Efecto de sistemas alternativos para el control del pH del medio de cultivo sobre la competencia de complejos cúmulus-ovocitos bovinos madurados in vitro
Fecha de publicación: 22/10/2021

El pH de los medios de cultivo es vital para las actividades celulares, por lo tanto, es un factor que debe ser controlado durante los procedimientos de producción in vitro de embriones. Este estudio tuvo como objetivo evaluar dos alternativas para el control del pH del medio de cultivo durante la maduración in vitro (MIV) de complejos cúmulus-ovocitos (CCOs) bovinos

Influence of L-carnitine on lipid metabolism of buffalo cumulus-oocyte complexes matured in either fetal bovine serum or fatty acid-free bovine serum albumin
Fecha de publicación: 15/12/2020

Consequences of oocyte supplementation with l-carnitine may vary depending on species-specific cellular lipid profile, level of mitochondrial activity, or even on ipid availability in culture medium. This study aimed to evaluate l-carnitine supplementation on competence and gene expression of enzymes related to lipid metabolism in oocytes and cumulus cells from buffalo COCs matured in the presence or absence of fetal bovine serum (FBS). COCs were matured in vitro in FBS (10%) or bovine serum albumin fatty acid-free (BSA-FAF) (0.4%) and with or without supplementation with l-carnitine (3.03 mM). COCs matured in the presence of FBS or BSA-FAF were fertilized and cultured, then supplemented with l-carnitine during in vitro maturation or in vitro embryo culture. Finally, in vivo mature and immature COCs were included for gene expression analysis. COCs matured in culture medium with FBS in the presence of l-carnitine produced a lower blastocyst rate (p ≤ 0.05) compared to controls. In turn, the blastocyst rate from COCs matured with BSA-FAF in the presence of l-carnitine was similar to controls (p > 0.05), and higher than FBS + L-carnitine treated COCs (p ≤ 0.05). Addition of l-carnitine during embryo culture showed no differences in blastocyst production between experimental groups and controls (p > 0.05). In cumulus cells, gene expression of ACACA, SCD and FASN was upregulated in COCs matured in the presence of BSA-FAF + L-carnitine, while all genes in oocytes were significantly expressed upregulated by COCs matured in vivo, and only BSA-FAF + L-carnitine group showed similar expression of the FASN gene. In conclusion, the consequences of l-carnitine supplementation during in vitro maturation of buffalo COCs on oocyte competence vary depending on presence or absence of FBS in culture. With FBS, l-carnitine impairs oocyte competence, while in its absence, gene expression suggests adequate lipid metabolism and increased oocyte competence.

Modulation of phosphatidylinositol 3-kinase activity during in vitro oocyte maturation increases the production of bovine blastocysts
Fecha de publicación: 03/08/2020

This study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus–oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.

Partial inhibition of nitric oxide synthase activity stimulates the nuclear maturation progression of bovine cumulus–oocyte complex in vitro in the presence of hemisections of the follicular walls
Fecha de publicación: 15/05/2020

This study aimed to assess the effects of the inhibition of nitric oxide synthase (NOS) on events that modulate bovine in vitro oocyte maturation. Cumulus–oocyte complexes (COCs) were cultured with hemisections (HSs) of the follicular walls in a maturation medium supplemented with different concentrations (0.1–10.0 mM) of Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Controls consisted of COCs cultured in the presence (+HSs) or absence of HSs (–HSs) with no additional l-NAME supplementation. The following parameters were assessed: oocyte nuclear maturation stage; cumulus cell (CC) membrane integrity; nitrate/nitrite, progesterone, and estradiol concentrations in the culture medium at 22 h of cultivation; and the concentrations of cGMP and cAMP in COCs during the first hour of maturation. The addition of 1.0 mM l-NAME increased the percentage of oocytes that reached metaphase II (MII) and the percentage of intact CCs (P < 0.05). All l-NAME concentrations reduced the nitrate/nitrite concentrations (P < 0.05), but none affected steroid concentrations compared with control +HSs (P > 0.05). The addition of 1.0 mM l-NAME reduced cGMP concentrations at 3 h and increased cAMP concentrations in the first hour of culture (P < 0.05). Our findings suggest that the NOS/NO/cGMP pathway participates in meiosis progression (MI to MII) of the bovine oocytes matured in vitro in the presence of hemisections of the follicular walls. Lastly, the mechanisms that lead to the progression of meiosis after NOS inhibition do not involve changes in steroid production.

Importance of lipid metabolism on oocyte maturation and early embryo development: Can we apply what we know to buffalo?
Fecha de publicación: 10/12/2019

The knowledge about the biological events that regulate lipid metabolism in oocytes and embryos in buffalo is scarce. Lipogenesis, lipolysis, transport and oxidation of fatty acids (FAs) occur in gametes and embryonic cells of all mammalian species, as an intrinsic component of energy metabolism. In oocytes and cumulus cells, degradation of lipids is responsible for the production of ATP that is essential for the metabolic processes that lead to oocyte maturation in in vivo and in vitro culture conditions. Similarly, throughout embryo development, blastomeres have the capacity to use exogenous and/or endogenous lipid reserves to serve as an energy source necessary for early embryonic development. In addition, supplementation of culture media with L-carnitine to promote lipid metabolism during in vitro oocyte maturation and early embryonic development leads to an improved embryo quality. The limited scientific evidence available in buffalo indicates there is relatively greater oocyte lipid content as compared with many other species that undergoes a dynamic distribution during folliculogenesis and follicle maturation and that has a positive effect on oocyte maturation and embryo development when there is L-carnitine supplementation of the media. Advances in the understanding of the biological peculiarities of lipid metabolism, and the consequences of its alteration on the quality of buffalo gametes and embryos, therefore, are necessary to design specific culture media and laboratory procedures as a strategy to increase in vitro-derived embryo production rates.