Synergistic effects of glial cell line-derived neurotrophic factor and base-medium on in vitro culture of testicular tissue derived from prepubertal collared peccary.

We evaluated the influence of different media plus various concentrations of Glial cell line-derived neurotrophic factor (GDNF) during the in vitro culture (IVC) of testicular tissues from prepubertal collared peccary. Testes from 5 individuals were collected, fragmented and cultured for 28 days (34°C and 5% CO2). Culture media were Dulbecco's modified essential medium (DMEM) or stem cell serum free media (StemPro-34™ SFM), both supplemented with various concentrations of GDNF (0, 10, or 20 ng/mL). Fragments were cultured on the flat surface of 0.75% agarose gel and were evaluated every 7 days for fragment area, histomorphology, cellular viability, and proliferative activity. Data were expressed as mean ± standard error and analyzed by Kruskal-Wallis's and Tukey test. Fragments area decreased over the 28 days-culture, regardless of the treatment. For morphology, the StemPro-37 SFM medium plus 10 ng/mL GDNF provided higher scores at all time points in comparison to DMEM using any GDNF concentration (p < .05). After 28 days, similar cellular viability (~70%) was observed in all treatments (p > .05). For proliferating cell nuclear antigen assay, only DMEM plus 10 ng/mL GDNF improved (p < .05) cellular proliferation on Days 14 and 28. Looking at argyrophilic nucleolar organizing regions, after 28 days, there were no differences among treatments regarding cell proliferative capacity for both spermatogonia and Sertoli cells (p > .05). In summary, the DMEM and StemPro-34 SFM are adequate medium for IVC of prepubertal peccary testicular tissue. Supplementation with GDNF, especially at a 10 ng/mL concentration, appears to be essential for the maintenance of cell survival and proliferation.

Comparison between commercial media and TRIS-egg yolk extender in the refrigerated storage of collared peccary semen at 17°C.

To assist in the conservation of collared peccary, it is important to strengthen semen processing protocols. The aim of this study was to compare the effects of different commercial extenders (BTS; NUTRIXcell+ and PRIMXcell Ultra) and TRIS + egg yolk on the functional and morphological aspects of collared peccary semen stored at 17 °C for 48 hours. Ten ejaculates obtained by electroejaculation were divided into 4 aliquots and diluted in the respective extenders, then stored in a biological incubator at 17 °C for 12, 24, 36, and 48 hours. The samples were evaluated for kinetic parameters, membrane functionality, membrane integrity, mitochondrial activity, morphology, and sperm-binding capacity. At the end of storage (48 h), promising results were found for motility parameters, with TRIS + egg yolk (71.0 ± 4.6%) being more efficient than NUTRIXcell+ (38.9 ± 10.9%) (P < 0.05) and similar to BTS (42.9 ± 11.9%) and PRIMXcell Ultra (46.8 ± 10.8%). The results for membrane integrity and mitochondrial activity were around ∼30-50%, with TRIS being the only extender to preserve both parameters (58.9 ± 5.3 and 59.2 ± 5.6%) for up to 48 hours, respectively (P < 0.05). Finally, the extenders could guarantee 60% membrane functionality and ∼ 60-70% normal sperm morphology, as well as similar binding capacity among the groups. In conclusion, TRIS + egg yolk is effective in preserving the sperm parameters of collared peccary semen at 17 °C for 48 hours, while PRIMXcell Ultra and BTS are viable alternatives for this purpose.

Prenatal post-implantation development of collared peccaries (Pecari tajacu Linnaeus, 1758).

Given the importance of information on intrauterine development in diagnosing anomalies in the gestational development of the species for the development of assisted reproduction technologies as well as understanding the autonomy and responsiveness of the newborn, the aim of the present study was to describe the external morphology of collared peccary conceptuses. For this study, two conceptuses were used per gestational age of 25-120 days post-copulation (dpc) and neonates with 145 dpc, totalling 22 animals. Females were euthanised, and embryos/foetuses were examined, measured, and photographed. During the first third of the gestational period (25-50 dpc, n = 8), a marked body curvature, brain vesicles, somites, internal organs, placid lens, auricular protrusion and limb buds are noted. In the second third of the gestational period (51-100 dpc, n = 10), foetuses lose their body curvature, displaying greater anatomical definition, including skeletal, external ears, nostrils, eyelids and tactile hair formation and cranial suture closure. In addition, dorsal scent gland and genital tubercle differentiation were visualized at 50 days post-copulation. In the third of the gestational period (101-145 dpc, n = 4), the organs become completely formed, alongside skin darkening, eyelid opening, dental eruption, dorsal odorous gland development, sexual organ externalization, and fanero attachment development. These data allowed for the construction of a prenatal growth curve, providing comparative anatomy information for ungulates and further contributing towards rational reproductive management and reproductive biotechnologies for this species.

Serum starvation is as efficient as roscovitine on the cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Fibroblast cycle synchronization in G0/G1 is an essential step for nuclear reprogramming by cloning or induced cells to pluripotency. Considering the diversity among rodents and the ecological and scientific importance of these animals, we compared the contact inhibition, serum starvation, and 10 µM of roscovitine as methods of synchronization of red-rumped agouti fibroblasts. The effects of each protocol were evaluated on the percentage of cycle phase, morphology, viability, and apoptosis levels. The results showed that culturing the cells to serum starvation for 24 h (75.9%), 48 h (81.6%), 72 h (86.2%), 96 h (84.0%), and 120 h (83.7%) 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 (31.4%). Also, this effect was not different between the times of 48 and 120 h (P > 0.05). A similar response was observed for cells cultured with roscovitine for 12 h (86.9%), 24 h (74.8%), and 48 h (81.7%), with a higher percentage of synchronized cells in G0/G1 compared to cells not submitted to any synchronization treatment (52.2%). Nevertheless, this effect was best evidenced at 12 h (P < 0.05). Also, the contact inhibition for 24-120 h could not synchronize cells in G0/G1, with values ranging from 70.9 to 77.9% (P > 0.05). Moreover, no difference was observed for morphology, viability, and apoptosis levels in any synchronization method (P > 0.05). Therefore, serum starvation is as efficient as roscovitine on cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Enhancing Titanium Disk Performance through In-Pack Cold Atmospheric Plasma Treatment.

While titanium dental implants have already been clinically established, ongoing research is continuously being conducted to advance the fields of osseointegration and bacterial resistance, seeking further improvements in these areas. In this study, we introduce an innovative method for treating titanium surfaces within tightly sealed packaging. Specifically, titanium discs, enclosed in surgical-grade packaging, underwent treatment using cold atmospheric plasma (CAP). The surfaces were thoroughly characterized in terms of wettability, crystalline structure, and chemical composition. Hemocompatibility analyses were conducted using blood diluted in sodium citrate (1:9) exposed to titanium discs for 30 min inside a CO2 incubator at 37 °C. Subsequently, various blood parameters were evaluated, including prothrombin time (PT), activated partial thromboplastin time (APTT), and platelet adhesion. Microbiological analyses were also performed using Pseudomonas aeruginosa (ATCC 27853) for 4 h at 37 °C. The treatment with CAP Jet resulted in a reduction in contact angle without causing any changes in the crystalline structure. No statistically significant differences were observed in the blood parameters. The plasma-treated samples exhibited lower PT and APTT values compared to those of the control group. The surfaces treated with CAP Jet showed increased platelet activation, platelet density, and thrombus formation when compared with the untreated samples. Moreover, the treated surfaces demonstrated lower bacterial colony formation compared with other surfaces.

Gonadal Morphology of Stillborn Hawksbill Sea Turtle (Eretmochelys imbricata) Hatchlings.

This study aimed to investigate sexual dimorphism in stillborn hawksbill sea turtles (Eretmochelys imbricata) through gonadal morphological characterizations. Macroscopic, light microscopy, and transmission electron analyses were performed for 30 gonad-mesonephros complexes. Female gonads were spindle-shaped and present a translucent whitish appearance with a grainy texture. Male gonads were approximately ovoid with a smooth opaque white surface. A primary sexual difference concerns different marrow structures, with females presenting organized cellularity featuring oocytes, lacunae, and blood vessels, while males presented a distinct organizational medulla pattern marked by testicular cords extending throughout the gonad length. Ultrastructurally, female's stroma presented interstitial cells and an abundant cytoplasm rich in electrodense droplets and large oval germline cells, with a conspicuous and noncentral nucleus. Males, on the other hand, presented testicular cord cells containing small amounts of heterochromatin and approximately triangular apical and basal cytoplasms with an evident nucleolus characteristic of support cells. Additionally, there were cells with a large spherical nucleus compared with the cell size and a relatively scarce cytoplasm, identified as gonocytes. These findings indicate that macroscopic, microscopic, and ultrastructural evaluations are effective and reliable techniques for the sexual identification of stillborn E. imbricata hatchlings.

Morphohistometric and steroidogenic parameters during testicular and epididymal differentiation in cavy (Galea spixii) fetuses.

Sexual differentiation and steroidogenic mechanisms have an important impact on postnatal gonadal phenotypic development. Thus, establishing the activities that lead to male phenotypic development can provide a better understanding of this process. This study examined the prenatal development of cavies to establish morphological and histometric development patterns and protein and enzyme immunolocalization processes that are responsible for androgen synthesis in the testes and epididymis. Histological and histometric analyses of the diameter of the seminiferous cords and epididymal ducts of male fetuses on Days 25, 30, 40, and 50 were performed, as well as immunohistochemistry of the steroidogenic enzymes 5α-reductase and 17ß-HSD, the androgen receptor, and the anti-Müllerian hormone (AMH). Our findings showed a cellular grouping of gonocytes from Day 30 onward that was characteristic of the seminiferous cord, which was not present in the lumen at any of the studied dates. From Day 50 onward, the differentiation of the three anatomical regions of the epididymis was evident, the head (caput), body (corpus), and tail (cauda), with tissue distinctions. Furthermore, the diameters of the seminiferous cords and epididymal ducts significantly increased with age. On Day 50, the tail showed the greatest diameter of the three regions. The Sertoli and Leydig cells exhibited AMH immunoreactivity at all dates. In addition, the Leydig cells and epididymal epithelial tissue were immunopositive for 5α-reductase, 17ß-HSD, and the androgen receptor; therefore, these factors influenced the development and maintenance of the testis and epididymis during cavy prenatal development.

Antillean manatee (Trichechus manatus manatus Linnaeus, 1758) Tongue Morphology and Adaptive Herbivorous Implications.

Morphological study of the tongue is an interesting way of understanding evolutionary processes associated with feeding habits. Therefore, the aim of the present study was to describe the tongue morphology of the Antillean manatee and to understand possible morphological relationships with its way of capturing food. Macroscopic dissections and light and scanning electron microscopy analyses of seven manatee tongues were performed. The tongue in Antillean manatees is a muscular and robust organ, divided into apex, body, and root. It is firmly adhered to the floor of the oral cavity. Lingual papillae were distributed over the entire tongue surface. They were identified as filiform papillae concentrated in the apex. Fungiform papillae were present on the apex and lateral regions. Foliate papillae were located on the dorsolateral portion of the root. Lentiform papillae were located across the dorsal tongue surface. The mucosa was lined by a keratinized stratified squamous epithelium presenting compound tubuloacinar glands and taste buds in the foliate papillae. The tongue of the Antillean manatee is similar to other Sirenia species, both of which share a completely herbivorous diet.

Placental development in the early stages of red-rumped agouti pregnancy (Dasyprocta leporina Linnaeus, 1758).

BACKGROUND: Hystricomorpha rodents display a similar placentation model to humans. The present study was carried out considering the scarcity of information concerning the placental development in agouti. OBJECTIVE: Describe the microscopy of the placenta, subplacenta and yolk sac of agoutis in early pregnancy and report on the inversion of the yolk sac. METHODS: Fifteen females between the 14th-32nd day of gestation were used following euthanasia. Gestational buttons were collected, fixed, processed, stained to optical microscopy or immunohistochemistry. RESULTS: Chorioallantoic placenta (CP) ranged from conical to a half-sphere, as follows: from the 14th to 17th day, the CP displays an inverted "V" shape, predominantly formed by cytotrophoblasts; from 20 to 22 days, formed almost entirely by cytotrophoblasts; at 28 days, a half sphere, with distinct lobes and interlobular area, numerous maternal gaps delimited by syncytiotrophoblasts and trophoblast giant cells; at 32 days, globose and undergoing the maturation process. Subplacenta, located between decidua and CP, initially presents septa consisting of simple columnar epithelium and after 17 days, comprising stratified epithelium. Visceral yolk sac (VYS) is attached to two CP projections between 14 and 17 days, formed by a simple cubic epithelium and inverted. Between 20 and 22 days, the epithelium displays apical villous projections with cytoplasmic vacuoles and a vascularized mesoderm. After the 24th day, the VYS near the placenta is pleated, very vascularized and villous, with decreased villi sizes further away from the placenta. CONCLUSION: The agouti CP displays similar characteristics to other hystricomorpha, including placenta lobulation, a subplacenta and an inverted vitelline placenta.

Abdominal aortic branches of red-rumped agouti (Dasyprocta leporina Linnaeus, 1758).

Understanding the cardiovascular system is fundamental in diagnosing pathologies and interpreting exams, such as contrast radiographs. In this context, the present study describes the collateral abdominal aorta artery branches of red-rumped agouti. Ten red-rumped agoutis, six males and four females, were assessed. The vascular system was perfused with Neoprene 450 latex coloured with a yellow pigment, dissected and analysed. Three euthanized animals were perfused with a barium sulfate solution (1 g mL-1 ) associated with latex Neoprene 450 at a 1:3 ratio to obtain contrast-enhanced radiographs. The abdominal aorta emitted the celiac artery, which in turn originated the left gastric, hepatic and splenic arteries. The second collateral branch comprised the cranial mesenteric artery, followed by the renal arteries, which emitted the adrenal arteries, with the caudal emergence of the gonadal arteries. The caudal mesenteric artery appeared in a caudal direction. The abdominal aorta divided after reaching the pelvic cavity entrance, originating the right and left common iliac arteries. Before its bifurcation, the abdominal aorta dorsocaudally emitted its last collateral branch, the median sacral artery. The collateral branches of the aorta, therefore, resemble previously described rodent patterns, with few variations.

Synergistic effects of follicle-stimulating hormone and epidermal growth factor on in vitro maturation and parthenogenetic development of red-rumped agouti oocytes.

Although oocyte in vitro maturation (IVM) is routinely used for in vitro embryo production in mice and rats, its use in wild rodents remains unexplored. Evidence suggests that hormone and growth factor supplementation influence oocyte meiotic resumption. This study evaluated the synergistic effects of follicle-stimulating hormone (FSH) and epidermal growth factor (EGF) on the IVM and parthenogenetic development of red-rumped agouti oocytes. Initially, we evaluated the IVM rates, mature oocyte quality, oocyte morphometry, and early embryonic development during IVM in the presence of 10, 50, and 75 mIU/mL FSH. No differences among the FSH concentrations were observed for IVM rates, oocyte morphometry, cumulus cell expansion, and viability. Although oocytes matured with 50 mIU/mL FSH showed a higher rate of cumulus expansion index (CEI), only oocytes matured with 10 mIU/mL FSH resulted in morulae after chemical activation (7.9% ± 4.2%). Thus, 10 mIU/mL FSH was used for further experiments. We subsequently evaluated the synergistic effects of 10, 50, and 100 ng/mL EGF and 10 mIU/mL FSH on the same parameters. No differences among the groups were observed in IVM rates, oocyte morphometry, and cumulus viability. Nevertheless, FSH with 10 ng/mL EGF showed a CEI superior to that of the other groups. Furthermore, oocytes matured with FSH alone or with both FSH and 10 or 50 ng/mL EGF developed morulae after activation (5.8%-8.3%). In conclusion, oocytes matured with 10 mIU/mL FSH and 10 ng/mL EGF are recommended for use in red-rumped agouti oocyte IVM, as they positively influence embryonic development.

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.

Modulation of Alveolar Macrophage Activity by Eugenol Attenuates Cigarette-Smoke-Induced Acute Lung Injury in Mice.

This study investigates the role of eugenol (EUG) on CS-induced acute lung injury (ALI) and how this compound is able to modulate macrophage activity. C57BL/6 mice were exposed to 12 cigarettes/day/5days and treated 15 min/day/5days with EUG. Rat alveolar macrophages (RAMs) were exposed to CSE (5%) and treated with EUG. In vivo, EUG reduced morphological changes inflammatory cells, oxidative stress markers, while, in vitro, it induced balance in the oxidative stress and reduced the pro-inflammatory cytokine release while increasing the anti-inflammatory one. These results suggest that eugenol reduced CS-induced ALI and acted as a modulator of macrophage activity.

Extraembryonic membrane morphology in greater rheas (Rhea americana americana Linnaeus, 1758).

The greater rhea, Rhea americana, is a wild ratite of high scientific importance and significant and zootechnical value, especially considering the current development state of Brazilian poultry production, where research aimed at increasing the productivity of these animals has become extremely relevant. Studies concerning fetal attachments and embryonic development are paramount, as they can provide essential information concerning reproductive and nutritional animal management. However, a lack of information on greater rhea fetal morphology is noted. Therefore, the aim of the present study was to establish a standard model for fetal attachments in this species. Greater rhea eggs were incubated from 0 to 36 days, and macroscopic and microscopic embryonic attachment characterizations were performed. Histologically, all embryonic annexes exhibit germ layers, namely the ectoderm (outer layer), mesoderm (middle layer) and endoderm (inner layer). The findings indicate that greater rhea development patterns are similar to other birds.

Changes in Sperm Morphology, Morphometry, and Motility from the Epididymis to the Vas Deferens in Rheas (Rhea americana, Linnaeus, 1758).

The objective was to characterize morphological, morphometric, and ultrastructural changes in rhea spermatozoa between the epididymis and the vas deferens. Sperm samples were collected from the reproductive tracts of seven adult individuals and evaluated for sperm characteristics using brightfield microscopy as well as ultrastructural features using scanning electron microscopy (SM). Mean sperm count tended to increase in the vas deferens (378.0 ± 135.0 × 106) compared to the epididymis (201.0 ± 77.4 × 106). Percentages of motile sperm grew from 37.0 ± 4.9% in the epididymis to 58.5 ± 7.7% in the vas deferens. The proportion of normal spermatozoa was 75.6 ± 1.8% and most common defects were bent tails (9.7 ± 0.9%). However, these proportions were not different between epididymis and vas deferens. SM analysis revealed further features of rhea spermatozoa. Normal rhea spermatozoa were threadlike with an acrosome (0.95 ± 0.0 µm), head (7.53 ± 0.01 µm), midpiece (2.08 ± 0.01 µm), and tail (30.7 ± 0.06 µm). Lengths of sperm acrosome, head, midpiece, and tail were longer in the vas deferens compared to the epididymis. Our findings suggest that rhea spermatozoa undergo a maturation process during the passage from the epididymis to the vas deferens.

Antimicrobial activity and chemical profile of wood vinegar from eucalyptus (Eucalyptus urophylla x Eucalyptus grandis - clone I144) and bamboo (Bambusa vulgaris).

Microbial resistance to drugs is a public health problem; therefore, there is a search for alternatives to replace conventional products with natural agents. One of the potential antimicrobial agents is wood vinegar derived from the carbonization of lignocellulosic raw materials. The objectives of the present work were to evaluate the antibacterial and antifungal action of two kinds of wood vinegar (WV), one of Eucalyptus urograndis wood and another of Bambusa vulgaris biomass, and determine their chemical profile. The antimicrobial effect was assessed against Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enteritidis, Escherichia coli, Streptococcus agalactiae, and Candida albicans. The minimum inhibitory concentration and the minimum bactericidal and fungicidal concentrations were determined. Micrographs of the microorganisms before and after exposure to both kinds of wood vinegar were obtained by scanning electron microscopy. The chemical profile of the eucalyptus and bamboo vinegar was carried out by gas chromatography and mass spectrometry (GC/MS). Both types of WV presented significant antimicrobial activity, with the bamboo one having a higher efficiency. Both studied pyroligneous extracts seem promising for developing natural antimicrobials due to their efficiency against pathogens. GC/MS analyses demonstrated that the chemical profiles of both kinds of WV were similar but with some significant differences. The major component of the eucalyptus vinegar was furfural (17.2%), while the bamboo WV was phenol (15.3%). Several compounds in both WVs have proven antimicrobial activity, such as acetic acid, furfural, phenol, cresols, guaiacol, and xylenols. Together, they are the major in the chemical composition of the organic fraction of both WVs. Bamboo vinegar had a more expressive content of organic acids. Micrographs of microorganisms taken after exposure to both kinds of wood vinegar displayed several cell modifications. The potential of both types of wood vinegar as a basis for natural antimicrobial products seems feasible due to their proven effect on inhibiting the microorganisms' growth assessed in this experiment.

Heart morphology during the embryonic development of Podocnemis unifilis Trosquel 1948 (Testudines: Podocnemididae).

Cardiogenesis is similar in all vertebrates, but differences in the valvuloseptal morphogenesis among non-crocodilian reptiles, birds, and mammals are noted. The origin of mesenchymal structures such as valves that regulate the passage of blood and the formation of partial septa that prevent the complete mixing of oxygen-rich and low-oxygen blood present in adult chelonians are essential in the evolutionary understanding of complete septation, endothermy and malformations, even in mammals. In this context, this study analyzed the heart morphogenesis of Podocnemis unifilis (Testudines: Podocnemididae) from the 4th to the 60th day of incubation. We identified the tubular heart stage, folding of the cardiac tube and expansion of the atrial and ventricular compartments followed by atrial septation by the septum primum, ventricle septation by partial septa, outflow tract septation and the formation of bicuspid valves with cartilage differentiation at the base. The formation of the first atrial septum with the mesenchymal cap is noted during the development of the atrial septum, joining the atrioventricular cushion on the 17th day and completely dividing the atria. Small secondary perforations appeared in the mid-cranial part, observed up to the 45th day. Partial ventricle septation into the pulmonary, venous, and arterial subcompartments takes place by trabeculae carneae thickening and grouping on the 15th day. The outflow tract forms the aorticopulmonary and interaortic septa on the 16th day and the bicuspid valves, on the 20th day. Therefore, after the first 20 days, the heart exhibits a general anatomical conformation similar to that of adult turtles.

Influence of Intracellular Cryoprotectants on the Conservation of Dermal Somatic Tissues Derived from Antillean Manatees (Trichechus manatus manatus Linnaeus, 1758).

Cryopreservation of somatic tissue has been studied as a tool for the knowledge and conservation of endangered species, such as Antillean manatees. The use of vitrification protocols is an important step in the establishment of biological banks. To decrease the damage caused by this technique, a reduction in the concentration of cryoprotectants has been proposed. Therefore, we aimed to evaluate combinations and concentrations of intracellular cryoprotectants for the conservation of somatic tissues derived from Antillean manatees. Dulbecco's modified Eagle's medium, F-12 composed of 10% fetal bovine serum and 0.25 M sucrose, was supplemented with 3.0 M ethylene glycol (EG) plus 3.0 M dimethyl sulfoxide (DMSO), or 1.5 M EG plus 1.5 M DMSO or 3.0 M EG or 3.0 M DMSO, to produce four solutions for solid-surface vitrification. Noncryopreserved tissues were used as the controls. After warming, tissues derived from four Antillean manatees were evaluated for ultrastructure, histology, and in vitro culture. No differences were observed among the cryopreserved and noncryopreserved tissues in terms of ultrastructure. The dermis thickness of the cryopreserved fragments in solutions containing 3.0 M EG plus 3.0 M DMSO, 3.0 M EG, and 3.0 DMSO was similar to that of the control. Moreover, cryopreservation with 3.0 M EG plus 3.0 M DMSO maintained tissue proliferative capacity potential evaluated by quantification of nucleolar organizing regions. Nevertheless, none of the cryopreserved fragments were able to maintain the number of fibroblasts and the collagen percentage as compared with that of the noncryopreserved fragments. Also, none of the cryopreserved fragments in the different solutions were able to produce cells in vitro. In summary, even reducing the concentration of intracellular cryoprotectants as well as their association did not guarantee the maintenance of cells after in vitro culture. Further studies are needed to optimize the cryopreservation protocols in Antillean manatee somatic tissues.

Pronephros and mesonephros characterization during the embryonic development of the giant South American river turtle, Podocnemis expansa (Podocnemididae: Testudines).

This study aimed to describe pronephros and mesonephros morphology during the embryonic development of Podocnemis expansa. Eggs were collected on an artificial beach at Balbina, Amazonas, Brazil, during the entire incubation period (mean of 59 days). The kidney-gonad complex was processed using light microscopy and the mesonephros using transmission electron microscopy. The pronephros was present for the first time on stage 4, composed of external glomeruli devoid of a capsule, protruding into the coelomic cavity, and internally composed of a capillary network. The pronephros degenerated after development stage 15. The first sign of the appearance of the mesonephros occurred around stage 8, indicated by the early formation of renal corpuscles. The mesonephros comprised an renal corpuscles, neck segment, proximal tubule, intermediate segment, distal tubule, collector tubule, and collector duct. Ultrastructural analysis of the mesonephros brush border was done in the proximal tubule, and the presence of cells with structural characters indicative of secretory activity was detected in the juxtatubular region. Renal corpuscles and proximal tubules were the main components that underwent morphological alterations during mesonephros degeneration. The pronephros is a transient kidney, and the mesonephros became the functional embryonic kidney in P. expansa. Mesonephros degeneration occurs in the cranial-caudal direction, and histologically, the degeneration is identified by changes in the morphology of the renal corpuscle and proximal tubule. However, the mesonephros is still present after hatching.

Oropharyngeal cavity floor morphology in Eretmochelys imbricata (Testudines: Cheloniidae) hatchlings and evolutionary implications.

Morphological studies of the oropharyngeal cavity of chelonians have become an interesting tool in the understanding of evolutionary processes associated with feeding habits in aquatic animals and the transition from aquatic to terrestrial forms. In this context, the aim of the present study was to describe the oropharyngeal cavity floor morphology of hawksbill sea turtle (Eretmochelys imbricata) hatchlings. Ten dead hatchlings of undefined sex were obtained from nests hatched on the coast of the state of Rio Grande do Norte, Brazil. The heads of each specimen were fixed, dissected, and analyzed at the macroscopic and microscopic levels. The oropharyngeal cavity floor of the hawksbill sea turtle hatchlings is formed by the tongue, pharynx, floor muscles, and hyolingual skeleton, delimited in the rostral and lateral directions by a keratinized beak, called the rhamphotheca, and in the caudal region at the limit between the pharynx and the esophagus. The tongue muscles and the muscles that support the floor of the oral cavity comprise the following: m. hypoglossohyoideus, m. hypoglossoglossus, m. hyoglossus, m. genioglossus, m. constrictor laryngis, m. geniohyoideus pars lateralis, and m. intermandibularis. The oropharyngeal cavity floor mucosa is formed by keratinized stratified squamous epithelium and the lamina propria is formed by loose connective tissue. The floor mucosa is devoid of taste buds. We believe that the basic oropharyngeal cavity floor characteristics in hawksbill sea turtle hatchlings may comprise indications that these animals are plesiomorphic and that semiaquatic and terrestrial turtles may have undergone adaptations to feed out of water.