Avaliação dos efeitos da piometra na viabilidade de oócitos caninos imaturos pela utilização do azul cresil brilhante / Evaluation of the effects of pyometra on the viability of immature canine oocytes through the use of brilliant cresyl blue

The bitch is an experimental model of wild and even endangered canids. Therefore, the study of the factors that influence your fertility benefits scientific advances in both segments. Knowing that pyometra is one of the common uterine pathologies in bitches, this work aimed to evaluate the effects of pyometra on the morphology and competence of canine oocytes through the use of Azul Cresil Brilhante (ACB). For this purpose, 1197 canine oocytes that were divided into 2 groups classified as control (healthy) and treatment (pyometra). They were morphologically classified into grade 1 (G1), grade 2 (G2) and grade 3 (G3) and according to the ACB stain as ACB (+) and ACB (­) (not stained). Bitches in the healthy group had higher amounts of total oocytes (795) and better quality (495 oocytes G1) and competence (45 ± 9.8 ACB (+)). The use of ACB was useful to distinguish the competence of the studied oocytes and can be an auxiliary tool for choosing the best oocytes.

Influência da idade sobre a qualidade de oócitos imaturos caninos / Influence of age on the quality of immature canine oocyte

Among some factors that influence the quality of canine oocytes, age is well studied. Knowing that the morphological assessment of oocytes is a useful tool for this analysis, the use of new techniques such as the brilliant cresyl blue staining (BCB) increases this assessment. The objective of this work was to analyze the quantity and the oocyte quality of bitches in different age groups through the conventional morphological evaluation, in addition to the bright cresyl blue dye. For this purpose, 2.302 oocytes from 33 bitches were divided into four age groups from 1 to 3 years old; 4-6, 7-10 and over 10 years old and their oocytes were classified as G1, G2 and G3, as well as stained (BCB (+)) and non-stained (BCB (-)). The values were compared using the Kruskal test -Wallis and Dunn post-test, by which it was possible to obtain higher amounts of G1 and BCB (+) oocytes in the range of 1 to3 years, indicating that this range oocytes have a greater chance of success in fertilization processes and in vitro production, for example.

Influência da idade sobre a qualidade de oócitos imaturos caninos / Influence of age on the quality of immature canine oocyte

Among some factors that influence the quality of canine oocytes, age is well studied. Knowing that the morphological assessment of oocytes is a useful tool for this analysis, the use of new techniques such as the brilliant cresyl blue staining (BCB) increases this assessment. The objective of this work was to analyze the quantity and the oocyte quality of bitches in different age groups through the conventional morphological evaluation, in addition to the bright cresyl blue dye. For this purpose, 2.302 oocytes from 33 bitches were divided into four age groups from 1 to 3 years old; 4-6, 7-10 and over 10 years old and their oocytes were classified as G1, G2 and G3, as well as stained (BCB (+)) and non-stained (BCB (-)). The values were compared using the Kruskal test -Wallis and Dunn post-test, by which it was possible to obtain higher amounts of G1 and BCB (+) oocytes in the range of 1 to3 years, indicating that this range oocytes have a greater chance of success in fertilization processes and in vitro production, for example.(AU)

Primeira avaliação da obtenção e qualidade de oócitos post mortem de lhamas pelo azul cresil brilhante / First recovery and quality assessment of post mortem oocytes from lhamas by brilliant cresyl blue

This study aimed to demonstrate the applicability of the ovarian slicing technique in the recovery of post mortem oocytes in a llama (lama glama) victim of acute bronchopneumonia and the effectiveness of the brilliant cresyl blue (ACB) technique to select quality oocytes for a subsequent in vitro culture. The ovary-salpinge-hysterectomy (OSH) was performed 12 hours after the animal's death. The ovaries were transported refrigerated immediately to the laboratory in Leibovitz medium (L-15). The oocytes were identified, quantified under a stereomicroscopic and classified according to two methods: conventional morphological classification and ACB assay. 26 oocytes classified by conventional morphology were obtained: 10 oocytes grade 1, 12 oocytes grade 2 and 4 oocytes grade3. The use of the ACB dye demonstrated that 88% of the evaluated oocytes maintained their quality and were competent for the conservation of genetic or future material application in reproductive biotechnologies in this species. Further studies are suggested aiming to use selected oocytes with ACB to enhance the success of in vitro culture in this species.

Primeira avaliação da obtenção e qualidade de oócitos post mortem de lhamas pelo azul cresil brilhante / First recovery and quality assessment of post mortem oocytes from lhamas by brilliant cresyl blue

This study aimed to demonstrate the applicability of the ovarian slicing technique in the recovery of post mortem oocytes in a llama (lama glama) victim of acute bronchopneumonia and the effectiveness of the brilliant cresyl blue (ACB) technique to select quality oocytes for a subsequent in vitro culture. The ovary-salpinge-hysterectomy (OSH) was performed 12 hours after the animal's death. The ovaries were transported refrigerated immediately to the laboratory in Leibovitz medium (L-15). The oocytes were identified, quantified under a stereomicroscopic and classified according to two methods: conventional morphological classification and ACB assay. 26 oocytes classified by conventional morphology were obtained: 10 oocytes grade 1, 12 oocytes grade 2 and 4 oocytes grade3. The use of the ACB dye demonstrated that 88% of the evaluated oocytes maintained their quality and were competent for the conservation of genetic or future material application in reproductive biotechnologies in this species. Further studies are suggested aiming to use selected oocytes with ACB to enhance the success of in vitro culture in this species.(AU)

Perfil proteico de oócitos caninos / Protein profile of canine oocytes

O presente estudo foi conduzido para caracterizaro proteoma de oócitos caninos. Oócitos foram coletados de 120 cadelas e apenas os COCs grau 1 foram selecionados para o cultivo in vitro. Após o cultivo, os oócitos foram submetidos à extração de proteínas. As proteínas foram digeridas com tripsina e analisadas por espectrometria de massa. Trinta e quatro proteínas foram identificadas nos oócitos caninos. Estas proteínas foram agrupadas em três categorias de acordo com a sua função biológica, molecular e localização celular. Quanto ao processo biológico, foram encontradas diversas proteínas envolvidas no ciclo celular, fertilização, regulação da transcrição e via de sinalização. A análise da ontologia do gene revelou alta porcentagem de proteínas envolvidas na atividade de ligação. Com base na análise da rede proteína-proteína usando a plataforma STRING, observou-se que a vimentina apresentou interações com as CASP3, CASP6, CASP7 e CASP8, envolvidos na apoptose. O componente de complemento C3, interagiu com receptores do complemento, como CR1 e CR2. A proteína de ligação retinol 4 interagiu com precursores de retinol. Actina esteve intimamente relacionada com as proteínas cofilinas 1e 2. A queratina 10 interagiu com a proteína CDK9 relacionada ao processo de sinalização celular. Essas proteínas são essenciais para o desenvolvimento completo de oócitos e fertilização. O presente estudo contém a primeira descrição da composição proteica dos oócitos caninos. A construção de bibliotecas de proteínas de oócitos, para cada espécie, estabelecerá as bases para a compreensão e o mapeamento dos eventos cruciais que definem a competência dos oócitos.

The present study was conducted to characterize the major proteome of canine oocytes. Ovaries were collected from 120 bitches and only Grade 1 COCs were selected for in vitro culture. After in vitro maturation, oocytes were subjected to protein extraction. Proteins were then trypsin-digested and analyzed by tandem mass spectrometry. Thirty-four proteins were identified in the canine oocytes. These proteins have been grouped into three different categories according to their biological, molecular function and cellular localization. With regard to biological process, we found many proteins involved in cell cycle, fertilization, transcription regulation and signaling pathway. The gene ontology analysis also revealeda high percentage of proteins involved in binding activity. Based on protein–protein network analysis using STRING platform, we found that vimentin presents links with CASP3, CASP6, CASP7 and CASP8, which are involved in apoptosis. Complement component C3, interacted with complement receptors, such as CR1 and CR2. Retinol-binding protein 4 interacted with retinol precursors. Actin potentially interacted with cofil in protein 1 and 2. Keratin 10, in turn, had interacted with CDK9, which are involved in pathway signaling. These proteins are essentials for the complete oocyte development and fertilization. In summary, the present study contains the first description of the main protein composition of canine oocytes. Construction of libraries of oocyte proteins, for each especies, will set the foundations for understanding and mapping the crucial events that define oocyte competence.

Achados necroscópicos em onça-pintada com tumor mamário / Necroscopic findings in jaguar with mammary tumor

With the increasing longevity of captive animals, chronic or senility-related diseases, as neoplasias, are even more often. The objective is to report the mainly anatomic and pathologic findings in jaguar (Pantera onca) with presumptive diagnostic of mammary carcinoma. The animal was taken to Ceará State University (UECE), were the necropsy proceeds. There was presence of two neoplasm tumor masses of large dimensions, one in the left mammary chain and the other one adhered to the rectus abdominis muscle, besides metastasis in inguinal lymph nodes, lungs, left ovary and adrenal, omentum and around the abdominal aorta, whose findings agree with what the literature says. With there being said, we can conclude that it was a high malignancy neoplasia, whose the large dimensions compromised the vascularization of the affected organs. Therefore, understanding the etiopathogeny of neoplasias is important to improve the prognostics and therapeutic conducts, and increase the quality of life in the affected animals.

Perfil proteico de oócitos caninos / Protein profile of canine oocytes

O presente estudo foi conduzido para caracterizaro proteoma de oócitos caninos. Oócitos foram coletados de 120 cadelas e apenas os COCs grau 1 foram selecionados para o cultivo in vitro. Após o cultivo, os oócitos foram submetidos à extração de proteínas. As proteínas foram digeridas com tripsina e analisadas por espectrometria de massa. Trinta e quatro proteínas foram identificadas nos oócitos caninos. Estas proteínas foram agrupadas em três categorias de acordo com a sua função biológica, molecular e localização celular. Quanto ao processo biológico, foram encontradas diversas proteínas envolvidas no ciclo celular, fertilização, regulação da transcrição e via de sinalização. A análise da ontologia do gene revelou alta porcentagem de proteínas envolvidas na atividade de ligação. Com base na análise da rede proteína-proteína usando a plataforma STRING, observou-se que a vimentina apresentou interações com as CASP3, CASP6, CASP7 e CASP8, envolvidos na apoptose. O componente de complemento C3, interagiu com receptores do complemento, como CR1 e CR2. A proteína de ligação retinol 4 interagiu com precursores de retinol. Actina esteve intimamente relacionada com as proteínas cofilinas 1e 2. A queratina 10 interagiu com a proteína CDK9 relacionada ao processo de sinalização celular. Essas proteínas são essenciais para o desenvolvimento completo de oócitos e fertilização. O presente estudo contém a primeira descrição da composição proteica dos oócitos caninos. A construção de bibliotecas de proteínas de oócitos, para cada espécie, estabelecerá as bases para a compreensão e o mapeamento dos eventos cruciais que definem a competência dos oócitos.(AU)

The present study was conducted to characterize the major proteome of canine oocytes. Ovaries were collected from 120 bitches and only Grade 1 COCs were selected for in vitro culture. After in vitro maturation, oocytes were subjected to protein extraction. Proteins were then trypsin-digested and analyzed by tandem mass spectrometry. Thirty-four proteins were identified in the canine oocytes. These proteins have been grouped into three different categories according to their biological, molecular function and cellular localization. With regard to biological process, we found many proteins involved in cell cycle, fertilization, transcription regulation and signaling pathway. The gene ontology analysis also revealeda high percentage of proteins involved in binding activity. Based on protein–protein network analysis using STRING platform, we found that vimentin presents links with CASP3, CASP6, CASP7 and CASP8, which are involved in apoptosis. Complement component C3, interacted with complement receptors, such as CR1 and CR2. Retinol-binding protein 4 interacted with retinol precursors. Actin potentially interacted with cofil in protein 1 and 2. Keratin 10, in turn, had interacted with CDK9, which are involved in pathway signaling. These proteins are essentials for the complete oocyte development and fertilization. In summary, the present study contains the first description of the main protein composition of canine oocytes. Construction of libraries of oocyte proteins, for each especies, will set the foundations for understanding and mapping the crucial events that define oocyte competence.(AU)

Achados necroscópicos em onça-pintada com tumor mamário / Necroscopic findings in jaguar with mammary tumor

With the increasing longevity of captive animals, chronic or senility-related diseases, as neoplasias, are even more often. The objective is to report the mainly anatomic and pathologic findings in jaguar (Pantera onca) with presumptive diagnostic of mammary carcinoma. The animal was taken to Ceará State University (UECE), were the necropsy proceeds. There was presence of two neoplasm tumor masses of large dimensions, one in the left mammary chain and the other one adhered to the rectus abdominis muscle, besides metastasis in inguinal lymph nodes, lungs, left ovary and adrenal, omentum and around the abdominal aorta, whose findings agree with what the literature says. With there being said, we can conclude that it was a high malignancy neoplasia, whose the large dimensions compromised the vascularization of the affected organs. Therefore, understanding the etiopathogeny of neoplasias is important to improve the prognostics and therapeutic conducts, and increase the quality of life in the affected animals.(AU)

Avaliação da atividade da proteína quinase P34Cdc2 na maturação in vitro de oócitos caninos / Evaluation of p34cdc2 kinase activity in the in vitro maturation of canine oocytes

O controle do ciclo celular é regulado por uma cascata de eventos coordenados que podem atuar influenciando na expressão ou repressão da atividade de proteínas relacionadas a retomada da meiose. Estudos indicam que a atividade dessas proteínas mostra-se tempo-dependente no processo de maturação in vitro (MIV). Esse trabalho teve o objetivo de avaliar a cinética da atividade quinase p34cdc2 durante a MIV de oócitos caninos. Ciência Animal 27(1), 2017. Os ovários foram obtidos de 40 cadelas submetidas à ovário-salpingo-histerectomia (OSH) eletiva. Após a OSH, os ovários foram imediatamente transportados a uma temperatura de 4 ºC. No laboratório, os ovários foram seccionados em fatias finas ("slicing"), para a liberação dos complexos cumulus-oócito (COCs). Apenas os COCs grau 1 foram selecionados e colocados em meio de maturação por um período de 24, 48 e 72 h de maturação. Após o cultivo, os COCs foram colocados em placas contendo solução de hialuronidase 0.2% para a retirada completa das células do Cumulus. A atividade da proteína p34cdc2 foi detectada por ELISA. Com base nos resultados verifica-se que a atividade da proteína mostra-se tempo-dependente, atingindo o pico após 48h de MIV (p<0,01). Após 72h, a atividade demonstrou um decréscimo. Com base neste estudo pode-se concluir que a proteína quinase p34cdc2 desempenha uma função de suma importância na progressão da meiose em cadelas. Dessa forma, a compreensão melhor dessa proteína assim como de outra que participam do processo de maturação poderá contribuir para o estabelecimento de meios mais adequados que melhorem significativamente as taxas dematuração.

The control of the cell cycle is regulated by a cascade of coordinated events that can act by influencing the expression or repression of the activity of proteins related to meiosis resumption. Studies have indicated that the activity of these proteins is time-dependent in the in vitro maturation process (IVM). This work aimed to evaluate the kinetics of the p34cdc2 kinase activity during IVM of canine oocytes. Ovaries were obtained from 40 bitches submitted to elective ovary-salpingo-hysterectomy (OSH). After OSH, ovaries were immediately transported at temperature of 4 °C. In the laboratory, ovaries were sliced for the release of cumulus-oocyte complexes (COCs). Only grade 1 COCs were selected and placed in maturation medium for a period of 24, 48 and 72 h of maturation. After culturing, COCs were plated of 0.2% hyaluronidase solution for complete removal of cumulus cells. The activity of the p34cdc2 protein was detected by ELISA. Based on the results, it was observed that the activity of the protein is time-dependent, peaking after 48 hours of IVM (p 0.01). After 72 hours, activity declined. Based on this study, it could be concluded that the p34cdc2 protein kinase plays a very important role in the meiosis progression in bitches. Thus, a better understanding of this protein as well as of others that participate in the maturation process may contribute to the establishment of more adequate media to significantly improve maturation rates.

Avaliação da atividade da proteína quinase P34Cdc2 na maturação in vitro de oócitos caninos / Evaluation of p34cdc2 kinase activity in the in vitro maturation of canine oocytes

O controle do ciclo celular é regulado por uma cascata de eventos coordenados que podem atuar influenciando na expressão ou repressão da atividade de proteínas relacionadas a retomada da meiose. Estudos indicam que a atividade dessas proteínas mostra-se tempo-dependente no processo de maturação in vitro (MIV). Esse trabalho teve o objetivo de avaliar a cinética da atividade quinase p34cdc2 durante a MIV de oócitos caninos. Ciência Animal 27(1), 2017. Os ovários foram obtidos de 40 cadelas submetidas à ovário-salpingo-histerectomia (OSH) eletiva. Após a OSH, os ovários foram imediatamente transportados a uma temperatura de 4 ºC. No laboratório, os ovários foram seccionados em fatias finas ("slicing"), para a liberação dos complexos cumulus-oócito (COCs). Apenas os COCs grau 1 foram selecionados e colocados em meio de maturação por um período de 24, 48 e 72 h de maturação. Após o cultivo, os COCs foram colocados em placas contendo solução de hialuronidase 0.2% para a retirada completa das células do Cumulus. A atividade da proteína p34cdc2 foi detectada por ELISA. Com base nos resultados verifica-se que a atividade da proteína mostra-se tempo-dependente, atingindo o pico após 48h de MIV (p<0,01). Após 72h, a atividade demonstrou um decréscimo. Com base neste estudo pode-se concluir que a proteína quinase p34cdc2 desempenha uma função de suma importância na progressão da meiose em cadelas. Dessa forma, a compreensão melhor dessa proteína assim como de outra que participam do processo de maturação poderá contribuir para o estabelecimento de meios mais adequados que melhorem significativamente as taxas dematuração.(AU)

The control of the cell cycle is regulated by a cascade of coordinated events that can act by influencing the expression or repression of the activity of proteins related to meiosis resumption. Studies have indicated that the activity of these proteins is time-dependent in the in vitro maturation process (IVM). This work aimed to evaluate the kinetics of the p34cdc2 kinase activity during IVM of canine oocytes. Ovaries were obtained from 40 bitches submitted to elective ovary-salpingo-hysterectomy (OSH). After OSH, ovaries were immediately transported at temperature of 4 °C. In the laboratory, ovaries were sliced for the release of cumulus-oocyte complexes (COCs). Only grade 1 COCs were selected and placed in maturation medium for a period of 24, 48 and 72 h of maturation. After culturing, COCs were plated of 0.2% hyaluronidase solution for complete removal of cumulus cells. The activity of the p34cdc2 protein was detected by ELISA. Based on the results, it was observed that the activity of the protein is time-dependent, peaking after 48 hours of IVM (p 0.01). After 72 hours, activity declined. Based on this study, it could be concluded that the p34cdc2 protein kinase plays a very important role in the meiosis progression in bitches. Thus, a better understanding of this protein as well as of others that participate in the maturation process may contribute to the establishment of more adequate media to significantly improve maturation rates.(AU)

Influence of anestrus and diestrus stages of oocyte on meiotic competence in bitches / Influência das fases de anestro e diestro na competência meiótica oocitária de cadelas

The bitch has reproductive peculiarities that differentiate it from other species. Several experiments have been conducted to establish efficient protocols for maturation; however, the results appear to be unsatisfactory. In this respect, the reproductive female donor should be considered, since it can be a factor of variability of findings in this species. The objective of this study was to evaluate the relationship between the estrous cycle phases phase diestrus and anestrus on canine oocyte in vitro maturation (IVM). The ovaries were transported in sodium chloride 0.9% solution, and were cut into phosphate-buffered saline (PBS) and the cumulus-oocyte complexes (COCs) selected in tissue culture medium (TCM), supplemented with 199 HEPES. A total of 469 grade 1 oocytes were collected from bitches in anestrus and diestrus. These selected oocytes were transferred to the maturation medium for a period of 72 hours, then subjected to hyaluronidase solution and stained with Hoechst 33342 to assess nuclear configuration. The comparison of anestrus and diestrus phase showed no differences (p > 0.05) between the nuclear maturation stages. Thus, the phase of the estrous cycle did not influence the in vitro maturation of canine oocytes, increasing the rates of M-II in this species.(AU)

A cadela apresenta particularidades reprodutivas que a diferencia de outras espécies. Diversos experimentos têm sido realizados visando estabelecer protocolos eficientes para a maturação, entretanto os resultados mostram-se insatisfatórios. Nesse aspecto, a fase reprodutiva da fêmea doadora deve ser considerada, já que pode ser um fator de variabilidade dos achados ate então presenciados nessa espécie. O objetivo deste estudo foi avaliar a influencia das fases do ciclo estral (anestro e diestro) na maturação in vitro (MIV) de cadelas. Os ovários foram transportados em solução de cloreto de sódio 0,9% e seccionados em solução salina fosfato tamponado (PBS) e os complexos cumulus-oócito (COCs) selecionados em meio de cultura de tecidos (TCM) 199 suplementado com HEPES. Foram obtidos 469 oócitos grau 1 de cadelas em anestro e diestro. Esses oócitos selecionados foram transferidos para o meio de maturação por um período de 72 horas, sendo posteriormente submetidos a solução de hialuronidase e corados com HOESCHT 33342 para avaliação da configuração nuclear. A comparação das fases de anestro e diestro não revelou diferença (p > 0,05) entre os estágios de maturação nuclear. Dessa maneira, a fase do ciclo estral não influenciou na maturação in vitro de oócitos caninos, incrementando os índices de M-II nesta espécie.(AU)

Efeito do fator de crescimento epidermal (EGF) na maturação in vitro de oócitos caninos / Effect of epidermal growth factor (EGF) on in vitro maturation of canine oocyte

Este trabalho leve o objetivo de avaliar a influência do EGF na maturação in vitro (MIV) de cadelas. Realizou-se o transporte dos ovários em solução de cloreto de sódio 0,9%, que foram seccionados em solução salina fosfato temponado (PBS) e os complexos cumulus oócitos (COCs) selecionados em meio de cultura de tecidos (TCM) 199 suplementado com HEPES. Foram coletados 405 oócitos grau 1 de cadelas em anestro e diestro. Os oocitos provenientes das duas fases do ciclo estral foram submetidos a dois tratamentos: meio com adição de 10ng/mL do fator de crescimento epidermal (EGF) (T) e meio sem suplementação (C). Depois de 72 horas de maturação, os COCs foram desnudados, fixados e corados com HOESCHT 33342 para avaliação da maturação nuclear. Os oócitos obtidos dos ovários da fase de diestro do grupo T demonstraram maior porcentagem (18,8%) de oócitos no estágio de metáfase II (M-II), em relação ao grupo C (1,3%) (p < 0,01). Nos oócitos obtidos da fase de anestro houve diferença (p < 0,05) entre os grupos, observando-se menor parcela (4,1%) de oócitos no estágio de vesícula germinativa (VG) no grupo T, quando comparado ao grupo C (16,1%). Comparando-se as diferentes fases reprodutivas, em meios suplementados com EGF, foi observada diferença (p < 0,05) apenas nos oócitos obtidos da fase de diestro em relação ao estágio de M-II. Dessa maneira, a suplementação no meio de maturação na concentração de 10 ηg/mL, neste estudo, exerceu influência positiva apenas na MIV de oócitos caninos oriundos da fase de diestro, incrementando os índices de M-II nessa espécie.(AU)

This work aimed to evaluate the influence of EGF on canine oocyte in vitro maturation (IVM). We carried out the transport of the ovaries in sodium chloride 0.9% solution, which were cut into phosphate-buffered saline (PBS) and the cumulus oocyte complexes (COCs) selected in tissue culture medium (TCM), supplemented with 199 HEPES. A total of 405 grade 1 oocytes were collected from bitches in anestrus and diestrus. Oocytes from the two phases of estrous cycle were subjected to two treatments: medium with addition of 10 ηg/mL epidermal growth factor (EGF) (T) and medium without supplementation (C). After 72 h of maturation, COCs were denuded, fixed and stained with Hoechst 33342 to assess nuclear maturation. The oocytes obtained from the ovaries of the diestrus phase of the T group demonstrated higher percentage (18.8%) of oocytes at metaphase II stage (M-II) than C group (1.3%) (p < 0.01). The oocytes from anestrus phase demonstrated difference (p < 0.05) between the groups, observing smaller proportion (4.1%) of oocytes at the stage of germinal vesicle (GV) in group T compared to group C (16.1%). Comparing the different reproductive status in media supplemented with EGF difference (p < 0.05) was observed only in oocytes diestrus phase relative to the stage of M-II. Thus, supplementation on maturation medium at a concentration of 10 ηg/mL of EGF in this study had positive influence only on IVM of canine oocytes obtained from diestrus phase, increasing the levels of M-II in this species.(AU)

Influence of stages anestrous and diestrus in chromatin configuration in germinal vesicle of canine oocytes / Influência das fases de anestro e diestro sobre a configuração da cromatina em vesícula germinativa de oócitos caninos

In canine specie, oocyte maturation rates are low and the percentage of oocytes that remain in the stage of germinal vesicle (GV) regardless of culture conditions is high. During maturation oocyte undergoes modification and the GV chromatin remodeling manifested by changes in the configuration and positioning. The objective of this work is to evaluate the configuration and positioning of chromatin of oocytes in GV stage during anestrus and diestrus bitches. The ovaries of 33 females (20 bitches in anestrous and 13 in diestrus) were isolated, sliced and only cumulus-oocyte complexes (COCs) grade 1 were subjected to solution of 0.2% hyaluronidase for release in cumulus cells. After this process, the selected oocytes were stained and evaluated. From a total of 920 oocytes, 566 were classified as grade 1 and the stages of chromatin configuration identified as GV-1, GV-2, GV-3 and GV-4. The observed changes in chromatin configuration been characterized as a transition dispersed chromatin (GV-1, GV-2) for partially condensed (GV-3) until it reaches a fully condensed stage (GV-4). The data analyzed from the chromatin configuration showed a significant difference between the stages with a higher proportion of GV-1 and GV-2 for the anoestrus and GV-3 and GV-4 during diestrus. There is need for further studies to be able to have a proper understanding of the influence of chromatin configuration of oocytes in GV stage in resumption of meiosis and consequently in oocyte meiotic competence(AU)

Na espécie canina as taxas de maturação oocitária são baixas e a porcentagem de oócitos que permanecem em estagio de vesícula germinativa (VG), independente das condições de cultivo, e alta. Durante a maturação oocitária, a VG sofre modificação e remodelamento da cromatina, que se manifesta por alterações na sua configuração e posicionamento. Assim, o objetivo deste trabalho e avaliar a configuração e o posicionamento da cromatina de oócitos em estagio de VG durante o anestro e diestro de cadelas. Os ovários de 33 fêmeas (20 cadelas em anestro e 13 em diestro) foram isolados, fatiados e os complexos cumulus-oócitos (COCs) foram submetidos à solução de hialuronidase 0,2% para a liberação das células do cumulus. Apos esse processo, os oócitos selecionados foram corados, avaliados e apenas COCs grau 1 foram utilizados. De um total de 920 oócitos, 566 foram classificados como grau 1 e os estágios de configuração da cromatina identificados como VG-1, VG-2, VG-3 e VG-4. As alterações observadas na configuração da cromatina foram caracterizadas como transição de uma cromatina dispersa (VG-1, VG-2) para parcialmente condensada (VG-3) ate atingir um estagio totalmente condensado (VG-4). Os dados analisados da configuração da cromatina mostraram uma diferença significativa entre as fases de anestro e diestro, com maior proporção de VG-1 e VG-2 durante o anestro e de VG-3 e VG-4 durante o diestro. Ha necessidade de novos estudos para uma compreensão adequada da influencia da configuração da cromatina de oócitos no estagio de VG na retomada da meiose e na competência meiótica do oócito(AU)

Low-level laser on femoral growth plate in rats / Laser terapêutico de baixa potência na placa de crescimento de ratos

PURPOSE: To determine the influence of low-level laser therapy on femoral growth plate in rats. METHODS: Thirty male Wistar rats aged 40 days were divided into two groups, G1 and G2. In G1 the area of the distal growth plate of the right femur was irradiated at one point using GaAlAs laser 830 nm wavelength, output power of 40 mW, at an energy density of 10 J/cm². The irradiation was performed daily for a maximum of 21 days. The same procedure was done in G2, but the probe was turned off. Five animals in each group were euthanized on days 7, 14 and 21 and submitted to histomorphometric analysis. RESULTS: In both groups the growth plate was radiographically visible at all moments from both craniocaudal and mediolateral views. On the 21st day percentage of femoral longitudinal length was higher in G2 than G1 compared to basal value while hypertrophic zone chondrocyte numbers were higher in G1 than G2. Calcified cartilage zone was greater in G1 than in G2 at all evaluation moments. Angiogenesis was higher in G1 than in G2 at 14th and 21st days. CONCLUSION: The low-level laser therapy negatively influenced the distal femoral growth plate.(AU)

OBJETIVO: Determinar a influência do Laser Terapêutico de Baixa Potência sobre a placa de crescimento de ratos. MÉTODOS: Trinta ratos Wistar machos com 40 dias de idade foram divididos em dois grupos, G1 e G2. O grupo G1 foi submetido à irradiação com laser GaAlAs 830 nm, potência de saída de 40 mW, e densidade de energia de 10 J/cm2. A irradiação foi aplicada diariamente por um período máximo de 21 dias. O mesmo procedimento foi realizado no grupo G2, com a probe desativada. Cinco animais em cada grupo foram sacrificados nos dias 7, 14 e 21 e submetidas à análise histomorfométrica. RESULTADOS: Em ambos os grupos, o disco fisário esteve radiograficamente visível em todos os momentos nas incidências craniocaudal e médio-lateral. No 21º dia a porcentagem de comprimento longitudinal do fêmur foi maior em G1 que em G2 em relação ao valor basal, e o número de condrócitos da zona hipertrófica foi maior em G1 que em G2. A zona de cartilagem calcificada estava maior em G1 em relação a G2 em todos os momentos de avaliação. A angiogênese foi maior em G1 que em G2 nos 14º e 21º dias. CONCLUSÃO: A terapia com laser terapêutico de baixa potência influenciou negativamente o disco fisário distal do fêmur de ratos.(AU)

Expressão imunoistoquímica da cicloxigenase-2 (Cox2) e quantificação das regiões organizadoras de nucléolos (NORs) nos diferentes padrões histológicos do osteossarcoma canino / Expression immunohistochemistry of the cyclo-oxygenase 2 (Cox2) and quantification of the nucleolus organizing region (NOR) in different histological patterns of a canine osteosarcoma

O propósito deste trabalho foi avaliar a intensidade da cicloxigenase 2 (Cox2), as Regiões Organizadoras de Nucléolos Impregnadas pela Prata (AgNORs) e as figuras de mitose por campo nos diferentes tipos de osteossarcomas canino, a partir de um estudo retrospectivo de pouco mais de 10 anos dos casos de osteossarcoma canino que surgiram na rotina da clínica de pequenos animais da Universidade Federal de Viçosa e da Escola de Veterinária da Universidade Federal de Minas Gerais. As 78 ocorrências encontradas foram classificadas histologicamente e 6 casos de cada grupo classificados como osteossarcomas osteoblásticos, condroblásticos, fibroblásticos e combinados (mistos) foram submetidos às avaliações histoquímicas e imunoistoquímicas do estudo. Os osteossarcomas teleangectásico, de células gigantes e pobremente diferenciados não foram avaliados em decorrência do pequeno número de casos encontrados. Os resultados mostraram diferenças estatística (p<0,05) nas expressões da Cox2 e nas proteínas AgNORs entre os diferentes osteossarcomas, no entanto, esta diferença não acorreu na contagem das figuras de mitoses. Em todas as avaliações, o osteossarcoma osteoblástico que surgiu com 53% dos casos, foi o que apresentou maior número de NORs, de figuras de mitoses por campo e de casos positivos com maior contagem de células imunomarcadas. Desta forma, pode-se sugerir que o osteossarcoma osteoblástico apresenta maior agressividade em relação aos demais tipos de osteossarcomas avaliados neste trabalho, porém, por falta de informações e padronização nos dados clínicos, não foi possível associar estes achados com a sobrevida e desenvolvimento tumoral, correlacionando-os com a agressividade dos diferentes tipos de osteossarcomas canino

The purpose of this study was to assess the intensity of the cyclo-oxygenase 2 (Cox2), as well as the nucleolus organizing regions impregnated with silver (AgNORs) and mitosis figures in different canine oteosarcomas. The material used for the analysis was obtained from a retrospective study of approximately 10 years, of canine oteosarcomas recorded in the clinics routine at Federal University of Viçosa and at Federal University of Minas Gerais. The 78 cases of osteosarcomas recorded were histologically analysed and 6 cases of each group were classified as being osteoblastics, condroblastics, fibroblastics and combined cases. This material was submitted to both histochemistry and immunohistochemistry evaluation. Telangiectatic giant cell type and poorly differentiated osteosarcomas were not evaluated due to the small number of observed cases. The results presented statistical differences (p<0,05) for the Cox2 expressions and for the AgNORs proteins among the different osteosarcomas, however no difference was found for the mitosis figures counting. Considering all the evaluations, the osteoblastic osteosarcoma recorded in 53 % of the cases, presented the highest number of NORs and mitosis figures by field, and also it showed the highest number of positive cases with the higher immunomarked cells counting. The results suggest that the osteoblastic osteosarcoma is more aggressive than the other kinds of osteosarcoma evaluated in this study, but because of the lack of both information and standardized clinics data, it is not possible at this point, to connect these findings with the over life and tumor development, and not even to correlate the agressivity of the different kinds of canine osteosarcomas