RESUMO

We tested FSHp, eCG and FSHp + eCG to establish ovum pick-up (OPU) and in vitro maturation method in spotted paca. Eight healthy adult females were subjected to each of four treatments to stimulate ovarian follicular growth. All females were subjected to a hormonal protocol using a single dose of 45 mg of injectable progesterone and single intramuscular injection of 0.075 mg d-cloprostenol on day 6. Ovarian stimulation was carried out as follows: in Group TFE (FSHp and eCG), animals were treated with a single dose of 80 mg of FSHp and 200 IU of eCG intramuscularly on day 6 after the application of progesterone; in Group TF (FSHp), they were treated with a single dose of 80 mg of FSHp intramuscularly on day 6 after application of progesterone; in Group treatment eCG, they were treated with 200 IU of eCG intramuscularly on day 6 after application of progesterone; and in Group TC (saline solution), 1 ml of saline solution was administered to control does. The OPU was performed between 22 and 26 hr after gonadotropin treatments. All recovered oocytes were placed into maturation media and incubated for 24 hr. There were no differences among the mean number of observed follicles, aspirated follicles and oocytes recovered per treatment. Oocyte maturation rates did not differ among groups, except, TF and treatment eCG oocytes had greater maturation rates than TC oocytes. In this study, gonadotropin administration failed to superovulate treated does and increase oocyte retrieval efficiency. Despite the feasibility of the procedure, further studies are needed to develop and refine hormonal protocols for oocyte recovery and in vitro maturation in this species.

Assuntos

RESUMO

The objective of this study was to evaluate the conception rate of crossbred heifers (n=50) and cows (n=50) inseminated with sexed and conventional semen between 18 and 24 hours after estrous detection. The synchronization protocol of the estrous cycle started on day zero (D0) by inserting the intravaginal device with 1g progesterone (Sincrogest® Ourofino, Brazil) and injecting 2 mg of estradiol benzoate, intramuscularly (Sincrodiol® Ourofino, Brazil). On the fifth day (D5), 200 IU of equine chorionic gonadotrophin was injected intramuscularly (Folligon®, Intervet, Brazil). On the eighth day (D8), after removing the progesterone device, 500 g of sodium cloprostenol was injected intramuscularly (Sincrocio®, Ourofino, Brazil). After that, the animals were checked for estrus 3 times daily, and inseminated 18 to 24 hours after estrus detection. Pregnancy diagnosis was performed 30 to 40 days after insemination. Conception rate did not differ (P> 0.05) according to animal category, but was higher for conventional semen compared to sexed semen when evaluating the total of animals and lactating cows (P < 0.05). Artificial insemination of heifers with sexed semen 18 to 24 hours after estrus detection was effective, however, conventional semen was more efficient in lactating cows.(AU)

Considerando os benefícios do uso de sêmen sexado e também os danos causados pelo processo de separação dos espermatozoides, o objetivo do presente estudo foi avaliar a taxa de concepção de novilhas (n=50) e vacas (n=50) mestiças inseminadas com sêmen sexado e convencional após 18 a 24 horas a observação do cio. O protocolo de sincronização do ciclo estral consistiu em inserção de dispositivo intravaginal com 1g de progesterona (Sincrogest® Ourofino, Brasil) e aplicação intramuscular de 2mg de benzoato de estradiol (Sincrodiol® Ourofino, Brasil) no dia zero (D0). No quinto dia (D5), foi realizada uma aplicação intramuscular de 200UI de gonadotrofina coriônica equina (Folligon®, Intervet, Brasil). No oitavo dia (D8), o dispositivo de progesterona foi retirado, e aplicado por via intramuscular 500g de cloprostenol sódico (Sincrocio®, Ourofino, Brasil). A partir deste momento, o estro foi observado 3 vezes ao dia e os animais foram inseminados 18 a 24 após a detecção do cio. O diagnóstico de gestação foi realizado 30 a 40 dias após a inseminação. Não foi observada diferença na taxa de concepção de acordo com a categoria animal (P > 0,05), entretanto, animais inseminados com sêmen convencional apresentaram melhor taxa de concepção do que com sêmen sexado quando se avaliou o total de animais e vacas lactantes (P < 0,05). A inseminação artificial de novilhas com sêmen sexado 18 a 24 horas após detecção de estro mostrou-se eficaz, entretanto, para vacas lactantes não foi observada a mesma eficiência ao se comparar com o sêmen convencional.(AU)

Assuntos

RESUMO

The objective of this study was to evaluate the conception rate of crossbred heifers (n=50) and cows (n=50) inseminated with sexed and conventional semen between 18 and 24 hours after estrous detection. The synchronization protocol of the estrous cycle started on day zero (D0) by inserting the intravaginal device with 1g progesterone (Sincrogest® Ourofino, Brazil) and injecting 2 mg of estradiol benzoate, intramuscularly (Sincrodiol® Ourofino, Brazil). On the fifth day (D5), 200 IU of equine chorionic gonadotrophin was injected intramuscularly (Folligon®, Intervet, Brazil). On the eighth day (D8), after removing the progesterone device, 500 g of sodium cloprostenol was injected intramuscularly (Sincrocio®, Ourofino, Brazil). After that, the animals were checked for estrus 3 times daily, and inseminated 18 to 24 hours after estrus detection. Pregnancy diagnosis was performed 30 to 40 days after insemination. Conception rate did not differ (P> 0.05) according to animal category, but was higher for conventional semen compared to sexed semen when evaluating the total of animals and lactating cows (P <0.05). Artificial insemination of heifers with sexed semen 18 to 24 hours after estrus detection was effective, however, conventional semen was more efficient in lactating cows.

Considerando os benefícios do uso de sêmen sexado e também os danos causados pelo processo de separação dos espermatozoides, o objetivo do presente estudo foi avaliar a taxa de concepção de novilhas (n=50) e vacas (n=50) mestiças inseminadas com sêmen sexado e convencional após 18 a 24 horas a observação do cio. O protocolo de sincronização do ciclo estral consistiu em inserção de dispositivo intravaginal com 1g de progesterona (Sincrogest® Ourofino, Brasil) e aplicação intramuscular de 2mg de benzoato de estradiol (Sincrodiol® Ourofino, Brasil) no dia zero (D0). No quinto dia (D5), foi realizada uma aplicação intramuscular de 200UI de gonadotrofina coriônica equina (Folligon®, Intervet, Brasil). No oitavo dia (D8), o dispositivo de progesterona foi retirado, e aplicado por via intramuscular 500µg de cloprostenol sódico (Sincrocio®, Ourofino, Brasil). A partir deste momento, o estro foi observado 3 vezes ao dia e os animais foram inseminados 18 a 24 após a detecção do cio. O diagnóstico de gestação foi realizado 30 a 40 dias após a inseminação. Não foi observada diferença na taxa de concepção de acordo com a categoria animal (P > 0,05), entretanto, animais inseminados com sêmen convencional apresentaram melhor taxa de concepção do que com sêmen sexado quando se avaliou o total de animais e vacas lactantes (P < 0,05). A inseminação artificial de novilhas com sêmen sexado 18 a 24 horas após detecção de estro mostrou-se eficaz, entretanto, para vacas lactantes não foi observada a mesma eficiência ao se comparar com o sêmen convencional.

Assuntos

RESUMO

As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos.

Assuntos

RESUMO

Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA.

Assuntos

RESUMO

A suplementação de bovinos leiteiros com fontes de ácidos graxos poli-insaturados (AGPs) é uma prática utilizada para aumentar o nível energético das dietas, além de proporcionar efeitos positivos nas funções reprodutivas de importantes tecidos, incluindo hipotálamo, hipófise, ovários e útero. O trabalho foi conduzido com objetivo de avaliar as condições reprodutivas do pós-parto, número de folículos, presença de corpo lúteo (CL), concentração de progesterona (P), quantidade de oócitos viáveis e totais e a produção in vitro de embriões (PIVE) de doadoras multíparas da raça Holandesa suplementadas com dieta rica em AGPs protegido (principalmente ácido linoleico - n-6) e não protegido (principalmente ácido linolênico - n-3) durante o pré e pós-parto. As dietas foram fornecidas por 30 dias pré-parto e 60 dias pós-parto. As doadoras foram divididas aleatoriamente em 3 grupos: Controle (n=6), Megalac-E® (n=5; suplementados com fonte de gordura protegida 100 g/doadora/dia no pré-parto e 300 g/doadora/dia no pós-parto) e linhaça (n=5); fonte de gordura não protegida contendo 1 kg/doadora/dia no pré-parto e 1,5 kg/ doadora/dia no pós-parto). Os animais foram submetidos à aspiração folicular (OPU) nos dias 30, 45 e 60 pós-parto. Os oócitos recuperados foram selecionados e os viáveis submetidos aos procedimentos da PIVE. Os dados foram analisados pelo método dos quadrados mínimos utilizando análise de variância pelo procedimento GLM. As diferenças entre as médias foram comparadas pelo teste de Tukey com significância de 5%. Não foi detectado efeito de tratamento de suplementação, de dias de aspirações pós-parto e das interações sobre as variáveis: quantidade de oócitos viáveis, taxa de oócitos viáveis, número de clivagem, de PIVE, taxa de doadoras com CL concentração de P. (P>0,05). No entanto, foi observado maior número de folículos e de oócitos totais no grupo suplementado com linhaça em relação ao grupo Megalac-E® e Controle (P<0,05). A suplementação com AGPs não aumentou o número de oócitos viáveis, a PIVE e o retorno à ciclicidade.

The supplementation of dairy cattle with sources of polyunsaturated fatty acids (PUFA) can be used to increase the energy level of the diet in addition to having positive effects on reproductive functions of important tissues including the hypothalamus, pituitary, ovaries and uterus. The aims of this study were to evaluate the reproductive conditions of the postpartum, number of follicles, corpus luteum (CL) presence, concentration of progesterone (P4), aspirated oocytes, amount of viable oocytes and in vitro production of embryos (IVPE) of the Holstein multiparous donors supplemented with rich diet in protected PUFA (especially linoleic acid - n-6) and non-protected (especially linolenic acid - n-3) during pre and postpartum. The diets had been given for pre-partum during 30 d and postpartum 60 d. The donors were divided into three groups: Control (n=6), Megalac-E® (n=5; supplemented with protected fat source 100 g/donor/day in pre-partum and 300 g/donor/day in postpartum) and linseed (n=5; supplemented with fat source unprotected containing 1 kg/donor/day pre-partum and 1.5 kg/donor/day in postpartum). The animals were submitted to ovum pick-up (OPU) on days 30, 45 and 60 d postpartum. The recovered oocytes were selected and the viable ones were submitted to IVPE procedures. The data were analyzed by the method of least squares variance using the GLM protocol. The differences between averages were compared by Tukey test with 5% significance. There was no detectable effect of treatment, aspirations of postpartum days and interactions on variables: CL presence, concentration of P4, amount of viable oocytes, viable oocytes rate, IVPE and embryos production rate. However, was observed in the group supplemented with linseed more follicles and total oocytes than Megalac-E® and Control group. Supplementation with PUFA didn't increase the number of viable oocytes and IVPE.

Assuntos

RESUMO

Despite extensive efforts, low efficiency is still an issue in bovine somatic cell nuclear transfer (SCNT). The hypothesis of our study was that the use of cytoplasts produced by chemically assisted enucleation (EN) would improve nuclear reprogramming in nuclear transfer (NT)-derived embryos because it results in lower damage and higher cytoplasm content than conventional EN. For that purpose, we investigated the expression of two X-linked genes: X inactive-specific transcript (XIST) and glucose 6-phosphate dehydrogenase (G6PD). In the first experiment, gene expression was assessed in day-7 female blastocysts from embryonic cell NT (ECNT) groups [conventional, ECNT conv; chemically assisted, ECNT deme (demecolcine)]. Whereas in the ECNT conv group, only one embryo (25%; n=4) expressed XIST transcripts, most embryos showed XIST expression (75%; n=4) in the ECNT deme group. However, no significant differences in transcript abundance of XIST and G6PD were found when comparing the embryos from all groups. In a second experiment using somatic cells as nuclear donors, we evaluated gene expression profiles in female SCNT-derived embryos. No significant differences in relative abundance (RA) of XIST transcripts were observed among the groups. Nonetheless, higher (p<0.05) levels of G6PD were observed in SCNT deme and in vitro-derived groups in comparison to SCNT conv. To know whether higher G6PD expression in embryos derived from SCNT chemically assisted EN indicates higher metabolism in embryos considered of superior quality or if the presence of higher reactive oxygen species (ROS) levels generated by the increased oxygen consumption triggers G6PD activation, the expression of genes related to stress response should be investigated in embryos produced by that technique.

Assuntos

RESUMO

Progestágenos para a sincronização de estro têm sido utilizados como rotina em propriedades com melhor controle reprodutivo. A produção de radicais livres, estresse oxidativo, está relacionada com os níveis de progesterona circulante. O estresse oxidativo é responsável pela agressão à membrana celular, causando lise e a formação de lipoperóxidos. Neste trabalho, buscou-se avaliar o efeito compensatório de antioxidantes (vitaminas C e E) associados ao implante de progesterona exógena (P4), utilizada em protocolos de sincronização de estro em fêmeas bovinas. Vinte e cinco fêmeas bovinas foram divididas aleatoriamente em cinco grupos: 1) controle sem implante de P4; 2) implante de P4; 3) implante de P4 + vitamina C e E; 4) implante de P4 + vitamina E; 5) implante de P4 + vitamina C. Avaliaram-se a lipoperoxidação lipídica, através da mensuração de substâncias reativas ao ácido tiobarbitúrico (TBA-RS), e a enzima glutationa peroxidase (GSHpx), nos dias zero e sete do protocolo de sincronização de estro. A utilização de vitamina E, neste experimento, apresentou uma maior taxa de prenhez; entretanto, existe a necessidade de validar esses resultados, antes que seu uso seja preconizado em fêmeas bovinas sincronizadas com P4.(AU)

Estrus synchronization with progesterone is commonly used in farms with better reproductive control. Free radicals production, i.e. oxidative stress, is associated with progesterone levels. The oxidative stress is responsible for aggression to the cellular membrane, leading to a lise and lipoperoxide formation. In this work, the antioxidant compensatory effect (Vitamins C and E) associated with the exogenous progesterone implant (P4), used in estrus synchronization protocols in cattle, was evaluated. Twenty-five cows were randomly selected in 5 different groups: 1) control without P4, 2) control with P4, 3) P4 + vitamin C and E, 4) P4 + vitamin E, 5) P4 + vitamin C. The lipid lipoperoxidation was measured trough Thiobarbituric acid reactive substances (TBA-RS) and Glutatione Peroxidase enzyme (GSHpx) at days 0 and 7 of the estrus synchronization protocol. The use of vitamin E, in this experiment, showed a better pregnancy rate, however, the results must be validated before orienting the use of the vitamin in cows synchronized with P4.(AU)

Assuntos

RESUMO

Progestágenos para a sincronização de estro têm sido utilizados como rotina em propriedades com melhor controle reprodutivo. A produção de radicais livres, estresse oxidativo, está relacionada com os níveis de progesterona circulante. O estresse oxidativo é responsável pela agressão à membrana celular, causando lise e a formação de lipoperóxidos. Neste trabalho, buscou-se avaliar o efeito compensatório de antioxidantes (vitaminas C e E) associados ao implante de progesterona exógena (P4), utilizada em protocolos de sincronização de estro em fêmeas bovinas. Vinte e cinco fêmeas bovinas foram divididas aleatoriamente em cinco grupos: 1) controle sem implante de P4; 2) implante de P4; 3) implante de P4 + vitamina C e E; 4) implante de P4 + vitamina E; 5) implante de P4 + vitamina C. Avaliaram-se a lipoperoxidação lipídica, através da mensuração de substâncias reativas ao ácido tiobarbitúrico (TBA-RS), e a enzima glutationa peroxidase (GSHpx), nos dias zero e sete do protocolo de sincronização de estro. A utilização de vitamina E, neste experimento, apresentou uma maior taxa de prenhez; entretanto, existe a necessidade de validar esses resultados, antes que seu uso seja preconizado em fêmeas bovinas sincronizadas com P4.

Estrus synchronization with progesterone is commonly used in farms with better reproductive control. Free radicals production, i.e. oxidative stress, is associated with progesterone levels. The oxidative stress is responsible for aggression to the cellular membrane, leading to a lise and lipoperoxide formation. In this work, the antioxidant compensatory effect (Vitamins C and E) associated with the exogenous progesterone implant (P4), used in estrus synchronization protocols in cattle, was evaluated. Twenty-five cows were randomly selected in 5 different groups: 1) control without P4, 2) control with P4, 3) P4 + vitamin C and E, 4) P4 + vitamin E, 5) P4 + vitamin C. The lipid lipoperoxidation was measured trough Thiobarbituric acid reactive substances (TBA-RS) and Glutatione Peroxidase enzyme (GSHpx) at days 0 and 7 of the estrus synchronization protocol. The use of vitamin E, in this experiment, showed a better pregnancy rate, however, the results must be validated before orienting the use of the vitamin in cows synchronized with P4.

Assuntos