Investigating cryopreservation techniques for maintaining morphology and in vitro viability of cartilage and skin from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831) for conservation through biobanks.

Conservation of the genetic diversity through skin and cartilage biobanks represents an essential strategy for maintaining biodiversity. Biobanks for the wild species of the order Rodentia have been little studied. Considering that the cryopreservation technique has specific relationships with the tissue and species of interest, we propose investigating different techniques for preserving tissue integrity and cell viability after cartilage and skin culture from Spix's yellow-toothed cavies. Subsequently, two techniques [solid-surface vitrification (SSV) vs. slow freezing (SF)] were used for cartilage and skin cryopreservation. Tissues not subjected to cryopreservation were used as controls. All tissues were evaluated for morphology and proliferation by histological techniques. Moreover, fragments were cultured, and cells were evaluated for viability, proliferation, metabolism, and apoptosis. Regardless of the cryopreservation technique, no differences were observed for the thickness of the epidermis, dermis, skin, spinous and basal layers, fibroblasts, and proliferative activity regarding the number of nucleolar organizer regions (NOR). SSV ensured better maintenance of epidermal cells, normal chondrocytes, filled gaps, collagen fibers, proliferative activity by NOR area/cell, and reduced perinuclear halos and empty gaps compared to SF. SF ensured the conservation of corneum thickness compared to the control. Although both techniques promoted cell recovery after culture, cells from SF resulted in better subconfluence time and day with cell growth around fragments compared to SSV. In conclusion, both cryopreservation techniques resulted in viable cells after culture. However, SSV promoted better maintenance of tissue morphological integrity, and SF ensured the preservation of all cell quality parameters in Spix's yellow-toothed cavies.

Cryopreservation and passaging optimization for Galea spixii (Wagler, 1831) adult skin fibroblast lines: A step forward in species management and genetic studies.

BACKGROUND: In vitro culture of fibroblasts is a technique based on cell isolation, physiological characterization, and cryopreservation. This technique has not been described for Galea spixii, therefore, it can be used to learn about its cellular biology and genetic diversity. OBJECTIVE: We established fibroblast lines of six G. spixii individuals from several passages (second, fifth, eighth, and tenth) and cryopreserved them. METHODS: Fibroblasts recovered from skin biopsies were identified based on morphology, immunocytochemistry, and karyotyping. The cells were analyzed for morphology, ultrastructure, viability, proliferation, metabolism, oxidative stress, bioenergetic potential, and apoptosis before and after cryopreservation. RESULTS: After the eighth passage, the fibroblasts showed morphological and karyotypic changes, although their viability, metabolism, and proliferation did not change. An increase in oxidative stress and bioenergetic potential from the fifth to the eighth passages were also observed. Post cryopreservation, cell damage with respect to the ultrastructure, viability, proliferative rate, apoptotic levels, oxidative stress, and bioenergetic potential were verified. CONCLUSION: Fibroblasts up to the tenth passage could be cultured in vitro. However, cells at the fifth passage were of better quality to be used for reproductive techniques. Additionally, optimization of the cryopreservation protocol is essential to improve the physiological parameters of these cells.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines.

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

Use of a product based on wood vinegar of Eucalyptus clone I144 used in the control of bovine mastitis.

Bovine mastitis is one of the most frequent diseases in dairy cattle worldwide. The use of antiseptics in milking, if properly used, can lead to a reduction in potentially pathogenic microorganisms and their transmission between herds. Several medicinal plants have antiseptic potential, including eucalyptus (Eucalyptus spp.). Therefore, the objective of this study was to evaluate the effectiveness of wood vinegar from Eucalyptus urograndis clone GG I144 (EU) as an antiseptic in vitro and in vivo; in addition, to its cytotoxicity and antimicrobial resistance. Fifteen bovines were used, lactating females 3-6 years of age and divided into three groups of five animals each. The wood vinegar was placed in the teats of the animal for 28 days and collections of cellular debris were performed every 7 days. At the Veterinary Microbiology Laboratory (LAMIV) of UFERSA, the samples were processed and serial dilution was performed in Petri plates with plate count agar (PCA) at 37 °C. Cytotoxicity was verified based on morphological alterations and metabolic activity. Morphological changes were not observed in all cells incubated with 1 % pyroligneous extract. The in vitro data demonstrated antimicrobial activity against S. aureus, S. agalactiae, Salmonella, E. coli and P. aeruginosa. The bacteria of the genus Staphylococcus and Corynebacterium were resistant to penicillin (PEN), rifampicin (RIF), nitrofurantoin (NIT), erythromycin (ERI), and ciprofloxacin (CIP). The extract was used in vivo in the post-dipping of dairy cows, which reduced the microbiological load present in the mammary glands from 4.74 to 2.54 CFU, indicating its future use as an antiseptic.

Efficiency of Pyroligneous Extract from Jurema Preta (Mimosa tenuiflora [Willd.] Poiret) as an Antiseptic in Cats (Felis catus) Subjected to Ovariosalpingohysterectomy.

Pyroligneous extract of Jurema preta (Mimosa tenuiflora [Willd.] Poiret) was evaluated for its efficacy as a cutaneous antiseptic in cats (Felis catus) that were subjected to ovariosalpingohysterectomy. For this purpose, 30 cats without a defined breed were sterilized and divided into two groups. The first group was the positive control, treated with 0.5% chlorhexidine-alcohol solution, and the second group was treated with 20% pyroligneous extract of M. tenuiflora. Regardless of age and sex, all animals had visible healing at similar times. A significant reduction in bacterial growth was observed in animals treated with the extract, and no cytotoxicity was observed in the feline epithelial cells. In addition, surgical wounds of cats treated with M. tenuiflora extract exhibited improved healing. On agar plates, treatment with both chlorhexidine and M. tenuiflora extract resulted in the inhibition zones for all bacterial strains isolated from surgical wounds. Therefore, M. tenuiflora extract is demonstrated to have antiseptic effects on the surgical wounds of cats undergoing ovariosalpingohysterectomy.