Different Methods for Seminal Plasma Removal and Sperm Selection on the Quality and Fertility of Collared Peccary Sperm.

Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll® gradient densities (PG 45-90% and PG 35-70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45-90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35-70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45-90% was used for fertilization. PG 45-90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2-26.3%) and morula (8.1-10.5%) rates did not differ between the groups. Therefore, PG 45-90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.

Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas.

The loss of wild biodiversity has prompted the development of cryobanks, such as those of somatic cells. This is the reality of Pumas, wild felids of ecological importance that suffer from anthropogenic actions, population decline, and subsequent loss of genetic diversity. Somatic cell banks are a strategy for conserving population diversity. We compared different concentrations and types of intracellular cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG) associated with 0.2 M of sucrose (SUC) in the cryopreservation of the somatic cells of captive Pumas. The cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and varying concentrations of DMSO and EG in the absence or presence of SUC. The cells were analyzed for morphological characteristics, viability, proliferative activity, metabolic activity, and apoptosis levels. Cells maintained similar fusiform morphology before and after cryopreservation. There was no difference in viability, regardless of the reduction in the concentration and type of intracellular cryoprotectants and sucrose. Similarly, proliferative activity, metabolic activity, and apoptosis levels were not altered by the composition of the cryoprotectants. In summary, we demonstrate that reducing the concentration of DMSO or EG ensures adequate cryopreservation of Puma somatic cells, regardless of the presence of SUC.

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.