Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification

Abstract

RESUMO.-[Acúmulo de lipídios intracitoplasmáticos, desenvolvimento e criotolerância de embriões bovinos produzidos in vitro e tratados com diferentes concentrações de forskolin antes da vitrificação.] Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5μM (grupo Forsk 2,5), 5,0μM (grupo Forsk 5,0) ou The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5μM (Forsk 2.5 group), 5.0μM (Forsk 5.0 group) or 10.0μM (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri--Ingá ® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P<0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P<0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 μM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.

The role of forskolin as a lipolytic stimulator during in vitro oocyte maturation and the in vitro embryo production of livestock

“…Parallel remarks were noted in cattle embryos by Meneghel et al. (2017) in which various levels of forskolin addition (2.5, 5 or 10 µM) to the embryo in vitro culture medium before vitrification did not induce substantial changes in the rate of blastocyst development between the treated and control groups. Similarly, Sanches et al.…”

“…Increasing the level of cAMP mediated by the stimulation of forskolin can activate the downstream degradation pathway, increasing calcium signalling in pancreatic ductal epithelial cells (PDEC), but the inhibition of IBMX leaves calcium free to bind with calcein (Albuz et al., 2010). It was documented that the pre‐treatment with forskolin (5.0 μM) for 24 hr before vitrification is influential in decreasing the intracytoplasmic lipid level and, therefore, enhances cryotolerance of IVP bovine embryos (Meneghel et al., 2017). Moreover, those cAMP modulators also sustained gap junction activity, increased cAMP production and transzonal projections (TZP) function (Thongkittidilok et al., 2020).…”

“…Moreover, those cAMP modulators also sustained gap junction activity, increased cAMP synthesis and transzonal projections (TZP) function (Meneghel et al., 2017). Hereafter, it was reported that some of the constructive impacts of lipid modulation and transcript abundance might represent an enhancement triggered by cAMP administration in those precise populations of oocytes (Monteiro et al., 2017).…”

“…On the other hand, it was depicted that elevated concentration of forskolin (10 µM) had an undesirable influence on embryo development (Meneghel et al., 2017). Contrariwise, forskolin (5 µM) displayed an increase (72.2%) in the rate of blastocoel re‐expansion after warming compared with that of the control (46.2%).…”

“…The feat of using forskolin/IBMX meaningfully improved embryo development and has been evidenced in different animals (Gilchrist et al., 2015; Richani et al., 2019). Supplementation with 5.0 μM forskolin (54.7%) to in vitro culture medium had higher blastocyst rate compared with control group (44.9%) (Meneghel et al., 2017). Moreover, this paper also indicated that only the rise in equine blastocysts formed per cleaved oocyte was found to be significantly different between the supplementation group (59%) and control group (35%) (Monteiro et al., 2017).…”