Comparative effect of cryoprotectant combinations on the conservation of somatic cells derived from jaguar, Panthera onca, towards the formation of biologic banks.

Due to the reduction of the jaguar population, the formation of somatic cell cryobanks represents an interesting tool for its conservation. Nevertheless, the success of these cryobanks depends on the cryoprotectants used in cryopreservation. We evaluated the effects of the intracellular cryoprotectants (10% dimethyl sulfoxide, DMSO; 10% ethylene glycol, EG) in the absence or presence of an extracellular cryoprotectant (0.2 M sucrose, SUC) on the morphology, confluence, viability, and metabolism of somatic cells derived from five jaguars belonging to Brazilian zoos. The morphology was presented in a descriptive manner, while the confluence, viability and metabolic activity were presented as means and compared using statistical tests. Non-cryopreserved cells were used as control and compared to frozen/thawed cells using cryoprotectants. No difference was observed for the morphology and confluence among non-cryopreserved and cryopreserved cells, regardless of the cryoprotectants. Only cryopreserved cells in EG (45.8%±12.9) had a reduction in their viability when compared to non-cryopreserved cells (97.8%±1.1). Only cryopreserved cells in DMSO with SUC (76.0%±2.7) or absence of SUC (77.0%±3.7) maintained their metabolic activity after thawing, when compared to non-cryopreserved cells (100.0%±6.7). Therefore, combinations of DMSO in the absence and presence of SUC were efficient in the cryopreservation of somatic cells of jaguars.

Evaluation of the damage caused by in vitro culture and cryopreservation to dermal fibroblasts derived from jaguars: An approach to conservation through biobanks.

Ex-situ conservation strategies such as the formation of somatic cell banks are valuable tools for the conservation of jaguars, whose population has been declining in recent years. Once properly established, these cells can be successfully leveraged for future applications. We aimed to assess the effects of in vitro culture and cryopreservation on the establishment of fibroblasts derived from jaguars. Initially, we identified five dermal fibroblastic lines using morphology and immunophenotyping assays; these lines were then subjected to two experiments. In the first experiment, the viability, metabolism, and proliferative activity of cells at different passages (first, third, and tenth) were evaluated. In the second experiment, the cells were cryopreserved and the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptosis were evaluated after one, three, and ten passages. Noncryopreserved cells were used as controls. The in vitro culture after first, third, and tenth passages and cryopreservation conditions did not affect the proliferative activity and viability. However, cells cultured until tenth passage and frozen/thawed cells showed reduced metabolism. In addition, cryopreserved cells showed higher levels of intracellular ROS and altered ΔΨm when compared with those of noncryopreserved cells. Finally, frozen/thawed cells cultured after ten passages showed reduced proliferative activity and number of viable cells than did frozen/thawed cells cultured after one and three passages. In summary, we have shown that viable fibroblasts can be established from jaguar skin and that although these cells do not show altered viability and proliferative activity, they do undergo damage during extended culture and cryopreservation.

Evaluation of different skin regions derived from a postmortem jaguar, Panthera onca (Linnaeus, 1758), after vitrification for development of cryobanks from captive animals.

Biological resource banks represent valuable tools for the conservation of species vulnerable to extinction, such as the jaguar. Cryobanks of skins have the potential to safeguard rare genotypes, allowing the potential exploitation of biological samples in animal multiplication technologies and the study of genetic variability. Determination of the most suitable skin regions for tissue conservation can help increase the efficiency of cryobanks and the storage of biological samples. To this end, we evaluated the effects of vitrification of skin tissues from the ear, caudal, and femoral regions of a post-mortem jaguar belonging to a zoo in Brazil. Non-vitrified and vitrified samples were evaluated and compared using quantitative methods, focusing on skin thickness, cell quantification, number of perinuclear halos, collagen and elastic density, and proliferative activity. No differences were observed in skin thickness, number of perinuclear halos, elastic density, and proliferative activity between non-vitrified and vitrified tissues in skin from any region. However, vitrified tissues derived from femoral skin showed a reduction in the number of fibroblasts, epidermal cells and collagen density compared to non-vitrified tissues. In summary, the ear and caudal regions provided the best conservation of somatic tissues derived from jaguars, and skin samples from these regions are therefore the most suitable for the formation of cryobanks.

Quantitative and descriptive histological aspects of jaguar (Panthera onca Linnaeus, 1758) ear skin as a step towards formation of biobanks.

Skin of mammals vulnerable to extinction, such as the jaguar, is used as a source of material in conservation strategies. The composition of skin is not uniform among species, and the ability to distinguish similarities in skin morphology in animal groups is fundamental in the application of skin tissue for use in biobanks. The aim of our study was to evaluate the structure, composition and capacity for culture of ear skin from the yellow and black jaguars. Both qualitative and quantitative methods were used, focusing on skin thickness, cell quantification and distribution, collagen density, proliferative activity and viability. Histomorphometrical study of the skin showed a total thickness of 273.2 and 274.6 µm for the yellow and black jaguars, respectively. Melanocytes and fibroblasts were, respectively, 9.7 and 23.0 for the yellow jaguar and 11.3 and 26.8 for the black jaguar. A collagen density of 67.0% and 49.0% was observed for yellow and black jaguars, respectively. Both animals presented a proliferative activity varying between 1.20 and 1.30. All tissues could promote cellular detachment, reaching subconfluence in 10-15 days. This kind of information from histomorphometrical features and cell cultures can be essential for a more targeted application of ear skin cryopreservation in this species, as such information will enable understanding the action of substances on tissues during the conservation process.

Single point incremental forming of a facial implant.

BACKGROUND: The investigation draws from the fundamentals of the mechanical behaviour of titanium grade 2 to the design and fabrication of facial implants by means of single point incremental forming. OBJECTIVES: To provide knowledge on the capabilities and limitations of a new manufacturing technology to fabricate low-cost, patient-specific medical implants. STUDY DESIGN: Rapid fabrication of a simplified model of a facial implant. METHODS: Circle grid analysis and its graphical representation in the fracture forming limit diagram combined with finite element modelling are utilized to identify the failure limits and to assist the overall design of the facial implants. RESULTS: Fabrication of facial implants without and with failure by cracking due to excessive thinning of the sheet from where the implant is to be cut. CONCLUSIONS: Identification of the major operative parameters that influence fabrication of sound facial implants by means of single point incremental forming. CLINICAL RELEVANCE: Reduce the gap between production engineers and the medical community by presenting a state-of-the-art manufacturing technology to produce low-cost, patient-specific medical implants.

Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs of Triatoma klugi Carcavallo et al, Triatoma vandae Carcavallo et al and Triatoma williami Galvão et al (Hemiptera: Reduviidae).

Ultrastructural analysis of the ventral region of the head--rostrum, buccula and stridulatorium sulcus--of 1st, 3rd and 5th instars of Triatoma klugi Carcavallo et al, Triatoma vandae Carcavallo et al, and Triatoma williami Galvão et al, are described in here. Morphological differences in the analyzed structures for all three Triatoma species studied were detected under scanning electron microscopy, allowing their grouping by their morphological similarities. Species-specific differences at each nymphal development stage were analyzed as well.

Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs of Triatoma klugi Carcavallo et al, Triatoma vandae Carcavallo et al and Triatoma williami Galvão et al (Hemiptera: Reduviidae)

Ultrastructural analysis of the ventral region of the head - rostrum, buccula and stridulatorium sulcus - of 1st, 3rd and 5th instars of Triatoma klugi Carcavallo et al, Triatoma vandae Carcavallo et al, and Triatoma williami Galvão et al, are described in here. Morphological differences in the analyzed structures for all three Triatoma species studied were detected under scanning electron microscopy, allowing their grouping by their morphological similarities. Species-specific differences at each nymphal development stage were analyzed as well.