Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin / Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta

Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin / Gene expression and in vitro nuclear maturation in bovine cumulus oocyte complexes maturated in a medium supplemented with bovine fetal serum or bovine serum albumin

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta

Background: The expansion and mucification of granulosa cells of the cumulus oophorus-oocyte complex (COC) is observed during the oocyte in vitro maturation (IVM) as a result of the intense synthesis of extracellular matrix (ECM) components. These changes in cumulus aspect are indicative of maturation and may be influenced by oocyte-related factors and by IVM conditions. The objectives of the present study were (i) to assess the expression of gene transcripts that codify for the proteins hyaluronan synthase-2 (HAS2), link protein 1, connexin 43 and -actin in bovine cumulus oophorus-oocyte complexes (COCs) before and after IVM, and (ii) to determine nuclear maturation rates of oocytes submitted to IVM. Materials, Methods & Results: Bovine COCs obtained from abattoir-derived ovaries were analyzed and selected for morphological aspects and divided in three experimental groups: G1, COCs submitted to IVM; G2, COCs submitted to IVM in medium supplemented with 10% fetal bovine serum (FBS); and G3, COCs submitted to IVM in medium supplemented with bovine serum albumin (BSA). After extraction of the messenger RNA (mRNA) of COCs, cDNA was extracted and fragments of the gene transcripts were amplified using the reverse transcription (RT) and the polymerase chain reaction (PCR). The RT-PCR products were electrophoresed in agarose gels and amplification intensity was quantified to obta

Is cardiac tissue more susceptible than lung to oxidative effects induced by chronic nasotropic instillation of residual oil fly ash (ROFA)?

The current study aimed to determine the role of oxidants in cardiac and pulmonary toxicities induced by chronic exposure to ROFA. Eighty Wistar rats were divided into four groups: G1 (10 µL Saline), G2 (ROFA 50 µg/10 µL), G3 (ROFA 250 µg/10 µL) and G4 (ROFA 500 µg/10 µL). Rats received ROFA by nasotropic instillation for 90 days. After that, they were euthanized and bronchoalveolar lavage (BAL) was performed for total count of leukocytes, protein and lactate dehydrogenase (LDH) determinations. Lungs and heart were removed to measure lipid peroxidation (MDA), catalase (CAT) and superoxide dismutase (SOD) activity. BAL presented an increase in leukocytes count in G4 in comparison to the Saline group (p = 0.019). In lung, MDA level was not modified by ROFA, while CAT was higher in G4 when compared to all other groups (p = 0.013). In heart, G4 presented an increase in MDA (p = 0.016) and CAT (p = 0.027) levels in comparison to G1. The present study demonstrated cardiopulmonary oxidative changes after a chronic ROFA exposure. More specifically, the heart tissue seems to be more susceptible to oxidative effects of long-term exposure to ROFA than the lung.

Expressão gênica das células do cumulus oophorus de bovinos após a vitrificação

Um dos desafios da criobiologia continua sendo o desenvolvimento de um método que proporcione a manutenção da viabilidade após a criopreservação de oócitos imaturos na espécie bovina. A vitrificação tem sido a metodologia que proporciona resultados de sobrevivência após a criopreservação mais promissores para complexos  cumulus-oócito (CCOs) imaturos bovinos. Entretanto, a ação dos crioprotetores sobre as células do cumulus oophorus, no que diz respeito à regulação da expressão de genes importantes nesta fase, ainda é pouco compreendida. O objetivo do trabalho foi avaliar a expressão gênica das proteínas ácido hialurônico sintase 2 (HAS2), link protein (HAPLN1), conexina 43 (GJA1) e proteína de choque térmico HSP70-1 (HSP70-1) em células do cumulus oophorus de oócitos imaturos bovinos submetidos a exposição e/ou vitrificação na solução crioprotetora (SV) composta por 20% de etileno glicol (EG) + 20% dimetil sulfóxido (DMSO) + 0,5M de sacarose. Os CCOs foram selecionados e distribuídos em 4 grupos experimentais: G1, CCOs não submetidos a maturação in vitro (MIV); G2, CCOs submetidos à MIV; G3, CCOs maturados após a exposição à SV; e G4, CCOs maturados após a vitrificação com a SV. A MIV foi realizada em TCM 199, suplementado com soro de égua em estro, à 39oC, 5% de CO2 e máxima umidade relativa, por 22 a 24 horas. A extração do RNA das amostras de células do cumulus foi realizad

Expressão gênica das células do cumulus oophorus de bovinos após a vitrificação

Um dos desafios da criobiologia continua sendo o desenvolvimento de um método que proporcione a manutenção da viabilidade após a criopreservação de oócitos imaturos na espécie bovina. A vitrificação tem sido a metodologia que proporciona resultados de sobrevivência após a criopreservação mais promissores para complexos  cumulus-oócito (CCOs) imaturos bovinos. Entretanto, a ação dos crioprotetores sobre as células do cumulus oophorus, no que diz respeito à regulação da expressão de genes importantes nesta fase, ainda é pouco compreendida. O objetivo do trabalho foi avaliar a expressão gênica das proteínas ácido hialurônico sintase 2 (HAS2), link protein (HAPLN1), conexina 43 (GJA1) e proteína de choque térmico HSP70-1 (HSP70-1) em células do cumulus oophorus de oócitos imaturos bovinos submetidos a exposição e/ou vitrificação na solução crioprotetora (SV) composta por 20% de etileno glicol (EG) + 20% dimetil sulfóxido (DMSO) + 0,5M de sacarose. Os CCOs foram selecionados e distribuídos em 4 grupos experimentais: G1, CCOs não submetidos a maturação in vitro (MIV); G2, CCOs submetidos à MIV; G3, CCOs maturados após a exposição à SV; e G4, CCOs maturados após a vitrificação com a SV. A MIV foi realizada em TCM 199, suplementado com soro de égua em estro, à 39oC, 5% de CO2 e máxima umidade relativa, por 22 a 24 horas. A extração do RNA das amostras de células do cumulus foi realizad

Diagnóstico ultra-sonográfico do momento ovulatório em búfalas (Bubalus bubalis) inseminadas no estro espontâneo e induzido

The aim of this experiment was to determine the interval between the beginning of the spontaneous or induced oestrus and the ovulation in females buffaloes using ultrasonography. This will be useful in the determination of the most proper moment for the pre-fixed artificial insemination. In the reproductive season, autumn in the South of Brazil (march-june), 132 clicling females were divided in 3 groups: Group A: 53 females were treated with auricular subcutaneous implant of norgestomet or intravaginal device of progesterone. In the moment that the devices were removed, 250mg of cloprostenol were applied for intravulvar submucosis (ivsm). Group B: 48 buffaloes females were treated twice with 250mg of cloprostenol for ivsm with interval of 11 days. Group C: 31 buffaloes females remained without any treatment (control group). All of them were inseminated in the moment that was observed the biggest diameter of pre-ovulatory follicle determinated by ultrasonography. In the 3 groups, there was significative difference between pregnant and non-pregnant females in the oestrus-ovulation interval and in the A.I.-ovulation interval. The pregnancy rates were 41.5%, 52.1% and 54.8% in the groups A, B amd C, respectively. The variation in the oestrus-ovulation in buffaloes is the major obstacle to achieve high pregnancy rates using pre-fixed artificial insemination in spontaneous and induced oestrus.

O objetivo deste experimento foi determinar o intervalo entre o início do estro induzido ou espontâneo e a ovulação em fêmeas bubalinas (Bubalus bubalis) com o auxílio da ultra-sonografia, o que permitirá a determinação de um horário mais apropriado para a I.A. pré-fixada. Nos meses de março a junho, outono no sul do Brasil (época reprodutiva dos bubalinos), 132 fêmeas adultas ciclando foram divididas em 3 grupos experimentais: Grupo A - 53 fêmeas foram tratadas com implante subcutâneo de Norgestomet ou espiral intravaginal contendo progesterona. Na retirada dos dispositivos, foi aplicado 250mg de cloprostenol pela via intra-submucosa vulvar (i.s.m.v.), Grupo B - 48 búfalas foram tratadas com dupla aplicação de 250mg de cloprostenol pela via i.s.m.v. com intervalo de 11 dias e Grupo C - 31 búfalas foram consideradas testemunhas, sem tratamento. Todas as búfalas foram inseminadas no momento da observação do maior diâmetro do folículo pré-ovulatório, detectado por ultra-sonografia, durante o estro. Após o diagnóstico de prenhez, constatou-se, nos três tratamentos, que houve diferença significativa entre as búfalas prenhes e vazias no período compreendido entre o início do estro até o momento da ovulação e no período entre a I.A.e a ovulação. Os índices de prenhez foram de 41,5%, 52,1% e 54,8% nos grupos A, B e C, respectivamente. A variação no intervalo estro-ovulação nas búfalas é uma barreira para a obtenção de taxas de prenhez por I.A. pré-fixada comparáveis à monta natural, tanto no estro induzido através de progesterona e prostaglandina F2 alfa como no estro espontâneo.

Diagnóstico ultra-sonográfico do momento ovulatório em búfalas (Bubalus bubalis) inseminadas no estro espontâneo e induzido

The aim of this experiment was to determine the interval between the beginning of the spontaneous or induced oestrus and the ovulation in females buffaloes using ultrasonography. This will be useful in the determination of the most proper moment for the pre-fixed artificial insemination. In the reproductive season, autumn in the South of Brazil (march-june), 132 clicling females were divided in 3 groups: Group A: 53 females were treated with auricular subcutaneous implant of norgestomet or intravaginal device of progesterone. In the moment that the devices were removed, 250mg of cloprostenol were applied for intravulvar submucosis (ivsm). Group B: 48 buffaloes females were treated twice with 250mg of cloprostenol for ivsm with interval of 11 days. Group C: 31 buffaloes females remained without any treatment (control group). All of them were inseminated in the moment that was observed the biggest diameter of pre-ovulatory follicle determinated by ultrasonography. In the 3 groups, there was significative difference between pregnant and non-pregnant females in the oestrus-ovulation interval and in the A.I.-ovulation interval. The pregnancy rates were 41.5%, 52.1% and 54.8% in the groups A, B amd C, respectively. The variation in the oestrus-ovulation in buffaloes is the major obstacle to achieve high pregnancy rates using pre-fixed artificial insemination in spontaneous and induced oestrus.

O objetivo deste experimento foi determinar o intervalo entre o início do estro induzido ou espontâneo e a ovulação em fêmeas bubalinas (Bubalus bubalis) com o auxílio da ultra-sonografia, o que permitirá a determinação de um horário mais apropriado para a I.A. pré-fixada. Nos meses de março a junho, outono no sul do Brasil (época reprodutiva dos bubalinos), 132 fêmeas adultas ciclando foram divididas em 3 grupos experimentais: Grupo A - 53 fêmeas foram tratadas com implante subcutâneo de Norgestomet ou espiral intravaginal contendo progesterona. Na retirada dos dispositivos, foi aplicado 250mg de cloprostenol pela via intra-submucosa vulvar (i.s.m.v.), Grupo B - 48 búfalas foram tratadas com dupla aplicação de 250mg de cloprostenol pela via i.s.m.v. com intervalo de 11 dias e Grupo C - 31 búfalas foram consideradas testemunhas, sem tratamento. Todas as búfalas foram inseminadas no momento da observação do maior diâmetro do folículo pré-ovulatório, detectado por ultra-sonografia, durante o estro. Após o diagnóstico de prenhez, constatou-se, nos três tratamentos, que houve diferença significativa entre as búfalas prenhes e vazias no período compreendido entre o início do estro até o momento da ovulação e no período entre a I.A.e a ovulação. Os índices de prenhez foram de 41,5%, 52,1% e 54,8% nos grupos A, B e C, respectivamente. A variação no intervalo estro-ovulação nas búfalas é uma barreira para a obtenção de taxas de prenhez por I.A. pré-fixada comparáveis à monta natural, tanto no estro induzido através de progesterona e prostaglandina F2 alfa como no estro espontâneo.