T. V. G. Silva

Effect of triiodothyronine on developmental competence of bovine oocytes

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development.

Characterization of folliculogenesis and the occurrence of apoptosis in the development of the bovine fetal ovary.

The aim of this research was to perform in situ quantification, morphometry evaluation, and apoptosis analysis of ovarian follicular wall cells in mechanically isolated follicles obtained from ovaries of bovine fetuses (Bos taurus indicus) between 3 and 9 months of age. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The number of isolated follicles increased from 3 months onward (102.5 ± 141.1, mean ± SEM), peaked at 6 months (12855.0 ± 9030.1), and then decreased by 7 months (3208.7 ± 3249.5), consistent with atresia occurring at these stages. Follicular density was greatest at 4 months, consistent with a sudden boost in follicular activity independent of a corresponding increase in ovarian size. Antral follicles were first observed at 5 months. As fetal age increased, there was a tendency for the percentage of primordial and primary follicles to decrease, and the percentage of secondary follicles to increase. However, the high variability (P < 0.05) for all follicle populations up to 5 months of age precluded further interpretation of these results. Oocyte diameter increased from the primordial (23.6 ± 4.4 μm) to the secondary follicular stages (38.0 ± 14.9 μm). Apoptosis was observed in ovaries from all fetal ages analyzed. We concluded that preantral follicles could be isolated from bovine fetuses by 3 months of age, with apoptosis affecting ovarian follicular dynamics throughout fetal life.

Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates

Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.