Ovum pick-up and in vitro maturation in spotted paca (Cuniculus paca-Linnaeus, 1766).

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.

Conception rate of lactating cows and heifers (Bos taurus X Bos indicus) for sexed and conventional semen in artificial insemination / Taxa de concepção de vacas lactantes e novilhas (Bos taurus X Bos indicus) com o uso de sêmen sexado e convencional na inseminação artificial

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.

Chemically induced enucleation of activated bovine oocytes: chromatin and microtubule organization and production of viable cytoplasts.

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.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos.

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.

Chemically assisted enucleation results in higher G6PD expression in early bovine female embryos obtained by somatic cell nuclear transfer.

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.