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Despite the cytotoxicity and embryotoxicity previously reported artesunate is a recommended drug to treat malaria for adults, children, and women in the first trimester of pregnancy. To address the putative effects of artesunate on female fertility and preimplantation embryo development, when the pregnancy is not detectable yet, artesunate was added to the oocyte in vitro maturation and in vitro embryo development of bovine. Briefly, in experiment 1 the cumulus-oocyte complexes (COCs) were in vitro matured for 18 h with 0.5, 1, or 2 µg/mL of artesunate or not (negative control) and then checked for nuclear maturation and subsequent embryo development. In experiment 2, the COCs were in vitro matured and fertilized without artesunate, which was added (0.5, 1, or 2 µg/mL) from the 1st to the 7th day of embryo culture along with a negative and a positive control group with doxorubicin. As a result, the use of artesunate on oocyte in vitro maturation did not differ from the negative control (p > 0.05) regarding nuclear maturation, cleavage, and blastocyst formation. Also, artesunate on in vitro embryo culture did not differ from negative control (p > 0.05) regarding cleavage and blastocyst formation, except for positive control, with doxorubicin (p < 0.05). In conclusion, under the conditions investigated, there was no evidence of artesunate toxicity on oocyte competence and the preimplantation period of in vitro embryo development in the bovine model, however, artesunate use still should be taken carefully as the outcome of implantation after oocytes and blastocysts exposure to artesunate remains unknown. HIGHLIGHTS: Artesunate added to in vitro maturation did not impair oocyte competence in bovine. Artesunate added to in vitro culture did not affect cleavage and blastocyst formation. No evidence of artesunate toxicity in oocytes and embryos of bovine was found. [ABSTRACT FROM AUTHOR]

Summary: Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT. Different CPs were evaluated in regard to proliferation rate, senescence level, and chromosome stability, as well as for POU5F1 (POU class 5 homeobox 1) mRNA expression levels. In total, 9 of 24 CPs (37.5%) were successfully expanded in vitro up to the fourth passage and shown to proliferate following cryopreservation, at which time cell analyses were performed. The use of a CP with low senescence level, normal karyotype, and highest POU5F1 expression levels did not improve embryo development rates or quality following SCNT. As previously suggested, this study supports the notion that levels of POU5F1 expression in the donor nucleus do not impact the SCNT results. Notably, the single-cell seeding approach used herein to isolate CPs may be extended to the evaluation of additional predictor markers of reprogrammability success for SCNT in future experiments. [ABSTRACT FROM AUTHOR]

Background: Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP. Methods: The presence of the high affinity melatonin receptors was investigated through an autoradiographic binding assay, using the non-permeable ligand [[125I]-iodomelatonin (17 pM) in embryos. The kind of melatonin site was investigated in oocytes and embryos by immunocytochemistry. In vitro fertilized bovine embryos produced from in vitro maturated oocytes supplemented with melatonin (0.0001 to 1000 nM) were analysed to determine their cleavage and blastocyst formation rates. Results: The [125I]-iodomelatonin (17 pM) binding in blastocysts was blocked by pre-incubation with melatonin (30000 nM), showing the presence of the high affinity melatonin receptors. MT1, MT2 and NQO2 immunoreactivity was observed in oocytes. MT1 immunoreactivity was observed in hatched blastocysts, however MT2 and NQO2 were not observed in this embryonic stage. Melatonin (pM) triggered significant difference in both cleavage and blastocysts formation rates. Conclusions: The high affinity MT1 melatonin receptor must be taking part in IVM events; furthermore it is the first melatonin receptor to appear during bovine embryo development in vitro. [ABSTRACT FROM AUTHOR]