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.

Avaliação da eficiência de reutilização do CIDR® em protocolos de superovulação de ovelhas / Evaluation of efficiency of the reuse of CIDR® in sheep superovulation protocols

Sheep farming is an area of veterinary medicine of great importance at regional and national level, among the easily found breeds of economic relevance in northeastern Brazil are Morada Nova and Santa Inês. In addition, ultrasonography has been used systematically in animals superovulatory response to the sheep supervoulation protocols. In order to assess, based on the number of yellow bodies and number of viable embryos produced, the efficiency of the reuse of CIDR® in superovulation protocols in sheep using ultrasonography in color Doppler mode, a superovulatory protocol was performed in a total 40 sheep, with G-USED when 10 Morada Nova ewes and 10 Santa Inês ewes received reused CIDR® and G-NOVO when 10 Morada Nova ewes and 10 Santa Inês ewes received new CIDR®. The mean and standard error of yellow bodies was 10.35±1.33 for G-USED and 9.25±1.65 for G-NOVO, while the number of viable embryos was 6.50±2, 54 for G-USED and 8.25±1.49 for G-NEW. Thus, the reuse of CIDR® demonstrated efficiency in superovulation and did not interfere with the development of corpus luteum and the production of quality transferable embryos in sheep.

Avaliação do uso do CIDR novo e reutilizado no início e duração do estro em ovinos / Evaluation of the use of new and reused CIDR at the beginning and duration of estrus in sheep

Study evaluated the use of new and re-utilized CIDR® to increase the reproductive breeding efficiency of the Santa Inês (SI) and Morada Nova (MN). In the synchronization protocol, 40 sheep was divided into four groups: MN with re-utilized CIDR®; MN with new CIDR®; SI with re-utilized CIDR® and SI with new CIDR®. The protocollasted for 15 days. At D0, females received CIDR®. Between D7 and D9, ovarian superstimulation was inducedby two doses application with a 12-hour break of p-FSH. Estrus confirmation was evaluated with ruffians, and the duration determined with the first and last mounts. The ovulation was determined by B-mode ultrasound and color Doppler. All animals of the experiment showed signs of estrus after removal of the device and there was no difference between treatments (p>0.05). In Treatment 1 (T1, CIDR® used), the beginning of estrus was 22.3±1.53h and in Treatment 2 (T2, new CIDR®) was 22.9±1.64h. In estrus duration, T1 and T2 was similar (21.6±1.81 and 22.2±0.63, respectively). Conclusion: the treatments are efficient to induce and synchronize estrus, with satisfactory results in their duration.

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)

Avaliação da eficiência de reutilização do CIDR® em protocolos de superovulação de ovelhas / Evaluation of efficiency of the reuse of CIDR® in sheep superovulation protocols

Sheep farming is an area of veterinary medicine of great importance at regional and national level, among the easily found breeds of economic relevance in northeastern Brazil are Morada Nova and Santa Inês. In addition, ultrasonography has been used systematically in animals superovulatory response to the sheep supervoulation protocols. In order to assess, based on the number of yellow bodies and number of viable embryos produced, the efficiency of the reuse of CIDR® in superovulation protocols in sheep using ultrasonography in color Doppler mode, a superovulatory protocol was performed in a total 40 sheep, with G-USED when 10 Morada Nova ewes and 10 Santa Inês ewes received reused CIDR® and G-NOVO when 10 Morada Nova ewes and 10 Santa Inês ewes received new CIDR®. The mean and standard error of yellow bodies was 10.35±1.33 for G-USED and 9.25±1.65 for G-NOVO, while the number of viable embryos was 6.50±2, 54 for G-USED and 8.25±1.49 for G-NEW. Thus, the reuse of CIDR® demonstrated efficiency in superovulation and did not interfere with the development of corpus luteum and the production of quality transferable embryos in sheep.(AU)

Avaliação do uso do CIDR novo e reutilizado no início e duração do estro em ovinos / Evaluation of the use of new and reused CIDR at the beginning and duration of estrus in sheep

Study evaluated the use of new and re-utilized CIDR® to increase the reproductive breeding efficiency of the Santa Inês (SI) and Morada Nova (MN). In the synchronization protocol, 40 sheep was divided into four groups: MN with re-utilized CIDR®; MN with new CIDR®; SI with re-utilized CIDR® and SI with new CIDR®. The protocollasted for 15 days. At D0, females received CIDR®. Between D7 and D9, ovarian superstimulation was inducedby two doses application with a 12-hour break of p-FSH. Estrus confirmation was evaluated with ruffians, and the duration determined with the first and last mounts. The ovulation was determined by B-mode ultrasound and color Doppler. All animals of the experiment showed signs of estrus after removal of the device and there was no difference between treatments (p>0.05). In Treatment 1 (T1, CIDR® used), the beginning of estrus was 22.3±1.53h and in Treatment 2 (T2, new CIDR®) was 22.9±1.64h. In estrus duration, T1 and T2 was similar (21.6±1.81 and 22.2±0.63, respectively). Conclusion: the treatments are efficient to induce and synchronize estrus, with satisfactory results in their duration.(AU)

Influência do hepes na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro / Influence of hepes on in vitro maturation of canine oocytes in different stages of the estrous cycle

Diferentemente de outras espécies domésticas, a maturação in vitro (MIV) de oócitos caninos apresenta sucesso limitado. O objetivo deste estudo foi avaliar o efeito do HEPES na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro. Os ovários foram coletados, isolados assepticamente e transportados refrigerados a uma temperatura de 4 ºC. Os complexos cumulus-oócito (CCOs), provenientes das duas fases do ciclo estral, foram submetidos a dois tratamentos: meio TCM-199 com adição de 25 mM de HEPES (GT) e meio sem suplementação (GC). Depois de 72 horas de maturação, os CCOs foram desnudados, fixados e corados com HOESCHT 33342 para avaliação da maturação nuclear. Os oócitos obtidos da fase de anestro e diestro do GT demonstraram, em relação ao grupo GC, maior frequência de oócitos nos estágios de M-II (p<0,01). Comparando-se os diferentes status reprodutivos, observou-se que os oócitos obtidos da fase de diestro apresentaram índices maiores de QVG e M-II. Nossos resultados demonstraram que o HEPES preserva a viabilidade e morfologia oocitária, indispensáveis para a aquisição da competência meiótica, potencializando as taxas de M-II, e que os oócitos obtidos da fase de diestro estão mais aptos a completarem a maturação oocitária.

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The aim of this study was to evaluate the effect of HEPES on the in vitro maturation of dog oocytes in anestrus and diestrus. Ovaries were collected, aseptically isolated and transported refrigerated at a temperature of 4ºC. Cumulus-oocyte complexe (COCs) from the two phases of estrous cycle were subjected to two treatments: medium TCM-199 with addition of 25 mM HEPES (TG) and medium without supplementation (CG). After 72 h of maturation, COCs were denuded, fixed and stained with Hoechst 33342 to assess nuclear maturation. The oocytes obtained from the anestrus and diestrus phases of the TG showed a higher frequency of oocytes in metaphase II (M-II) (p <0.01) stage.Comparing the different reproductive status, it was observed that the oocytes obtained from the diestrus phase presented higher rates of GVBD (germinal vesicle breakdown) and M-II. Our results showed that HEPES preserves the viability and oocyte morphology essential for the acquisition of meiotic competence, increasing the M-II rates and that the oocytes obtained from the diestrus phase are better able to complete oocyte maturation.

Influência do hepes na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro / Influence of hepes on in vitro maturation of canine oocytes in different stages of the estrous cycle

Diferentemente de outras espécies domésticas, a maturação in vitro (MIV) de oócitos caninos apresenta sucesso limitado. O objetivo deste estudo foi avaliar o efeito do HEPES na maturação in vitro de oócitos caninos obtidos de cadelas em diestro e anestro. Os ovários foram coletados, isolados assepticamente e transportados refrigerados a uma temperatura de 4 ºC. Os complexos cumulus-oócito (CCOs), provenientes das duas fases do ciclo estral, foram submetidos a dois tratamentos: meio TCM-199 com adição de 25 mM de HEPES (GT) e meio sem suplementação (GC). Depois de 72 horas de maturação, os CCOs foram desnudados, fixados e corados com HOESCHT 33342 para avaliação da maturação nuclear. Os oócitos obtidos da fase de anestro e diestro do GT demonstraram, em relação ao grupo GC, maior frequência de oócitos nos estágios de M-II (p<0,01). Comparando-se os diferentes status reprodutivos, observou-se que os oócitos obtidos da fase de diestro apresentaram índices maiores de QVG e M-II. Nossos resultados demonstraram que o HEPES preserva a viabilidade e morfologia oocitária, indispensáveis para a aquisição da competência meiótica, potencializando as taxas de M-II, e que os oócitos obtidos da fase de diestro estão mais aptos a completarem a maturação oocitária.(AU)

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The aim of this study was to evaluate the effect of HEPES on the in vitro maturation of dog oocytes in anestrus and diestrus. Ovaries were collected, aseptically isolated and transported refrigerated at a temperature of 4ºC. Cumulus-oocyte complexe (COCs) from the two phases of estrous cycle were subjected to two treatments: medium TCM-199 with addition of 25 mM HEPES (TG) and medium without supplementation (CG). After 72 h of maturation, COCs were denuded, fixed and stained with Hoechst 33342 to assess nuclear maturation. The oocytes obtained from the anestrus and diestrus phases of the TG showed a higher frequency of oocytes in metaphase II (M-II) (p <0.01) stage.Comparing the different reproductive status, it was observed that the oocytes obtained from the diestrus phase presented higher rates of GVBD (germinal vesicle breakdown) and M-II. Our results showed that HEPES preserves the viability and oocyte morphology essential for the acquisition of meiotic competence, increasing the M-II rates and that the oocytes obtained from the diestrus phase are better able to complete oocyte maturation.(AU)

Sinoscopia como método de diagnóstico e tratamento de sinusite pós-traumática em equino / Synoscopy as a diagnostic method and treatment of post-traumatic synusitis in equin

Um cavalo de 6 anos apresentou secreção nasal unilateral e forte odor associado ao baixo desempenho atlético. Após avaliação clínica e exames de sinoscopia, confirmou-se sinusite primária, com grande quantidade de conteúdo purulento de muco nos seios paranasais esquerdos, além de fratura no osso nasal, sendo a possível causa da patologia. Com o auxílio do endoscópio e uma sonda de Foley, foram realizadas lavagens para remover o conteúdo, associando antibiótico com mucolítico diluído no soro. Como tratamento pós-operatório, foi formulada antibioticoterapia sistêmica, bem como lavagens diárias através da sonda com a mesma solução utilizada na cirurgia.

A 6-year-old horse presented unilateral nasal discharge and strong odor associated with poor athletic performance. After clinical evaluation and exams of sinoscopy, a primary sinusitis was confirmed, with a large amount of mucus-purulent content in the left paranasal sinuses, as well as a fracture in the nasal bone, being the possible cause of the pathology. With the aid of the endoscope and a Foley probe, washes were performed to remove the contents, associating antibiotic with mucolytic diluted in serum. As postoperative treatment a systemic antibiotic therapy was formulated, as well as daily washes through the probe with the same solution used in the surgery.

Sinoscopia como método de diagnóstico e tratamento de sinusite pós-traumática em equíno / Synoscopy as a diagnostic method and treatment of post-traumatic synusitis in equin

Um cavalo de 6 anos apresentou secreção nasal unilateral e forte odor associado ao baixo desempenho atlético. Após avaliação clínica e exames de sinoscopia, confirmou-se sinusite primária, com grande quantidade de conteúdo purulento de muco nos seios paranasais esquerdos, além de fratura no osso nasal, sendo a possível causa da patologia. Com o auxílio do endoscópio e uma sonda de Foley, foram realizadas lavagens para remover o conteúdo, associando antibiótico com mucolítico diluído no soro. Como tratamento pós-operatório, foi formulada antibioticoterapia sistêmica, bem como lavagens diárias através da sonda com a mesma solução utilizada na cirurgia.(AU)

A 6-year-old horse presented unilateral nasal discharge and strong odor associated with poor athletic performance. After clinical evaluation and exams of sinoscopy, a primary sinusitis was confirmed, with a large amount of mucus-purulent content in the left paranasal sinuses, as well as a fracture in the nasal bone, being the possible cause of the pathology. With the aid of the endoscope and a Foley probe, washes were performed to remove the contents, associating antibiotic with mucolytic diluted in serum. As postoperative treatment a systemic antibiotic therapy was formulated, as well as daily washes through the probe with the same solution used in the surgery.(AU)

Testicular morphological and ultrasonographic characterization of male gray brocket deers (Mazama gouazoubira) in different reproductive status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention.Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance ofmonitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotalcircumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocketdeer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods & Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicularvolume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes weresubjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD).Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity -EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen wasanalyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and threewere oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or KruskalWallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parametersusing Pearson’s correlation for parametric variables and Spearman’s correlation for non-parametric variables. F+ grouppresented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testescross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). Nosignificant differences were observed between the groups regarding...

Testicular morphological and ultrasonographic characterization of male gray brocket deers (Mazama gouazoubira) in different reproductive status

Background: Gray brocket deer (Mazama gouazoubira) populations have been declining due to human intervention.Yet, only a few studies have assessed ultrasonographic testicular characteristics in cervids. Considering the relevance ofmonitoring testicular size, blood flow, and parenchyma, the present study aims to establish baseline information on scrotalcircumference, testicular volume, and spectral Doppler parameters, to describe differences among adult male gray brocketdeer in different reproductive status, and to correlate ultrasound parameters with testes size measurements.Materials, Methods & Results: Six adult male gray brocket deers were used in the study. Scrotal circumference and testicularvolume were measured. B mode ultrasound images of testes (longitudinal and cross-sectional views) and epididymes weresubjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel standard deviation (PSD).Using spectral Doppler, supratesticular artery blood flow velocities (peak systolic velocity - PSV, end diastolic velocity -EDV, time-average maximum velocity - TAMAX, resistivity - RI and pulsatility indices - PI) were obtained. Semen wasanalyzed through total motility, vigor, and concentration tests. Three animals were normospermic (F+ group) and threewere oligo/azoospermic (F- group). Groups were compared using were compared using a one-way ANOVA or KruskalWallis followed by Student-Newman-Keuls (SNK) test. Ultrasound parameters were correlated to testes size parametersusing Pearsons correlation for parametric variables and Spearmans correlation for non-parametric variables. F+ grouppresented significantly higher scrotal circumference (14.57 ± 1.19 cm), testicular volume (26.18 ± 4.94 cm3), and testescross-sectional NPV (69.88 ± 24.00) and PSD (10.78 ± 3.42) than group F- (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). Nosignificant differences were observed between the groups regarding...(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.

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)

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.

Influence of epidermal growth factor (EGF) supplementation at different times of in vitro maturation of canine oocytes / Influência da suplementação do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos

The aim of this study was to evaluate the influence of epidermal growth factor (EGF) on in vitro maturation of canine oocytes at different times of the process. Ovaries were collected from 55 bitches considered healthy and aseptically isolated, immersed in physiological solution (0.9% NaCl) and transported under refrigeration. Grade 1 cumulus-oocyte complexes (COCs) were selected and divided into two groups: control group (CG) and treatment group (TG). In CG 698 grade I COCs were placed in 4-well plates containing TCM-199 medium supplemented with 25 mM HEPES, 100 IU/mL penicillin, 100 mg/mL streptomycin, 26 mM sodium bicarbonate, 1.5 mM sodium pyruvate, 2.9 mM sodium lactate pentahydrate, 0.6 mM cysteine, 0.03 IU/mL hCG, 0.5 µg/mL FSH, 20 µg/mL estrogen at 38.5ºC in a humidified atmosphere of 5% CO2 in times of 24 h, 48 h, and 72 h. In TG 547 COCs received the same maturation medium plus 10 ηg/mL EGF. Logistic regression models (SAS, 2011) were constructed in order to estimate the chances of oocytes being observed at nuclear maturation stages in different culture times (24 h, 48 h, and 72 h). Based on the results found EGF-supplemented medium showed 2.56 times more chances of having an oocyte at metaphase I (M-I) than medium without EGF (p < 0.0001). The results of this study demonstrated that the time of 72 h showed 5.88 times more chances of having an oocyte at metaphase II (M-II) compared to time of 24 h (p = 0.0001) and 7.69 times more chance than time of 48 h (p = 0.0001). The chances of finding an oocyte at M-II were also 9.09 times higher in medium supplemented with EGF than in medium without EGF (p = 0.0001). Thus, these results demonstrated the essential importance of EGF at different moments of oocyte maturation, being a key component for the acquisition of meiotic competence in bitches, increasing the M-I and M-II rates.(AU)

O objetivo deste estudo foi avaliar a influência do fator de crescimento epidermal (EGF) em diferentes momentos da maturação in vitro de oócitos caninos. Os ovários foram coletados de 55 cadelas consideradas sadias e isolados assepticamente, imersos em solução fisiológica e transportados refrigerados. Os complexos cumulus-oócito (COCs) grau 1 foram selecionados e divididos em dois grupos, denominados grupo controle (GC) e grupo tratamento (GT). No GC, 698 COCs grau I foram cultivados em placas de quatro poços contendo meio TCM-199 suplementado com 25 mM de HEPES, 100 UI/mL de penicilina, 100 mg/mL de estreptomicina, 26 mM de bicarbonato de sódio, 1,5 mM de piruvato de sódio, 2,9 mM de lactato de sódio penta hidratado, 0,6 mM de cisteína, 0,03 UI/mL de hCG, 0,5 µg/mL de FSH, 20 µg/mL de estrógeno em estufa úmida a 38ºC, 5% de CO2 nos períodos de 24h, 48 h e 72 h . Já no GT, 547 COCs receberam o mesmo meio de maturação acrescido de 10 ηg/mL do EGF. Modelos de regressão logística foram elaborados para estimar as chances do oócito ser observado nos estágios de maturação nuclear em diferentes tempos de cultivo. Com base nos resultados encontrados, o meio suplementado com EGF demonstrou 2,56 vezes mais chances de ter um oócito no estágio de metáfase I (M-I) do que o meio sem EGF (p < 0,0001). Os resultados desse estudo demonstraram também que o tempo de 72 h mostrou 5,88 vezes mais chances de ter um oócito no estágio de metáfase II (M-II) do que o tempo de 2 h (p = 0,0001) e 7,69 vezes mais chance do que o tempo de 48h (p = 0,0001). As chances de se encontrar um oócito em M-II também foram 9,09 vezes maiores no meio suplementado com EGF do que no meio sem EGF (p = 0,0001). Dessa forma, estes resultados demonstraram a importância essencial do EGF em diferentes momentos da maturação oocitária, sendo componente chave para a aquisição da competência meiótica nas cadelas, aumentando os índices de M-I e M-II.(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.(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)