Sperm cells require time to adjust to lower temperatures. While some studies have established stabilization periods ranging from two to four hours for ovine semen, the exploration of longer periods of stabilization remains limited. The objective of the study is to evaluate the 12-hour refrigeration curve during ovine semen cryopreservation and the impact of a diluent medium containing egg yolk and liposomes. Eight mixed-breed rams (½ Dorper and ½ Santa Inês) provided two ejaculates each, divided into two groups. Group A used a commercial egg yolk-based diluent (Botu-Bov® - Botupharma Ltda, Brazil), while Group B used a commercial liposome-based diluent (OptiXcell®, IMV Technologies, France). Semen was packaged in French straws, cooled, cryopreserved, and thawed for analysis. Group A exhibited superior values (P < 0.05) in progressive motility (MP), progressive linear velocity (VSL), straightness (STR), and linearity (LIN) post-thawing and after 3 hours at 37°C (TTR). Conversely, Group B showed higher (P < 0.05) values for path velocity (VAP), curvilinear velocity (VCL), lateral head displacement amplitude (ALH) post-thawing, and VAP, VSL, VCL, and ALH after TTR. Flow cytometry revealed Group A's higher (P > 0.05) plasma and acrosomal membrane integrity and membrane stability. However, Group B exhibited greater (P > 0.05) superoxide anion generation and lipid peroxidation, indicative of higher oxidative stress. In conclusion, the egg yolk-based diluent outperformed the diluent containing liposomes in sperm kinetic parameters evaluated by CASA, although liposomes showed increased oxidative stress, 12 hours of refrigeration at 5.0°C is an alternative viable for semen cryopreservation in sheep.
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Artificial insemination with cooled-shipped semen is the primary method used in the equine breeding industry; yet, sperm quality and fertility can be suboptimal for some stallions when standard techniques are used. Therefore, there is a critical need to develop alternative approaches for these stallions.
To assess sperm quality parameters and fertility of cooled-stored stallion semen processed by SpermFilter® or centrifugation and resuspended in three extenders.
Controlled and field study.
In Experiment 1, semen was collected from 21 stallions classified as having good ('Good-coolers', n = 8) or poor ('Bad-coolers', n = 13) semen cooling. The semen was extended at 30 million spermatozoa/mL in a skimmed milk-based (SM) diluent, and refrigerated for 24 h. Then, the cooled-stored semen was processed through SpermFilter® or centrifugation, and the resulting sperm pellets were resuspended in SM, SM containing pentoxifylline (SM-P), or an egg yolk-based (EY) extender. Unprocessed cooled-stored semen served as control. Sperm motility parameters, plasma membrane integrity (PMI), and mitochondrial membrane potential (HMMP) were assessed in cooled-semen pre- and post-processing. Experiment 2, cooled semen from 9 stallions classified as Bad-coolers was used to inseminate 18 embryo donor mares at 66 cycles (Unprocessed, n = 22; SpermFilter®/SM-P, n = 16; or SpermFilter®/EY, n = 28). Data were analysed with a mixed model and Tukey's as posthoc, and logistic regression.
Processed semen resuspended in EY had superior sperm motility compared to unprocessed, SM and SM-P (p < 0.0001). Semen processed by SpermFilter® resuspended in SM-P was similar to EY (p > 0.05). Pellet resuspension with EY and SM-P improved the HMMP of Bad-cooler stallions (p = 0.0010). Semen processed by SpermFilter® had superior PMI to centrifuged semen (p < 0.0001). Mares inseminated with SpermFilter®/SM-P (50%, 8/16) or SpermFilter®/-EY (68%, 9/28) had higher pregnancy rates than mares bred with unprocessed semen (14%, 3/22) (p < 0.001).
Low number of mares in the fertility trial.
Sperm quality and fertility of Bad-cooler stallions can be enhanced by SpermFilter® and pellet resuspension with either EY or SM-P.
© 2024 EVJ Ltd.

Ovarian cancer (OC) is the most lethal gynecologic malignancy, and melatonin has shown various antitumor properties. Herein, we investigated the influence of melatonin therapy on energy metabolism and mitochondrial integrity in SKOV-3 cells and tested whether its effects depended on MT1 receptor activation. SKOV-3 cells were exposed to different melatonin concentrations, and experimental groups were divided as to the presence of MT1 receptors (melatonin groups) or receptor absence by RNAi silencing (siRNA MT1+melatonin). Intracellular melatonin levels increased after treatment with melatonin independent of the MT1. The mitochondrial membrane potential of SKOV-3 cells decreased in the group treated with the highest melatonin concentration. Melatonin reduced cellular glucose consumption, while MT1 knockdown increased its consumption. Interconversion of lactate to pyruvate increased after treatment with melatonin and was remarkable in siRNA MT1 groups. Moreover, lactate dehydrogenase activity decreased with melatonin and increased after MT1 silencing at all concentrations. The UCSC XenaBrowser tool showed a positive correlation between the human ASMTL gene and the ATP synthase genes, succinate dehydrogenase gene (SDHD), and pyruvate dehydrogenase genes (PDHA and PDHB). We conclude that melatonin changes the glycolytic phenotype and mitochondrial integrity of SKOV-3 cells independent of the MT1 receptor, thus decreasing the survival advantage of OC cells.

Metastatic prostate cancer (mPCa) is resistant to several chemotherapeutic agents. Brachydin A (BrA), a glycosylated flavonoid extracted from Fridericia platyphylla, displays a remarkable antitumoral effect against in vitro mPCa cells cultured as bidimensional (2D) monolayers. Considering that three-dimensional (3D) cell cultures provide a more accurate response to chemotherapeutic agents, this study investigated the antiproliferative/antimetastatic effects of BrA and the molecular mechanisms underlying its action in mPCa spheroids (DU145) in vitro. BrA at 60-100 μM was cytotoxic, altered spheroid morphology/volume, and suppressed cell migration and tumor invasiveness. High-content analysis revealed that BrA (60-100 µM) reduced mitochondrial membrane potential and increased apoptosis and necrosis markers, indicating that it triggered cell death mechanisms. Molecular analysis showed that (i) 24-h treatment with BrA (80-100 µM) increased the protein levels of DNA disruption markers (cleaved-PARP and p-γ-H2AX) as well as decreased the protein levels of anti/pro-apoptotic (BCL-2, BAD, and RIP3K) and cell survival markers (p-AKT1 and p-44/42 MAPK); (ii) 72-h treatment with BrA increased the protein levels of effector caspases (CASP3, CASP7, and CASP8) and inflammation markers (NF-kB and TNF-α). Altogether, our results suggest that PARP-mediated cell death (parthanatos) is a potential mechanism of action. In conclusion, BrA confirms its potential as a candidate drug for preclinical studies against mPCa.

An extreme halophilic archaeon, strain SGH1, is a novel microorganism isolated from endolithic microbial communities colonizing halites at Salar Grande, Atacama Desert, in northern Chile. Our study provides structural, biochemical, genomic, and physiological information on this new isolate living at the edge of the physical and chemical extremes at the Atacama Desert. SGH1 is a Gram-negative, red-pigmented, non-motile unicellular coccoid organism. Under the transmission electron microscope, strain SGH1 showed an abundant electro-dense material surrounding electron-lucent globular structures resembling gas vacuoles. Strain SGH1 showed a 16S rRNA gene sequence with a close phylogenetic relationship to the extreme halophilic archaea Haloterrigena turkmenica and Haloterrigena salina and has been denominated Haloterrigena sp. strain SGH1. Strain SGH1 grew at 20-40°C (optimum 37°C), at salinities between 15 and 30% (w/v) NaCl (optimum 25%) and growth was improved by addition of 50 mM KCl and 0.5% w/v casamino acids. Growth was severely restricted at salinities below 15% NaCl and cell lysis is avoided at a minimal 10% NaCl. Maximal concentrations of magnesium chloride and sodium or magnesium perchlorates that supported SGH1 growth were 0.5 and 0.15M, respectively. Haloterrigena sp. strain SGH1 accumulates bacterioruberin (BR), a C50 xanthophyll, as the major carotenoid. Total carotenoids in strain SGH1 amounted to nearly 400 μg BR per gram of dry biomass. Nearly 80% of total carotenoids accumulated as geometric isomers of BR: all-trans-BR (50%), 5-cis-BR (15%), 9-cis-BR (10%), 13-cis-BR (4%); other carotenoids were dehydrated derivatives of BR. Carotenogenesis in SGH1 was a reversible and salt-dependent process; transferring BR-rich cells grown in 25% (w/v) NaCl to 15% (w/v) NaCl medium resulted in depigmentation, and BR content was recovered after transference and growth of unpigmented cells to high salinity medium. Methanol extracts and purified BR isomers showed an 8-9-fold higher antioxidant activity than Trolox or β-carotene. Both, plasma membrane integrity and mitochondrial membrane potential measurements under acute 18-h assays showed that purified BR isomers were non-toxic to cultured human THP-1 cells.
Copyright © 2020 Flores, Hoyos, Venegas, Galetovic´, Zúñiga, Fábrega, Paredes, Salazar-Ardiles, Vilo, Ascaso, Wierzchos, Souza-Egipsy, Araya, Batista-García and Gómez-Silva.