Vitrification of canine ovarian tissue using the Ovarian Tissue Cryosystem (OTC) device.

The present study evaluated the effect of Ovarian Tissue Cryosystem (OTC) on follicular morphology and density, as well as on stromal cell density of vitrified canine ovarian tissue. Canine ovarian fragments collected from adult female dogs in stages of the random oestrous cycle were fixed (FC, fresh control) or vitrified (VIT) with an OTC device. After vitrification and warming, the fragments were fixed for histological analysis. Overall, the mean percentage of normal pre-antral follicles decreased after vitrification procedure (FC: 74.5% ± 1.6% vs. VIT: 52.05% ± 1.5%). Although the rates of normal primordial (71.1% ± 1.8%) and secondary (0.7% ± 0.4%) follicles vitrified showed a reduction (p < .05), vitrification using OTC showed considerable preservation of follicles, when compared to the fresh control (81.1% ± 1.5% and 2.3% ± 0.6%, respectively). The mean follicular density was maintained after vitrification (FC: 199.65 ± 12.8 vs. VIT: 199.68 ± 10.8), whereas the stromal cell density decreased in the VIT group. Based on the results, we recommend the use of OTC for vitrification of canine ovarian tissue.

Cryoprotectant agents and cooling effect on embryos of Macrobrachium amazonicum.

SummaryThere are few reports of cryopreservation and injuries in Macrobrachium amazonicum embryos. Thus, the aim of this study was to analyze the effects of cryoprotectants agents and cooling on stage VIII of this species. Fertilized eggs from ovigerous females were removed from the incubation chamber, then placed in 10 ml Falcon tubes with a cryoprotectant solution and saline-free calcium solution. Thus, the embryos underwent a cooling curve of 1°C per min until reaching 5°C, and then were stored for 2 h. The tubes containing the embryos were washed to remove the cryoprotectant, acclimated for 5 min and then transferred to 50 ml incubators. At the end of the 24-h period, living embryos from each tube were counted and tabulated. A pool of embryos was fixed with 4% formaldehyde and then subjected to histology using 3-mm thick sections and stained with haematoxylin/eosin. Another pool was used for biometric analysis in which length, width and volume were analyzed. The cryoprotectants agents used were: dimethylsulfoxide (DMSO), methyl alcohol, ethylene glycol at 1, 5 and 10% and sucrose (0.5 M). Variance analysis was performed followed by Tukey's honest significant difference (HSD) test at 5% significance level. DMSO cryoprotectant affected embryo survival the least with rates of 71.8, 36.2 and 0% for concentrations of 1, 5 and 10%, respectively. Ethylene glycol caused 100% mortality at all the concentrations used. It was not possible to observe the interference of cooling and cryoprotectants on embryonic structures in this study.

Nephroprotective effects of (-)-α-bisabolol against ischemic-reperfusion acute kidney injury.

BACKGROUND: Ischemia/reperfusion (I/R) in kidney is commonly related to acute kidney injury (AKI), essentially through oxidative stress. (-)-α-Bisabolol is a sesquiterpene isolated from the essential oil of a variety of plants, including chamomile, which has important antioxidant activity. STUDY DESIGN: This study intends to evaluate the nephroprotective activity of (-)-α-bisabolol (Bis) in both in vivo and in vitro models of kidney I/R. METHODS: Male Wistar rats were submitted to right nephrectomy, followed by ischemia by clamping of the renal artery in the left kidney for 60min. and 48h of reperfusion. The animals were treated orally with Bis (100mg/kg) or vehicle for 24h after reperfusion, and placed in metabolic cages, to evaluate water consumption, diuresis, urinary osmolality, classic biochemical markers and urinary KIM-1 (kidney injury molecule-1). Additionally, the left kidney was collected for histological evaluation and determination of glutathione (GSH) and Thiobarbituric Acid Reactive Substances (TBARS) levels. Tubular epithelial cells LLC-MK2 were used to assess Bis effect on in vitro I/R, by MTT assay. It was performed the cellular respiration tests by flow cytometry: evaluation of the production of cytoplasmic reactive oxygen species by DCFH-DA assay and mitochondrial transmembrane potential analysis with the dye rhodamine 123. RESULTS: I/R caused alterations in diuresis, water intake, urinary osmolality, plasmatic creatinine, urea and uric acid, creatinine clearance, proteinuria and microalbuminuria. Treatment with Bis ameliorated all of these parameters. Also, KIM-1 level enhanced by I/R was also diminished in groups treated with Bis. The histological examination showed that Bis attenuated the morphological changes caused by I/R, markedly vascular congestion and intratubular deposits of proteinaceous material. Additionally, Bis was able to reduce the changes observed in TBARS and GSH levels in kidney tissue. In in vitro assay, Bis was capable to partially protect the cell lineage against cell damage induced by I/R. CONCLUSION: (-)-α-Bisabolol has a nephroprotective effect in kidney I/R, with antioxidant effect. Moreover, this result seems to be associated to a direct protective effect on tubular epithelia.