Forskolin effect on the cryosurvival ofin vitro-produced bovine embryos in the presence or absence of fetal calf serum

Abstract

The objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

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

“…the embryos but not in the oocytes, it was expected that the blastocyst yields were unaffected by supplementation of IVC medium with forskolin in various concentrations. These results are in agreement with data from Paschoal et al (2012), which did not observe differences in blastocyst yield of embryos treated with (46.3% and 34.2% in medium supplemented with or without FCS, respectively) or without 10μM forskolin (46.8% and 33.3% in medium supplemented with or without FCS, respectively) during 24 hours of IVC. Likewise, Sanches et al (2013) found no differences in blastocysts production rates after treatment of IVC embryos with 10μM forskolin for 48 hours (45.3%) compared to untreated embryos (39.9%).…”

“…Such observations highlight the importance of the post-fertilization culture environment for the quality of the resulting blastocyst. Although the term "quality" is very unspecific and the parameters that allow the classification of embryos in different quality grades were not well established until the present time, several studies have evaluated the quality in terms of specifics aspects like apoptosis rates, cytoplasmic accumulation of reactive oxygen species, mitochondrial membrane potential, lipid accumulation, resistance to freezing and even pregnancy rates after transfer to synchronized recipients (Accorsi et al 2016, Rocha-Frigoni et al 2014, Sanches et al 2013, Paschoal et al 2012, Pereira et al 2008. Thus, the present results demonstrated an increase in the quality of in vitro-produced bovine embryos treated with forskolin, measured in terms of cryotolerance.…”

“…Forskolin has been reported to increase the cryosurvival of porcine blastocysts produced in vitro by reducing their intracellular lipid content, especially triacylglycerols (Men et al 2006). Although data concerning the effect of forskolin on bovine IVP embryos are more scarce than for pig, recent studies demonstrated an improvement in bovine embryo survival and quality after vitrification (Paschoal et al 2012, Sanches et al 2013.…”

“…CAMP is an important signaling molecule that regulates the physiological functions of various systems (Seamon et al 1981, Ho & Shi 1982, Ek et al 1997, Arrese et al 1999, including the regulation of lipolysis (Lafontan & Berlan 1993). Although the delipidation induced by forskolin have improved cryotolerance of bovine embryos (Paschoal et al 2012, Sanches et al 2013, the effects of increasing cAMP levels are unspecific on the metabolism (Seamon et al 1981, Ho & Shi 1982, Ek et al 1997, Arrese et al 1999, so it is possible that forskolin can induce effects on embryonic cells that are still unknown (Sanches et al 2013). For this reason, it is necessary to establish the lowest effective dose of this drug for purpose of inducing lipolysis in IVP embryos.…”

Change in energy metabolism of in vitro produced embryos: an alternative to make them more cryoresistant?

“…Thus, supplementation with this substance in the in vitro culture medium has produced positive results regarding reduced intracytoplasmic lipids, resulting in better quality bovine embryos that overcome the damage caused by cryopreservation (SANCHES et al, 2013;PASCHOAL et al, 2014).…”