l-carnitine supplementation during vitrification or warming of in vivo-produced ovine embryos does not affect embryonic survival rates, but alters CrAT and PRDX1 expression.

l-carnitine is an antioxidant and ß-oxidation stimulator substance commonly used to improve metabolic performance of oocytes and embryos in in vitro systems. However, few studies have evaluated its beneficial effects in embryos produced in vivo. This study aimed to evaluate the effect of l-carnitine supplementation into vitrification or warming solutions on the post-warming character of day 6-7 in vivo-produced ovine embryos. l-carnitine (3.72 mM) was added to vitrification (Experiment 1) or warming solutions (Experiment 2). In experiments 1 and 2, the embryos were vitrified using straw and cryo-tip protocols, respectively. In vitro culture (IVC) of warmed embryos was performed for 72 h in order to evaluate survival rates, reactive oxygen species (ROS) levels, total cell number (TCN), number of apoptotic cells, apoptotic index evaluation, and gene expression analysis of carnitine palmitoyltransferase I and 2 (CPT1 and CPT2), carnitine O-acetyltransferase (CrAT), and peroxiredoxin-1 (PRDX1). In experiment 1, survival rate, ROS levels after 24 h of IVC, total cell number at 24 h and 72 h, apoptotic cells and apoptotic index at 72 h of IVC were similar in embryos vitrified in medium supplemented with LC or not. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to fresh embryos (FE); however, CrAT was downregulated (p < 0.05) in C1, and PRDX1 was downregulated (p < 0.05) in both the control (C1) and l-carnitine (LC1) groups, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p < 0.05) in C2, and CrAT was downregulated (p < 0.05) in LC2, in relation to FE. Although the short-term LC supplementation at 3.72 mM did not improve survival, and quality parameters of in vivo-produced ovine embryos, it could affect their quality at a molecular level. In conclusion, further investigations with different concentrations of LC and tools are needed for improvement of the efficiency of these strategies.

Effects of histone hyperacetylation on the preimplantation development of male and female bovine embryos.

Trichostatin A (TSA) induces histone hyperacetylation by inhibiting histone deacetylases and consequently increasing gene expression. The hypothesis was that TSA supplementation during the in vitro culture (IVC) of bovine embryos would increase the blastocyst rate, particularly in low-quality and female embryos. Oocytes were fertilised separately with X and Y spermatozoa and, 70 h after IVF, the IVC medium was supplemented with 5 nM and 15 nM TSA for 48 or 144 h. Incubation of female embryos with 5 nM and 15 nM TSA resulted in similar increases in acetylated histone H3K9 levels. However, to see comparable effects on acetylated histone H3K9 levels in male embryos, the culture medium needed to be supplemented with 15 nM TSA (as opposed to 5 nM TSA for female embryos). Treatment of male and female embryos with 5 nM TSA for 48 h or female embryos with 5 nM for 144 h had no effect on blastocyst rates, although 15 nM TSA compromised embryonic development. The terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay revealed increased apoptosis in female embryos treated with 5 nM TSA for 144 h, as well as in male and female embryos treated with 15 nM TSA for 48 h, but this increase in apoptosis was not observed in low-quality embryos. The results of the present study suggest that TSA treatment promotes histone hyperacetylation, but has no beneficial effects on the in vitro production of male and female bovine embryos during preimplantation development.