Endometrial prostaglandin F2α in vitro production and its modulation regarding dominant follicle position in cattle / Produção in vitro de PGF F2α endometrial e sua modulação referente ao folículo dominante em bovinos

Prostaglandin F2α (PGF2α) determines luteolysis in cattle, and the ability to manipulate its endogenous synthesis is indispensible for large-scale animal breeding. Estradiol (E2) and progesterone (P4) modulate several molecular pathways in endometrial cells, including the synthesis of PGF2α; however, its specific mechanisms are still not totally known. This study investigated the production in vitro and possible modulation of endometrial PGF2α due to a local effect of endogenous E2 in the ipsilateral uterine horn (UH) containing the dominant follicle (DF) or from P4 in ipsilateral horn containing the corpus luteum (CL). The PGF2α stimulators oxytocin (OT) and phorbol 12,13-dibutyrate (PDBu) were incubated with endometrial explants, and PGF2α content was measured. For that, cycling cows were synchronized, the development of DF and CL was examined by ultrasonography and on the seventh day of the estrous cycle, endometrial explants were collected and cultured in medium supplemented with 10-6 M PDBu or 10-6 M OT or non-supplemented. Media samples were collected immediately after treatment and 60 min later. Radioimmunoassay showed that the PGF2α content of the UH ipsilateral to the DF was 49% less than that of the contralateral UH (8.22 ± 0.95 vs. 12.24 ± 0.95 pg/mL/mg tissue, respectively; P < 0.01). However, the PGF2α levels did not differ between the UHs as a function of the CL position (9.46 ± 0.95 vs. 11 ± 0.95 pg/mL/mg; P > 0.05). The cellular stimulators promoted an increase in PGF2α synthesis (P < 0.02), and the effects differed among the animals (P < 0.04). The PGF2a production was higher in the explants treated with PDBu rather than OT (13.68 ± 1.16 vs. 10.01 ± 1.16 pg/mL/mg tissue, respectively; P < 0.05). In conclusion, PGF2α synthesis is modulated by the presence of the DF (local E2) but not the CL (local P4), and both PDBu and OT stimulated PGF2a synthesis.(AU)

A prostaglandina F2α (PGF2α) determina a luteólise em bovinos. A capacidade de manipular sua síntese endógena é indispensável para a produção animal em grande escala. O estradiol (E2) e a progesterona (P4) modulam diversas vias moleculares das células endometriais, incluindo a síntese de PGF2α; no entanto, pouco se sabe sobre seus mecanismos específicos. Este trabalho investigou a produção in vitro e a possível modulação da PGF2α endometrial devido a um efeito local do E2 endógeno no corno uterino ipsilateral ao folículo dominante (FD) ou da P4 no corno ipsilateral ao corpo lúteo (CL). Os estimuladores de PGF2α oxitocina (OT) e 12,23-dibutirato de forbol (PDBu) foram incubados com explantes endometriais, e o conteúdo de PGF2α foi mensurado. Para tal, vacas cíclicas foram sincronizadas, o desenvolvimento de FD e CL foi examinado por ultrassonografia, e no 17º dia do ciclo estral os explantes endometriais foram coletados e cultivados em meio ou suplementados com PDBu 10-6M ou 10-6M OT. As amostras de meio foram coletadas imediatamente após o tratamento e sessenta minutos depois. O radioimunoensaio mostrou que o conteúdo de PGF2α do corno ipsilateral ao FD foi 49% menor que o do corno contralateral (8,22 ± 0,95 vs. 12,24 ± 0,95 pg/mL/mg de tecido, respectivamente, P < 0,01). No entanto, os níveis de PGF2α não diferiram entre os cornos em função da posição do CL (9,46 ± 0,95 versus 11 ± 0,95 pg/mL/mg; P > 0,05). Os estimuladores celulares promoveram um aumento na síntese de PGF2α (P < 0,02), e os efeitos diferiram entre os animais (P < 0,04). A produção de PGF2α foi maior nos explantes tratados com PDBu em comparação à OT (13,68 ± 1,16 versus 10,01 ± 1,16 pg/mL/mg de tecido, respectivamente, P < 0,05). A conclusão obtida foi que a síntese de PGF2α é: modulada pela presença do FD (E2 local), mas não do CL (P4 local); e estimulada por PDBu e OT.(AU)

Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles / Desafios e perspectivas na produção in vitro de embriões bovinos: enfoque epigenético e em vesículas secretadas por células

This review aim to present some clinical problems found in IVP-derived animals focusing on NT procedures and to discuss the possible role of epigenetics in such process. Also, as cell-secreted vesicles have been reported as possible regulators of important physiological reproductive processes such as folliculogenesis and fertilization, it is also presented herein a new perspective of manipulating the pre-implantation period trough effector molecules contained in such vesicles.

Nesta revisão, apresentamos alguns problemas clínicos encontrados nos animais derivados de PIV, principalmente derivados de transferência de núcleo, e discutimos o possível papel da epigenética em tais processos. Além disso, uma vez que vesículas secretadas por células têm sido descritas como possíveis reguladores de processos reprodutivos fisiológicos importantes, tais como a foliculogênese e a fertilização, estas são aqui apresentadas como uma possível nova ferramenta para a manipulação do período embrionário pré-implantacional através de moléculas efetoras, contidas em tais vesículas.

Exploring the effects of second estrus synchronization and dietary flushing on the incidence of ovarian cysts in gilts by using exogenous gonadotropins / Explorando os efeitos da sincronização do segundo estro e flushing alimentar sobre a incidência de cistos ovarianos em marrãs utilizando gonadotrofinas exógenas

Estrus stimulation by exogenous gonadotropins (EG) in association with dietary flushing is an important tool for theimprovement of gilt reproductive performance. However, there is evidence associating both flushing and EG with a disturbance in the endocrine balance that could lead to increased ovarian cysts. The aim of this study was to evalu- ate whether flushing or EG might affect the ovulation rate and the incidence of ovarian cysts. Seventy-one gilts were randomly distributed into 2x2 factorial design with four treatments: flushing and hormone (wFwH); no flushing and hormone (nFwH); flushing without hormone (wFnH); and neither flushing nor hormone (nFnH). Gilts were slaughtered for macroscopic and histopathological ovary examination approximately five days after AI. The characterization of these cysts was performed by optical microscopy in the following: follicular cysts (FC), luteinizedcysts (LC) or cystic corpora lutea (CCL). The number of ovulations did not differ between treatments. There was no interaction between the factors in any analyzed variable. The frequency of gilts with CCL and LC was not affected by flushing and EG. No difference was found in the incidence of FC, with 12.5% and 5.88% in gilts from wFwH and nFwH treatments, respectively. There were no differences in the proportion of CCL between FC and LC (9.85 vs. 4.22 and 4.22%, respectively). In conclusion, the use of exogenous gonadotropins for second estrus synchronization in gilts, either alone or in association with dietary flushing, does not increase the incidence of ovarian cysts, nor does it decrease the ovulation rate.

A estimulação do estro por gonadotrofinas exógenas (GE) associada ao flushing alimentar é uma ferramenta importante na melhoria do desempenho reprodutivo de marrãs. Contudo, há evidência da associação do flushing com GE levando ao desequilíbrio no sistema endócrino que poderia levar ao aumento de cistos ovarianos. O objetivo deste estudo foi avaliar se o flushing ou GE pode afetar a taxa de ovulação e a incidência de cistos ovarianos. Setenta e uma marrãs foramdistribuídas aleatoriamente em arranjo fatorial 2x2 com quatro tratamentos: flushing e hormônio (cFcH); sem flushing e com hormônio (sFcH); com flushing e sem hormônio (cFsH) e sem flushing e hormônio (sFsH). Marrãs foram abatidas para exame macroscópico e histopatológico dos ovários, aproximadamente cinco dias após IA. A caracterização desses cistos foi realizada por microscopia óptica: cistos foliculares (CF), cistos luteinizados (CL) ou corpos lúteos císticos(CCL). O número de ovulações não diferiu entre os tratamentos. Não houve interação entre os fatores em qualquer variável analisada. A frequência de leitoas com CCL e CL não foi afetada pelo flushing e GE. Não houve diferença na incidência de CF, com 12,5% e 5,88 % em leitoas dos tratamentos cFcH e sFcH, respectivamente. Não foram obtidas diferenças na proporção de CCL entre CF e CL (9,85 vs. 4,22 e 4,22%, respectivamente). Em conclusão, a utilização de gonadotrofinas exógenas para sincronização do segundo estro de marrãs, isoladamente ou em associação com o flushing, não aumenta a incidência de cistos ovarianos e não diminui a taxa de ovulação.