Effects of somatic tissue cryopreservation on puma (Puma concolor L, 1771) tissue integrity and cell preservation after in vitro culture.

Somatic resource banks play a crucial role in the conservation of genetic diversity, allowing for the preservation of biological samples from different populations. Puma somatic cells can be recovered from these banks and used in assisted techniques toward enhancing their multiplication and conservation. In response to the population reduction of this ecologically importance species, we aimed to evaluate the capacity of cryopreservation of somatic tissues on the maintenance of the integrity and quality of the cells recovered after culture, with the aim of establishing a somatic tissue bank that will allow for the safeguarding of a wide genetic sampling of pumas. Cryopreservation increased the thickness of the corneum layer in the tissues, and the number of perinuclear halos and empty gaps. Nevertheless, cryopreservation was able to maintain normal fibroblast patterns, even showing an increase in the percentage of collagen fibers. Cryopreservation maintained the proliferative potential of the tissues and the parameters evaluated during in vitro culture, mainly regarding the viability, proliferative activity, and apoptosis levels. Nevertheless, cells from cryopreserved tissues showed decreased metabolism and mitochondrial membrane potential when compared to cells from non-cryopreserved tissues. In summary, we demonstrated for the first time that puma somatic tissues subjected to cryopreservation are viable and maintain tissue integrity, featuring minimal changes after warming. Although viable somatic cells are obtained from these tissues, they undergo alterations in their metabolism and mitochondrial membrane potential. Improvements in the conservation conditions of somatic samples are needed to increase the quality of somatic tissue banks in this species.

Characterization of seminal parameters, sperm morphometry, micromorphology, and ultrastructure in gray brocket deer (Mazama gouazoubira, Fischer, 1814).

Populations of gray brocket deer (Mazama gouazoubira) are declining; yet, knowledge on the reproductive biology of this species remains limited. Therefore, this study aimed to describe morphology, viability, membrane integrity, mitochondrial activity, morphometry, micromorphology, and ultrastructure of the gray brocket deer sperm. Three adult male gray brocket deer were used in the study. Semen collection was performed using electroejaculation. Semen were analyzed by evaluating pH, motilities, vigor, mass movement, volume, concentration, viability, membrane integrity, mitochondrial activity, morphology, and morphometry. Micromorphology and ultrastructure of sperm were analyzed using scanning and transmission electron microscopy (SEM and TEM), respectively. There was no significant difference among males regarding on pH, motilities, vigor, mass movement, volume, concentration, viability. High values for membrane integrity, mitochondrial activity, and normal sperm were observed. The most frequent defects were simple bent tail and bowed midpiece. The head length, and width, midpiece, and tail length were 8.5, 4.4, 11.5, and 41.3 µm, respectively. SEM sperm showed paddle-shaped heads, with apical ridge and serrated band on the equatorial segment. TEM revealed the nucleus, acrosome, plasma membrane, mitochondria sheath, proximal centrioles, segmented columns, axoneme, outer dense fibers, and fibrous sheath. SEM and TEM showed the presence of some abnormalities. These results are expected to provide baseline values of diverse semen parameters, contributing toward the development of reproductive biotechnologies for gray brocket deer and, other deer species at risk of extinction.

Morphology, morphometry, ultrastructure, and mitochondrial activity of jaguar (Panthera onca) sperm.

The jaguar is categorized as "Near Threatened". Conservation strategies, therefore, are needed which include use of reproductive biotechniques. For implementation of biotechnique use, the reproductive characteristics of the species must be understood, which is currently not the case. This study, therefore, aimed to describe the detailed morphology of jaguar sperm, and to evaluate the sperm mitochondrial activity. Five male adults were used. Slides stained with Rose Bengal were used for morphometric and morphological analyses. The length and the width of the sperm head were measured, as well as the length of the middle piece, the tail, and the total length. Scanning and transmission electron microscopy were used for ultrastructural analysis. Mitochondrial function was assessed using the marker 3,3'-diaminobenzidine (DAB). The results are expressed as means ± SEM. The most significant morphological abnormalities observed were head (9 ± 1.7%) and tail defects (12.5 ± 3.3%). The width and length of the head were 3.6 ± 0.03 µm and 4.9 ± 0.02 µm, respectively. The middle piece measured 9.7 ± 0.3 µm, the tail measured 54.5 ± 4.4 µm, and the total length of the sperm was 59.5 ± 0.1 µm. Electron-lucent regions and approximately 54 mitochondrial spirals in the middle piece were identified in the nucleus using electron microscopy. The greatest percentages of cells were classified as DAB I (46.6 ± 4.9%) and DAB II (38 ± 4.4%). The data provide detailed information on the sperm characteristics of jaguars and can support research on germplasm conservation for the species.

Seminal plasma and sperm proteome of ring-tailed coatis (Nasua nasua, Linnaeus, 1766).

Ring-tailed coati is listed as a species of least concern in the International Union for Conservation of Nature (IUCN) Red List, however, there has been a sharp decline in their population. The present study was conducted to evaluate the major proteins of both seminal plasma and sperm in ring-tailed coatis. Semen sample was collected from three adult coatis and evaluated for their morphological characteristics. Further, the sample was centrifuged to separate spermatozoa from seminal plasma, and then stored in liquid nitrogen. The seminal plasma and sperm proteins were subjected to one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry. Gene ontology and protein networks were analyzed using bioinformatics tools. Based on sperm concentration and average protein content of the semen, the concentration of protein/spermatozoon was found to be 104.69 ±â€¯44.43 µg. The analysis of SDS-PAGE gels showed 20.3 ±â€¯3.1 and 17 ±â€¯2 protein bands/lane for seminal plasma and sperm, respectively. In-gel protein digestion and peptide analysis by mass spectrometry revealed 238 and 246 proteins in the seminal plasma and sperm, respectively. The gene ontology analysis revealed that the proteins of seminal plasma mainly participated in cellular (35%) and regulatory (21%) processes. According to their cellular localization, seminal plasma proteins were categorized as structural (18%), extracellular (17%), and nuclear (14%) proteins with molecular functions, such as catalytic activity (43%) and binding (43%). The sperm proteins were also involved in cellular (38%) and regulatory (23%) processes, and mainly categorized as extracellular (17%), nuclear (13%), and cytoplasmic (10%) proteins. The major molecular functions of the sperm proteins were catalytic activity (44%) and binding (42%). These results indicated that the seminal plasma of ring-tailed coati has an array of proteins that can potentially modulate several sperm functions, from sperm protection to oocyte binding. However, further studies are necessary to interpret the roles of these major seminal plasma proteins in coatis.