Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines / Efeito de rolipram na maturação in vitro, expressão gênica e desenvolvimento embrionário em bovinos

The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.(AU)

O objetivo deste trabalho foi avaliar o efeito do rolipram durante a maturação de oócitos bovinos, expressão gênica e embriões produzidos in vitro. Os ovários bovinos foram coletados no matadouro. Os COCs foram selecionados e divididos em cinco grupos: controle 0 tempo; controle: MIV por 24 horas; tratamentos rolipram com bloqueio MIV por 24 horas em meio de maturação contendo 100, 150 e 200µM. Após 24 horas, todos os grupos foram recolocados em MIV por mais 24 horas. Subsequentemente COCs foram submetidos ao mesmo sistema MIV e fertilizados, sendo avaliada a taxa de clivagem e de blastocisto, além da expressão dos seguintes genes: Mater, BMP15 e Bax. Nenhuma diferença foi observada na expressão gênica. Dos oócitos avaliados logo após a aspiração folicular, 79,0% estavam em GV, GVBD, MI, enquanto 13,40% estavam em MII, e 7,60% em D/NI. A diferença significativa foi observada em diferentes concentrações (T100, T200 e T150µM) em oócitos que atingiram a fase MII em comparação aos tratamentos de controle (P=0,3). Diferenças foram observadas nas taxas de clivagem (P<0,5) entre T150 e T200 quando comparadas com as taxas do grupo C/24. Uma grande diferença foi observada na taxa de blastocisto (P<0,1) entre os tratamentos em relação ao grupo controle.(AU)

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos.

Embryo sexing is a powerful tool for livestock producers because it allows them to manage their breeding stocks more effectively. However, the cost of supplies and reagents, and the need for trained professionals to biopsy embryos by micromanipulation restrict the worldwide use of the technology to a limited number of specialized groups. The aim of this study was to couple a fast and inexpensive DNA extraction protocol with a practical biopsy approach to create a simple, quick, effective, and dependable embryo sexing procedure. From a total of 1847 sheep and cattle whole embryos or embryo biopsies, the sexing efficiency was 100% for embryo biopsies, 98% for sheep embryos, and 90.2% for cattle embryos. We used a primer pair that was common to both species and only 10% of the total extracted DNA. The whole protocol takes only 2 h to perform, which suggests that the proposed procedure can be readily applied to field conditions. Moreover, in addition to embryo sexing, the procedure can be used for further analyses, such as genotyping and molecular diagnosis in preimplantation embryos.

Comparative expression profiles of genes related to oocyte development in goats after long-term feeding with biodiesel castor industry residues.

The aim of this study was to determine whether the consumption of detoxified castor meal (DCM) by goats over a long period of time affects mRNA levels in oocytes, and in mural granulosa and cumulus cells. A total of 41 adult does were supplemented (DCM group, n=21) or not (control group, n=20) with detoxified castor meal (DCM) for a period of 500 days. Then, 13 and 12 does were randomly selected for slaughter from the DCM and control treatments groups, respectively, for the determination of the number of visible ovarian follicles, retrieved cumulus-oocyte complexes (COCs), and viable and non-viable oocytes. The relative expression levels for distinct genes were determined by quantitative PCR in viable immature oocytes prior to in vitro maturation (IVM), in oocytes attaining or not the metaphase stage after IVM, as well as in granulosa cells obtained upon oocyte collection, and in cumulus cells obtained after IVM. The number of follicles ≥4 mm did not differ between treatments (overall mean 23.3 ± 2.0) and no significant differences were observed in the recovery of viable, non-viable, or total mean numbers of oocytes (control group: 44.7 ± 4.6, DCM group: 54.9 ± 5.9, respectively) between control and DCM fed goats. The maturation rate was significantly higher for control than DCM oocytes (58.0% vs. 45.3%; P<0.05). The mRNA levels in immature COC for controls were significantly higher for GLUT1 and lower for HSP70 (P<0.05) than for DCM. Following maturation, MII oocytes from both treatments had mRNA levels that were significantly higher for GDF9 and lower for BMP15 than for NC oocytes (P<0.05). In cumulus cells, the mRNA levels were significantly higher for LHR, FSHR, LeptinR, and IGF1, and lower for MnSOD in the control group compared with the DCM group (P<0.05). In conclusion, the inclusion of DCM in goat feed for long periods of time changed gene expression in immature oocytes and in cumulus cells. This was reflected by a decrease in the in vitro oocyte maturation rate.