Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of Superovulatory Protocols on In Vitro Production of Nellore (Bos indicus) Embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The Mechanism of Oocyte Activation Influences the Cell Cycle-Related Genes Expression During Bovine Preimplantation Development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência do diâmetro e da fase folicular sobre a competência in vitro de oócitos obtidos de novilhas da raça Nelore

Avaliou-se o efeito do diâmetro e da fase do desenvolvimento folicular sobre a competência de oócitos para a produção in vitro de embriões bovinos. A primeira onda folicular foi sincronizada com progestógeno por nove dias e 24 horas após a sua retirada aplicou-se LH. Os ovários foram recuperados 60h (G-60), 96h (G-96) e 108h (G-108) após a ovulação induzida pelo LH. Os folículos foram dissecados ou aspirados e medidos e os oócitos recuperados e submetidos à maturação, fecundação e cultivo in vitro. Os ovários do G-60 apresentaram mais oócitos viáveis (graus I, II e III) (96,6%). A taxa de clivagem teve efeito significativo sobre o diâmetro folicular, sendo maior nos oócitos oriundos de folículos classe 3 (>7mm). Na taxa de produção de blastocisto observou-se interação diâmetro versus fase de desenvolvimento folicular. A taxa de produção de blastocisto foi maior em oócitos obtidos de folículos com diâmetros <5mm (classe 1) no G-60 (64,5%), de 5-7mm (classe 2) no G-96 (33,3%) e >7mm (classe 3) no G-108 (50%). Conclui-se que o diâmetro e a fase de desenvolvimento folicular influenciam a competência oocitária para o desenvolvimento in vitro. Nos estádios iniciais da onda folicular a produção de blastocisto foi maior em oócitos de folículos pequenos; com o avanço da onda, a produção de blastocistos foi maior em oócitos obtidos de folículos maiores.

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylammopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

Use of strontium for bovine oocyte activation

Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca2+ and Mg2+-free TALP for 6 h), ionomycin + strontium (IS), and strontium + ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 mu M for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to 1, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were similar for S, I, IS, and SI treatments (61.7-76.7, and 8.3-13.3%, respectively) and the same was observed for ICM, TE, and total cell number, and ICM/total cell ratio (22-25, 64-69, and 86-95, and 0.26-0.27). In conclusion, strontium may be efficiently applied for bovine oocyte activation at 20 mM in Ca2+-and Mg2+-free TALP medium for 6 h.

Effects of histone hyperacetylation on the preimplantation development of male and female bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of sire breed (Bos indicus versus Bos taurus) and interval from slaughter to oocyte aspiration on heat stress tolerance of in vitro-produced bovine embryos

Based on in vitro experiments, Bos indicus embryos were more resistant to heat stress (HS) than Bos taurus embryos. To increase knowledge regarding differences between Bos indicus and Bos taurus in resistance to HS, the primary objective of this study was to determine if tolerance to HS is due to the breed, origin of the oocyte, sperm, or both. Additionally, the influence of the interval between ovary acquisition (in the abattoir) and oocyte aspiration in the laboratory, on early embryo development was ascertained. Oocytes were collected from Nelore and Holstein cows in an abattoir; 4.0 or 6.5 h later, oocytes were aspired in the laboratory, and then matured and fertilized using semen from Nelore (N), Gir (GIR), or Holstein (H) bulls. Ninety-six h post insemination (hpi), embryos with >= 16 cells were divided in two groups: control and HS. In the control group, embryos were cultured at 39 degrees C, whereas in the HS group, embryos were subjected to 41 degrees C for 12 h, and then returned to 39 degrees C. Rates of cleavage, and formation of morula and blastocysts were higher (P < 0.05) for oocytes aspirated at 4.0 versus 6.5 h after ovaries were acquired. Heat stress decreased rates of blastocyst formation for all breeds (N X N; H x H; and H X GIR) and in both time intervals (4.0 and 6.5 h). However, N X N had higher cleavage rate (P < 0.05) in both time intervals when compared with H X H and H X GIR. In addition, Nelore oocytes fertilized with Nelore semen (N X N) had higher blastocyst yields (P < 0.05) in the control and HS group, when compared with the other two breeds (H X H and H X GIR). We concluded that the breed of origin of the oocyte was more important than that of the sperm for development of thermotolerance, because bull breed did not influence embryo development after HS, and in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the interval between ovary acquisition and oocyte aspiration. (C) 2011 Elsevier B.V. All rights reserved.

In vitro Culture of Bovine Embryos in Murine ES Cell Conditioned Media Negatively Affects Expression of Pluripotency-Related Markers OCT4, SOX2 and SSEA1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of the breed of bull (Bos taurus indicus vs. Bos taurus taurus) and the breed of cow (Bos taurus indicus, Bos taurus taurus and crossbred) on the resistance of bovine embryos to heat

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)