Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

High doses of lipid-core nanocapsules do not affect bovine embryonic development in vitro.

The improvement of in vitro embryo production by culture media supplementation has been a potential tool to increase blastocyst quality and development. Recently, lipid-core nanocapsules (LNC), which were developed for biomedical applications as a drug-delivery system, have demonstrated beneficial effects on in vitro embryo production studies. LNCs have a core composed of sorbitan monostearate dispersed in capric/caprylic triglyceride. Based on that, we firstly investigated if LNCs supplemented during in vitro oocyte maturation had affinity to the mineral oil placed over the top of the IVM media. Also, the effects of LNC supplementation in different concentrations (0; 0.94; 4.71; 23.56; 117.80 and 589.00µg/mL) during the in vitro maturation protocol were evaluated in oocytes and blastocysts by in vitro tests. LNCs seemed not to migrate to the mineral oil overlay during the in vitro oocyte maturation. Interestingly, LNCs did not show toxic effects in the oocyte in vitro maturation rate, cumulus cells expansion and oocyte viability. The highest LNCs concentration tested (589µg/mL) generated the lowest ROS and GSH levels, and reduced apoptosis rate when compared to the control. Additionally, toxic effects in embryo development and quality were not observed. The LNC supramolecular structure demonstrated to be a promising nanocarrier to deliver molecules in oocytes and embryos, aiming the improvement of the embryo in vitro development.