L. H. Aguiar

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.

Morphometric developmental pattern of bovine handmade cloned concepti in late pregnancy.

Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement.

Development of caprine preantral follicles after orthotopic autotransplantation of ovarian tissue: Short communication

Objective: The aim of this study was to evaluate the follicle morphology, density, development and hormone production after orthotopic autransplantation of fresh or vitrified goat ovarian tissue. Methods: Fresh and vitrified ovarian cortex was orthotopically autotransplanted for six months in two and three adults bilaterally ovariectomized goats, respectively. The animals were monitored during 196 days and blood samples collected. Results: It was observed that the percentage of morphologically normal preantral follicles (MNPF) after grafting of fresh ovarian tissue was similar to control. The follicular density in the fresh graft reduced significantly when compared to fresh control. unfortunately, after transplantation of vitrified tissue it was not possible to identified any follicles after recovery. Furthermore, the proportion of developing follicles was higher (P < 0.05) in the fresh auto-grafts than in control fragments. Moreover, progesterone plasma levels increased significantly from day 179 to day 195 of transplantation. Conclusion: In conclusion, orthotopic transplantation of fresh ovarian tissue was able to keep healthy the preantral follicles, as well as the restoration of goat endocrine function.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian SemiArid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.