% to 15%, and milk/cream ratio from 70% to 90%, as stipulated by the Simplex Centroide Design. All formulations result within the standards defined by current legislation. Ice creams showed higher content of palmitic, oleic and stearic fatty acids. They stood out as formulations with the highest concentration of co-products for presenting the best nutritional composition, considering the high levels of protein, protein fractions, lipids and lactose. The high concentration of whey and buttermilk concentration, as well as the control formulations, totaling Lactobacillus acidophilus six, were increased with 0.5% of, being three with the addition of 20% of cagaita pulp and three without the addition of pulp. Subsequently, all formulations were increased with 5% of inulin. Physical-composition, microbiological, fatty acid profile, quantitative descriptive analysis, rheology, texture profile analysis and confocal microscopy were performed. All formulations result within the standards defined by current legislation. Available probiotic and symbiotic ice creams were categorized into standard and premium ice creams by fat content. Formulations with cagaita pulp presented higher acidity, moisture and lower melting speed and higher instrumental hardness. The ice creams exhibited equivalence in relation to the semiquantification of image pixels, accompanied by confocal microscopy analysis, with the quantification of lipids and proteins. Ice cream with the addition of pulp, presented higher intensity of yellow color and citrus aroma. All ice creams are categorized as probiotic foods and source count 8 Log10 CFU / g, being great vehicles to provide probiotic culture and all its benefits when consumed.

ATIVIDADE ANTI-HELMÍNTICA IN VITRO DE NANOEMULSÕES DE CARVONA SOBRE Haemonchus contortus

A resistência anti-helmíntica tem dificultado o controle de nematoides gastrintestinais em pequenos ruminantes. Dessa forma, novas alternativas de controle estão sendo estudadas, como o nanoencapsulamento de compostos bioativos, na tentativa de maximizar a eficácia parasitária desses compostos. Este trabalho teve como objetivo avaliar a atividade ovicida, larvicida e adulticida de nanoemulsões de carvona produzida por diferentes métodos de homogeneização sobre Haemonchus contortus. Três nanoemulsões de R-carvona foram preparadas usando dois equipamentos de homogeneização (ultra homogeneizador e sonicador) e com e sem revestimento de alginato de sódio: nanoemulsões de R-carvona sem revestimento homogeneizada em sonicador (NSR-son) e homogeneizadas em ultra homogeneizador (NSR- ultra) e R-carvona com revestimento de alginato de sódio (NCR-ultra). Foi realizada a análise de estabilidade, eficiência de encapsulamento e caracterizações físico-químicas das nanoemulsões. Os efeitos das nanoemulsões foi avaliado sobre H. contortus multirresistente no teste de eclosão de ovos (TEO), teste de desenvolvimento larvar (TDL) e teste de motilidade em adultos (TMA). As alterações na cutícula induzidas em H. contortus adultos foram avaliadas por meio da microscopia eletrônica de varredura (MEV) e microscopia confocal de varredura a laser. Os resultados foram submetidos à análise de variância (ANOVA) e comparados pelo teste Tukey (P<0,05). A concentração efetiva para inibir 50% da eclosão dos ovos e do desenvolvimento larvar (CE50) foi calculada no programa SPSS 17.0. NSR-ultra e NCR-ultra, foram mais estáveis frente à sedimentação e cremeação, respectivamente. Os tamanhos de partículas foram de 281,1 nm (NSR-son), 152,7 nm (NSR-ultra) e 557,8 nm (NCR-ultra) e os potenciais zeta de -15 mV (NSR-son), -10,8 mV (NSR-ultra) e -24,2 mV (NCR-ultra). A eficiência de encapsulamento foi de 99,84±0,01%. A microscopia eletrônica de varredura nas nanoemulsões detectou aumento no tamanho, distribuição não uniforme das partículas e presença de estruturas esféricas agregadas a rede polimérica. No TEO, as CE50 de NSR-son, NSR-ultra e NCR-ultra foram de 0,19 mg/mL, 0,02 mg/mL e 0,17 mg/mL, respectivamente. No TDL foram de 0,29 mg/mL, 0,31 mg/mL e 0,95 mg/mL para NSR-son, NSR-ultra e NCR- ultra, respectivamente. A inibição da motilidade de adultos foi de 100%, após 12h de exposição a NSR-ultra e NCR-ultra, enquanto que para NSR-son foi de 79,16%. A microscopia confocal e de varredura detectaram alterações na cápsula bucal e danos cuticulares. Conclui-se que as nanoemulsões de R-carvona com e sem revestimento de alginato de sódio produzidas por diferentes métodos de homogeneização causaram alterações morfológicas e inibiram a eclosão dos ovos, o desenvolvimento larvar e a motilidade de H. contortus adulto, sendo promissoras para controle de infecções por nematoides gastrintestinais em pequenos ruminantes após avaliações futuras no que se refere à segurança toxicológica desses produtos assim como a comprovação da eficácia anti-helmíntica in vivo.

Anthelmintic resistance has made it difficult to control gastrointestinal nematodes in small ruminants. Thus, new control alternatives are being studied, such as the nanoencapsulation of bioactive compounds, in an attempt to maximize the parasitic efficacy of these compounds. This work aimed to evaluate the ovicidal, larvicidal and adulticidal activity of carvone nanoemulsions produced by different homogenization methods on Haemonchus contortus. Three R-carvone nanoemulsions were prepared using two homogenization equipment (ultra homogenizer and sonicator) and with and without sodium alginate coating: uncoated R-carvone nanoemulsions homogenized in sonicator (UNAlg-son) and homogenized in ultra homogenizer (UNAlg-ultra) and sodium alginate coated R-carvone (CNAlg-ultra). Stability analysis, encapsulation efficiency and physicochemical characterizations of the nanoemulsions were performed. The of the nanoemulsions were evaluated on multidrug resistant H. contortus in the egg hatch test (EHT), larval development test (LDT) and adult worm motility test (AWMT). Changes in cuticle induced in adult H. contortus were evaluated by means of scanning electron microscopy (SEM) and confocal laser scanning microscopy. The results were submitted to analysis of variance (ANOVA) and compared using the Tukey test (P<0.05). The effective concentration to inhibit 50% of egg hatching and larval development (EC50) was calculated using the SPSS 17.0 program. UNAlg-ultra and CNAlg-ultra were more stable against sedimentation and creamation, respectively. The particle sizes were 281.1 nm (UNAlg-son), 152.7 nm (UNAlg-ultra) and 557.8 nm (CNAlg-ultra) and the zeta potentials were -15 mV (UNAlg-son), - 10.8 mV (NSR-ultra) and -24.2 mV (CNAlg-ultra). The encapsulation efficiency was 99.84±0.01%. Scanning electron microscopy of the nanoemulsions detected an increase in size, non-uniform distribution of particles and presence of spherical structures aggregated to the polymeric network. In EHT, the EC50 of UNAlg-son, UNAlg-ultra and CNAlg-ultra were 0.19 mg/mL, 0.02 mg/mL and 0.17 mg/mL, respectively. In LDT they were 0.29 mg/mL, 0.31 mg/mL and 0.95 mg/mL for UNAlg- son, UNAlg-ultra and CNAlg-ultra, respectively. The adult motility inhibition was 100% after 12h of exposure to UNAlg-ultra and CNAlg-ultra, while for UNAlg-son it was 79.16%. Confocal and scanning microscopy detected changes in the buccal capsule and cuticular damage. It is concluded that R-carvone nanoemulsions with and without sodium alginate coating produced by different homogenization methods caused morphological changes and inhibited egg hatching, larval development and adult motility of H. contortus, being promising for control of infections by gastrointestinal nematodes in small ruminants after further evaluations regarding the toxicological safety of these products as well as proof of anthelmintic efficacy in vivo.

AVALIAÇÃO HISTOLÓGICA, MICROESTRUTURAL E DE BIOCOMPATIBILIDADE IN VIVO DE CARTILAGENS AURICULARES DE BOVINO TRATADAS EM SOLUÇÃO ALCALINA

Com o avanço de técnicas para a correção de defeitos ou perdas teciduais, busca-se a utilização de biomateriais que promovam o reparo com o mínimo de resposta inflamatória. Estudos anteriores realizados utilizaram diferentes meios de preparo e obtenção de cartilagens elásticas de bovinos, visando o uso como biomaterial. Entretanto, um dos grandes desafios relacionados ao uso dessas membranas, refere-se na ocorrência de reações imunogênicas indesejadas, no local de implantação. Este estudo objetivou caracterizar as propriedades físico-químicas, microestruturais e histológicas de cartilagens elásticas de bovinos tratadas em solução alcalina e sua biocompatibilidade in vivo. Para tanto, foram realizadas análises físico-químicas para a caracterização deste material. As análises térmicas realizadas, Termogravimetria (TGA) e a Calorimetria Exploratória Diferencial (DSC), verificaram as possíveis alterações do material frente as variações de temperatura em que foi submetido. As curvas da TGA demonstraram a variação da massa de cartilagens elásticas tratadas e não tratadas em função da temperatura e na DSC foi avaliado a temperatura de desnaturação do colágeno das amostras de cartilagem. As análises demonstraram que a cartilagem tratada apresentou características físico-químicas similares a cartilagem não tratada. Nas avaliações microestruturais, foi realizado a Microtomografia computadorizada 2D e 3D e a Microscopia de scanner a laser confocal. Na Micro-Ct bidimensional evidenciou que a região da cartilagem apresentou maior densidade em relação ao pericôndrio, e o tratamento alcalino foi efetivo na descelularização, pela presença de lacunas observadas na matriz extracelular entremeadas a estrutura do colágeno. A Micro-Ct tridimensional demonstrou que a cartilagem apresenta menor porosidade e poros com maior diâmetro e na Microscopia de scanner a laser notou-se que a cartilagem tratada apresenta uma rugosidade considerável, fatores que podem contribuir com a proliferação e adesão celular. Neste estudo também foram feitos o processamento histológico da cartilagem, demonstrando que o tratamento alcalino promoveu a descelularização tecidual, com a manutenção da arquitetura da matriz extracelular e da estrutura das fibras elásticas e de colágeno. Concluiu-se que o tratamento alcalino foi eficiente para promover a descelularização na cartilagem elástica. A última etapa do estudo consistiu em avaliar a biocompatibilidade in vivo de implantes de cartilagens elásticas tratadas com solução alcalina (CA) comparadas a tela de polipropileno (TP) em coelhos. Foi verificado que o grupo (CA) apresentou processo inflamatório menos intenso que o grupo (TP), no qual foi observado a formação de cápsula fibrosa ao redor dos implantes. Notou-se no grupo (CA) a presença de calcificação promovida pela osteoindução de osteoblastos em decorrência do processamento alcalino, o que pode ser considerado um viés de interesse para estudos posteriores, envolvendo a regeneração de tecidos ósseos, em que a aplicabilidade de tipo celular observado é viável.

With the advancement of techniques for the correction of tissue defects or losses, the use of biomaterials that promote repair with a minimum of inflammatory response is sought. Previous studies carried out used different means of preparing and obtaining elastic bovine cartilage, aiming at the use as biomaterial. However, one of the major challenges related to the use of these membranes, refers to the occurrence of unwanted immunogenic reactions at the implantation site. This study aimed to characterize the physical-chemical, microstructural and histological properties of elastic bovine cartilages treated in alkaline solution and their biocompatibility in vivo. Therefore, physical-chemical analyzes were carried out to characterize this material. The thermal analyzes performed, Thermogravimetry (TGA) and Differential Exploratory Calorimetry (DSC), verified the possible changes in the material in view of the temperature variations in which it was submitted. The TGA curves showed the variation in the mass of treated and untreated elastic cartilages as a function of temperature and in the DSC the temperature of the collagen denaturation of the cartilage samples was evaluated. The analyzes showed that the treated cartilage had physicochemical characteristics similar to untreated cartilage. In the microstructural evaluations, 2D and 3D computed microtomography and confocal laser scanner microscopy were performed. Two-dimensional Micro-Ct showed that the cartilage region showed higher density in relation to the perichondrium, and the alkaline treatment was effective in decellularization, due to the presence of gaps observed in the extracellular matrix interspersed with the collagen structure. The three-dimensional Micro-Ct showed that the cartilage has less porosity and pores with a larger diameter and in the laser scanner microscopy it was noted that the treated cartilage has considerable roughness, factors that can contribute to cell proliferation and adhesion. In this study, histological processing of cartilage was also performed, demonstrating that the alkaline treatment promoted tissue decellularization, with the maintenance of the architecture of the extracellular matrix and the structure of elastic and collagen fibers. It was concluded that the alkaline treatment was efficient to promote decellularization in the elastic cartilage. The last stage of the study consisted of evaluating the in vivo biocompatibility of elastic cartilage implants treated with alkaline solution (CA) compared to polypropylene (TP) mesh in rabbits. It was found that the group (CA) had a less intense inflammatory process than the group (TP), in which the formation of a fibrous capsule around the implants was observed. It was noted in the group (CA) the presence of calcification promoted by osteoinduction of osteoblasts due to alkaline processing, which can be considered a bias of interest for further studies, involving the regeneration of bone tissues, in which the cell-type applicability observed is feasible.

EFEITO DA ASSOCIAÇÃO DE TIABENDAZOL, LEVAMISOL E IVERMECTINA IN VITRO COM ACETATO DE CARVACRILA EM EMULSÃO COM E SEM MATRIZ DE ALGINATO DE SÓDIO SOBRE Haemonchus contortus

A resistência dos parasitos aos anti-helmínticos comerciais tem impulsionado a pesquisa de novas formas de controle, dentre essas o uso de biocompostos, associação de compostos e modificações de formulação. O objetivo do presente estudo foi avaliar o efeito da associação dos anti-helmínticos sintéticos, tiabendazol (TBZ), levamisol (LEV) e ivermectina (IVM) com acetato de carvacrila (AC) em emulsão com e sem matriz de alginato de sódio sobre Haemonchus contortus. As emulsões foram avaliadas quanto a estabilidade, tamanho de partícula, potencial Zeta, eficiência de encapsulamento e liberação. O efeito anti-helmíntico das emulsões foi avaliado no teste de eclosão de ovos (TEO) para AC e TBZ e teste de desenvolvimento larvar (TDL) para AC, IVM e LEV, utilizando H. contortus multirresistente, isolado Kokstad. O efeito das emulsões sobre os ovos foi visualizado por microscopia confocal de varredura a laser (MCL). Os resultados foram submetidos à análise de variância (ANOVA) e seguido pelo teste Tukey (P<0,05). A concentração efetiva para inibir 50% da eclosão dos ovos (CE50) no TEO foi calculada a partir do programa SPSS 17.0 para Windows. As emulsões foram avaliadas pelo índice de combinação (IC) por meio do programa CompuSyn 1.0. As emulsões de AC com TBZ (TBZ/AC) com e sem matriz apresentaram perfil multimodal, com sua maioria em escala nanométrica. O potencial Zeta mostrou maior estabilidade nas emulsões com matriz. A eficiência de encapsulamento da matriz de alginato de sódio em TBZ/AC foi de 80,25% e LEV/AC de 89,73%. No TEO, AC apresentou CE50 de 0,65 mg/mL, TBZ de 0,01 mg/mL, TBZ/AC sem matriz apresentou IC médio de 0,55 e TBZ/AC com matriz de 0,36, demonstrando sinergismo em ambos. No TDL, a IVM apresentou CE50 de 8,4 x 10-4 mg/mL, LEV 0,014 mg/mL e AC 0,38 mg/mL. IVM/AC apresentou IC médio de 0,75, LEV/AC sem matriz de 0,4 e LEV/AC com matriz 0,93. Na MCL dos ovos no tratamento com TBZ (0,05 mg/mL) não houve formação de larva. Nos ovos tratados com AC (8 mg/mL) evidenciou-se larva em formação eclodindo; em TBZ/AC sem matriz foi visualizada larva não completamente formada, cascas de ovos rompidas; nos ovos tratados com TBZ/AC com matriz evidenciou-se a ausência da estrutura da casca de ovos e larvas desenvolvidas e imóveis. Conclui-se que a emulsão TBZ/AC apresentou interação sinérgica e com a adição da matriz de alginato o efeito foi potencializado, com exceção da maior concentração testada. Além disso, a matriz trouxe estabilidade ao produto encorajando seu aperfeiçoamento para obtenção de eficácia mais elevada.

Parasite resistance to commercial anthelmintics has driven the search for new forms of control, including the use of biocompounds, the combination of compounds, and formulation modifications. The present study aimed to evaluate the effect of the association of synthetic anthelmintics, thiabendazole (TBZ), levamisole (LEV), and ivermectin (IVM) with carvacryl acetate (CA) in emulsion with and without sodium alginate matrix on Haemonchus contortus. Emulsions were evaluated for stability, particle size, Zeta potential, encapsulation efficiency, and release. The anthelmintic effect of the emulsions was evaluated in the egg hatch test (EHT) for CA and TBZ and larval development test (LDT) for CA, IVM, and LEV, using multiresistant H. contortus, isolated from Kokstad. The effect of emulsions on eggs was visualized by confocal laser scanning microscopy (CLSM). The results were submitted to analysis of variance (ANOVA) and followed by the Tukey test (P<0.05). The effective concentration to inhibit 50% of egg hatching (EC50) in the EHT was calculated using the SPSS 17.0 program for Windows. Emulsions were evaluated by the combination index (CI) using the CompuSyn 1.0 program. The emulsions of CA with TBZ (TBZ/CA) with and without matrix showed a multimodal profile, with most of them on the nanometric scale. Zeta potential showed greater stability in matrix emulsions. The encapsulation efficiency of the sodium alginate matrix in TBZ/CA was 80.25% and LEV/CA was 89.73%. In EHT, CA had EC50 of 0.65 mg/mL, TBZ of 0.01 mg/mL, TBZ/CA without matrix showed mean CI of 0.55, and TBZ/CA with the matrix of 0.36, demonstrating synergism in both. In LDT, IVM presented EC50 of 8.4 x 10-4 mg/mL, LEV 0.014 mg/mL and CA 0.38 mg/mL. IVM/CA had a mean CI of 0.75, LEV/CA without the matrix of 0.4, and LEV/CA with the matrix of 0.93. In the CLSM of eggs treated with TBZ (0.05 mg/mL), there was no larvae formation. Eggs treated with CA (8 mg/mL) showed larvae in formation, hatching; in TBZ/CA without matrix, larva not completely formed, broken eggshells were visualized; in eggs treated with TBZ/CA with matrix, the absence of eggshell structure and developed and immobile larvae was evident. It is concluded that the TBZ/CA emulsion showed a synergistic interaction and with the addition of the alginate matrix the effect was enhanced, except for the highest concentration tested. In addition, the matrix brought stability to the product, encouraging its improvement to obtain higher efficacy.

NANOEMULSÕES DE ALGINATO E ÓLEO ESSENCIAL DE Eucalyptus staigeriana: PRODUÇÃO, CARACTERIZAÇÃO, TOXICIDADE E ATIVIDADE ANTI-HELMÍNTICA SOBRE NEMATOIDES GASTRINTESTINAIS DE PEQUENOS RUMINANTES

O parasitismo por nematoides gastrintestinais (NGI) é uma grave ameaça a ovinocaprinocultura e com o aparecimento e disseminação da resistência anti-helmíntica (RA), novas alternativas de controle são necessárias. O óleo essencial de Eucalyptus staigeriana (OEEs) apresenta atividade anti-helmíntica, e o desenvolvimento de formulações visando o seu encapsulamento usando matriz de alginato de sódio (ALG) pode ser vantajoso para aumentar sua estabilidade e eficácia. Objetivando-se desenvolver emulsões de ALG e OEEs para o controle de NGI de pequenos ruminantes, a metodologia foi realizada em três fases. Na fase 1, cujo objetivo foi caracterizar uma população de Haemonchus contortus, nomeada Caucaia, quanto à RA, realizou-se a comparação entre testes fenotípicos de eclosão de ovos (TEO), desenvolvimento larvar (TDL) e redução de ovos nas fezes (TRCOF) e genotípico por qPCR para benzimidazois (BZs) e levamisol (LEV). Houve concordância entre TEO, TRCOF e qPCR para BZs, e entre TDL e qPCR para LEV. A população Caucaia foi considerada resistente a BZs, lactonas macrocíclicas, LEV e suspeita para monepantel. Na fase 2, objetivou-se desenvolver emulsões de ALG e OEEs, caracterizá-las e avaliar seus efeitos in vitro sobre eclosão de ovos e desenvolvimento larvar de H. contortus. Quatro emulsões foram preparadas com proporções variadas de Tween 80, OEEs e ALG 4%, caracterizadas físico-quimicamente e avaliadas no TEO e TDL. As emulsões foram estáveis, com tamanho de partícula predominantemente nanométrico e potencial zeta negativo. As emulsões foram mais efetivas que o OEEs no TEO, com concentração efetiva 50% (CE50) de 0,088 a 0,150 mg/mL para as emulsões e 0,308 mg/mL para OEEs. No TDL, emulsões e OEEs foram semelhantes, com CE50 variando de 3,91 a 4,60 mg/mL para emulsões e 4,17 mg/mL para OEEs. Portanto, emulsões de ALG e OEEs foram produzidas com sucesso, sendo a emulsão 2 (E2), com menor razão Tween 80/OEEs e ALG/OEEs, considerada a melhor e com potencial para o controle alternativo de H. contortus. Na fase 3, objetivou-se avaliar E2 in vitro contra H. contortus adultos, sua toxicidade aguda e atividade in vivo contra NGI. Foram realizados os testes de motilidade do adulto (TMA), avaliação por microscopia eletrônica de varredura (MEV) e confocal de varredura a laser (MCVL). A dose letal 50% (DL50) de E2 foi verificada e o TRCOF foi realizado contra NGI da população Caucaia. No TMA, OEEs e E2 inibiram a motilidade de H. contortus e nas concentrações de 0,4 e 0,6 mg/mL, E2 foi mais efetiva que o OEEs. As imagens de MEV e de MCVL demonstraram alterações estruturais, principalmente, enrugamento de cutícula. A DL50 estimada da emulsão foi superior a 2.000 mg/kg. No TRCOF, a eficácia do OEEs e de E2 foram similares, com 54,2% de redução no opg pós-tratamento. Portanto, E2 demonstrou efeito promissor contra H. contortus adultos in vitro, segurança toxicológica e atividade contra NGI in vivo. Dessa forma, foi possível produzir e caracterizar, de forma bem-sucedida, emulsões de ALG e OEEs, que demonstraram estabilidade e atividade anti-helmíntica contra NGI de pequenos ruminantes, com potencial para aplicação em futuros esquemas de tratamento alternativo.

Parasitism by gastrointestinal nematodes (GIN) is a serious threat to sheep and goat farming and due to emergence and spread of anthelmintic resistance (AR), new control alternatives are required. The essential oil of Eucalyptus staigeriana (EsEO) has anthelmintic activity, so the development of formulations aimed at its encapsulation using sodium alginate matrix (ALG) can be advantageous to increase its stability and effectiveness. Aiming to develop emulsions of ALG and EsEO to control GIN in small ruminants, the methodology was developed in three phases. In phase 1, whose objective was to characterize a population of Haemonchus contortus, named Caucaia, regarding AR, a comparison was made between phenotypic tests of egg hatch (EHT), larval development (LDT) and fecal egg reduction (FECRT) and genotypic by qPCR for benzimidazoles (BZs) and levamisole (LEV). There was agreement between EHT, FECRT and qPCR for BZs, and between LDT and qPCR for LEV. The Caucaia population was considered resistant to BZs, macrocyclic lactones, LEV and suspected for monepantel. In phase 2, the aim was to develop emulsions of ALG and EsEO, characterize them and evaluate their in vitro effects on egg hatching and larval development of H. contortus. Four emulsions were prepared by varying proportions between Tween 80, EsEO and 4% ALG, physicochemically characterized and evaluated on EHT and LDT. The emulsions were stable, with predominantly nanometric particle size and negative zeta potential. The emulsions were more effective than EsEO in EHT, with effective concentration 50% (EC50) ranging from 0.088 to 0.150 mg/mL for the emulsions and 0.308 mg/mL for EsEO. In LDT, emulsions and EsEO were similar, with EC50 ranging from 3.91 to 4.60 mg/mL for emulsions and 4.17 mg/mL for EsEO. Therefore, ALG and EsEO emulsions were successfully produced, and emulsion 2 (E2), with low levels of the ratio between Tween 80/EsEO and ALG/EsEO was considered the best and with potential for alternative control of H. contortus. In phase 3, the aim was to evaluate E2 in vitro against adult H. contortus, its acute toxicity and in vivo activity against GIN. Adult worm motility test (AWMT) was performed, evaluation by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The lethal dose 50% (LD50) of E2 was assessed and FECRT was performed against the Caucaia population GIN. In AWMT, EsEO and E2 inhibited the motility of H. contortus and at concentrations 0.4 and 0.6 mg/mL, E2 was more effective than EsEO. SEM and CLSM images showed structural changes, mainly cuticle wrinkling. The LD50 of the emulsion was estimated to be greater 2,000 mg/kg. In FECRT, the efficacy of EsEO and E2 were similar, with a 54.2% reduction in epg post-treatment. Therefore, E2 showed promising effect against adult H. contortus in vitro, toxicological safety and activity against GIN in vivo. In this way, it was possible to successfully produce and characterize emulsions of ALG and EsEO, which demonstrated stability and anthelmintic activity gainst GIN of small ruminants, with potential for application in future alternative treatment schemes.

Enhancement of pigment extraction from B. braunii pretreated using CO2 rapid depressurization

Extraction of compounds from microalgae requires cell disruption as a pretreatment to increase extraction yield. Botryococcus braunii is a microalga with a significant content of carotenoids and other antioxidant compounds, such as chlorophylls. Cell disruption of B. braunii using CO2 rapid depressurization was studied as a pretreatment for the extraction of carotenoid and chlorophyll pigments. We studied the effect of temperature (2149 °C) and pressure (613 MPa) during static compression on pigment recovery with supercritical CO2 at 40 °C, 30 MPa and solvent flow of 4.7 L NPT/min. Within the experimental region, the extraction yield of carotenoids and chlorophylls increased by 2.4- and 2.2-fold respectively. Static compression conditions of high pressure and low temperature increased the extraction of carotenoids and especially chlorophylls. We selected 21 °C and 13 MPa as the cell disruption condition, which produced 1.91 g/kg d.s. of carotenoids and 14.03 mg/kg d.s. of chlorophylls. Pretreated microalga gave a 10-fold higher chlorophyll extraction yield compared to the untreated sample. While for carotenoids and tocopherols were 1.25 and 1.14-fold higher, respectively. Additionally, antioxidant activity of pretreated microalga (33.22 mmol TE/kg oil) was significantly higher than the value for the untreated samples (29.11 mmol TE/kg oil) (p 0.05). Confocal microscopy images showed morphological differences between micro-colonies with and without disruption treatment, suggesting that partial cell disruption by rapid depressurization improved the extraction of microalga compounds.(AU)