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.

Cryopreservation efficiency of red-rumped agouti (Dasyprocta leporina) sperm obtained from different origins through epididymal retrograde flushing or electroejaculation.

This study investigated whether the origin of sperm (epididymal vs. ejaculate) affects the cryopreservation efficiency in agouti (Dasyprocta leporina). Five sexually mature agoutis underwent electroejaculation, resulting in obtaining four semen samples. After 15 days, the same animals were euthanized, and through retrograde flushing, sperm samples were obtained from the epididymis tails. In both collection methods, samples were evaluated for sperm parameters (sperm concentration, motility, vigor, membrane integrity, osmotic response, and morphology). Then, samples were diluted in ACP 109c, added with 20% egg yolk, and a final concentration of 6% glycerol. Finally, the samples were packaged in 0.25 mL straws and frozen in liquid nitrogen. After one week, samples were thawed and evaluated in the same way as fresh samples, with the addition of membrane integrity analysis using fluorescent probes (C-FDA/PI) and computerized analysis (CASA). Immediately after obtaining the sperm, samples obtained directly from the epididymis presented higher values (P ≤ 0.05) than those obtained by electroejaculation concerning the parameters of volume, sperm concentration, and total number of sperm (1,398.25 ± 206.0 x106 and 184.5 ± 78.0 x106 sperm). On the other hand, in the classical evaluation of the other sperm parameters and the computerized analysis (CASA) after thawing, such as total motility, no statistical differences were observed between sperm from both origins (ejaculate: 16.7 ± 8.2% and epididymal: 24.8 ± 12.0%, P > 0.05). This demonstrates the possibility of direct application of the cryopreservation protocol for agouti (D. leporina) sperm obtained via the epididymis or ejaculate.

Africanized honeybee (Apis mellifera) semen freezing using Tris-based and Collins extenders.

This study is aimed at evaluating the effect of different extenders on the cryopreservation of semen from Africanized honeybees (A. mellifera). Semen from honeybee drones from 10 different colonies was obtained by endophallus exposure technique and immediately evaluated for motility, viability using fluorescent probes, functional membrane integrity using the water test, and morphology. Samples from each colony were divided in three aliquots and subjected to a dilution ratio of 12:1 (diluent: semen) using Tris, Tris + egg yolk (Tris+EY), and Collins extender. Samples were cryopreserved and stored in liquid nitrogen for one week and then rewarmed and reevaluated. Immediate dilution provoked no significant effect on sperm motility and functional membrane integrity, regardless of the extender used; however, the greatest values (P < 0.05) for normal sperm morphology were found at the use of isolate Tris (69.3 ± 1.9%). After thawing, there were no significant differences among extenders with relation to the preservation of sperm motility, viability, and functional membrane integrity, but the Tris extender provided the highest post-thawing values (P < 0.05) for sperm normal morphology (49.2 ± 4.9%) while the Collins extender provoked the highest amounts (P < 0.05) of curled tail defects (67.5 ± 3.2%). Moreover, the Tris was the only extender at preserving the proportion of normal sperm after thawing similar to what was verified for fresh samples. In summary, we suggest the use of a Tris-based extender for the cryopreservation of Africanized honeybee semen.

Short-term preservation of canine sperm-binding ability and other metrics using the INRA-96 in comparison to Tris-egg yolk extender.

The use of assisted reproductive techniques, such as chilled semen, contributes to the maintenance and genetic improvement of canine breeding. The INRA-96 extender is a commercially available, chemically defined medium that was initially developed for the preservation of equine semen and exhibits preservation potential in the canine species. This research aims to evaluate the INRA-96 extender as an alternative for the short-term preservation of canine semen in terms of sperm quality parameters such as motility and kinetic parameters, integrity and functionality of the plasma membrane in fresh and chilled-rewarmed samples, as well as the sperm-binding ability using the perivitelline membrane of the chicken egg as an indicator of the fertilizing capacity of the preserved semen. A total of 18 ejaculates from 9 French bulldogs (two ejaculates per dog) were collected and divided into two aliquots that were diluted in Tris-egg yolk 20% (control) or INRA-96 to a final concentration of 100 × 106 sperm/mL. Samples were refrigerated in a biological incubator at 5°C and evaluated at 0, 24 and 48 h time points. Comparing the two treatments after 48 h of refrigeration, both extenders showed similar values (p < .5) for the majority of kinetic parameters, with the INRA-96 group promoting a total motility of 88.1 ± 2.9%. In addition, the morphology, integrity and functionality of the plasma membrane were preserved above 70% in this group. Dilution with INRA-96 also provided a significantly higher amount of sperm bound (256.2 ± 21.1) to the perivitelline membrane of the egg yolk compared to the sperm-binding rates (p < .05) achieved at the use of Tris-egg yolk (215.2 ± 21 bound spermatozoa) at 48 h. Our study proved similar functional properties of dog sperm cells treated with INRA-96 in comparison to commonly used home-made Tris-based extender during short-time storage.

In vitro culture of red-rumped agouti preantral follicles enclosed in fresh and vitrified ovarian tissues using TCM199 plus different pFSH concentrations.

Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.

Development of assays for the characterization of sperm motility parameters, viability, and membrane integrity in the epididymis and vas deferens of the greater rhea (Rhea americana).

The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean ± SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 ± 2.5%. The bromophenol blue staining technique revealed that 64.6 ± 5.2% sperm were viable, while that proportion was 72.1 ± 2.5% using eosin-nigrosine. An average of 77.6 ± 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 ± 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.

Biologia reprodutiva das abelhas africanizadas (Apis melífera) e biotecnologias aplicadas à sua conservação / Reproductive biology of Africanized bees (Apis mellifera) and biotechnologies applied to their conservation

A biodiversidade das abelhas africanizadas (Apis melífera) encontra-se sob grande ameaça e, neste cenário as investigações acerca da biologia reprodutiva, alguns aspectos ainda incipientes, bem como métodos aprimorados para a inseminação instrumental e criopreservação de gametas podem ser ferramentas preciosas para a conservação in situ e ex situ de subespécies e ecótipos. O entendimento e adoção de ferramentas como estas mencionadas, podem auxiliar na seleção de características de interesse para os criadores, assim como, nos esforços para a conservação de populações de abelhas ameaçadas.(AU)

The biodiversity of Africanized bees (Apis mellifera) is under great threat and, in this scenario, investigations about reproductive biology, some aspects are still incipient, as well as improved methods for instrumental insemination and cryopreservation of gametes can be precious tools for ex situ and in conservation. situ of subspecies and ecotypes can be contemplated. The understanding and adoption of tools such as those mentioned can help in the selection of characteristics of interest to breeders, as well as those committed to the conservation of endangered bee populations.(AU)

Recuperação, caracterização e criopreservação de espermatozoides epididimários de animais silvestres do bioma Caatinga / Recovery, characterization and cryopreservation of epididymal sperm from wild animals of the Caatinga biome

A Caatinga, bioma exclusivamente brasileiro, abriga grande diversidade biológica, porém sofre com graves ameaças ambientais. Por isso, é iminente a necessidade de desenvolvimento de técnicas voltadas para a conservação dos animais que nela habitam, bem como se seu germoplasma. Quando do súbito óbito de um animal biologicamente valioso, a recuperação de espermatozoides epididimários pode se apresentar como a única possibilidade para salvaguardar gametas. Ainda, esta biotécnicas configura-se em uma ferramenta possível de ser utilizada quando não há outra forma de se coletar o sêmen em determinada espécie. Os espermatozoides coletados podem ser armazenados por meio da criopreservação, e posteriormente utilizados em outras biotecnologias. Neste sentido, esta revisão tem como objetivo apresentar os aspectos da recuperação, caracterização e criopreservação de espermatozoides epididimários em animais silvestres com principal foco em espécies do bioma Caatinga, como os preás, cutias, catetos e emas.(AU)

The Caatinga, an exclusively Brazilian biome, is home to great biological diversity, but suffers from serious environmental threats. Therefore, there is an imminent need to develop techniques aimed at the conservation of the animals that inhabit it, as well as their germplasm. When the sudden death of a biologically valuable animal, the recovery of epididymal spermatozoa may present itself as the only possibility to safeguard gametes. Still, this biotechnique is a tool that can be used when there is no other way to collect semen in each species. Collected spermatozoa can be stored through cryopreservation, and later used in other biotechnologies. In this sense, this review aims to present aspects of recovery, characterization, and cryopreservation of epididymal spermatozoa in wild animals with a focus on species from the Caatinga biome, such as cavies, agoutis, collared peccary, and rheas.(AU)

In vitro culture of red-rumped agouti preantral follicles enclosed in fresh and vitrified ovarian tissues using TCM199 plus different pFSH concentrations

Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.(AU)

Microbiological load and preantral follicle preservation using different systems for ovarian tissue vitrification in the red-rumped agouti.

We evaluated the effect of open and closed systems used for ovarian tissue vitrification on the microbiological load and preservation of preantral follicles (PAFs) in the red-rumped agoutis. The ovaries from eight females were recovered and fragmented, with four cortexes fragments immediately fixed and evaluated (fresh group). The other fragments were processed for the solid-surface vitrification method (SSV) or an ovarian tissue cryosystem (OTC) using fetal calf serum, ethylene glycol, and sucrose as cryoprotectants, stored for two weeks, and rewarmed. Subsequently, fragments were subjected to a 24-h in vitro culture and assessed for microbiological load, PAF morphology, and DNA integrity. There was no fungal contamination; however, the vitrified samples from two individuals showed bacterial contamination of 79 200 colony forming units per milliliter (CFU)/mL for SSV and 3120 CFU/mL for OTC. From those samples, a total of eight different types of bacterial colonies were isolated and identified as coagulase-negative Staphylococci and Gram-positive bacilli. Regarding PAF morphology, both systems provided adequate preservation, with values higher than 70% normal follicles observed before and after culture. The TUNEL assay revealed that both SSV (52.39%) and OTC (41.67%) could preserve DNA integrity after vitrification and after 24 h of culture. In summary, both open and closed systems were equally efficient in preserving agouti ovarian tissues, especially concerning the preantral follicle morphology and DNA integrity; however, the OTC seems to provide a less adequate environment for bacterial proliferation.

Impacto de antimicrobianos nos parâmetros cinéticos de espermatozoides criopreservados de catetos / Impact of antimicrobials on the kinetic parameters of collared peccary frozen sperm

The objective was to verify the impact of the addition of antimicrobials on the kinetic parameters of sperm in the cryopreserved semen of collared peccaries. Ejaculates from 10 adult male, obtained by electroejaculation, were used. The samples had their kinetic parameters evaluated by computer analysis (CASA). Subsequently, they were cryopreserved in Tris plus egg yolk (20%) and glycerol (3%), whether or not (control) added gentamicin (70µg/mL) or the combination penicillin (1000 IU/mL) and streptomycin (1mgE/mL) (P+E). After one week, the samples were thawed and evaluated similarly to fresh semen. In fresh semen, total motility of 95.3±0.8% and 72.1±3.5% progressive motility were observed. After thawing, there were no differences between treatments, except for the cross-beat frequency (BCF) parameter, which was negatively influenced by P+E, in relation to fresh semen (p <0.05). In conclusion, it is suggested the use of gentamicin as an antimicrobial for the cryopreservation of semen from peccaries.

Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation.

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions.

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca).

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ±â€¯7.7%) and membrane functionality (60.5 ±â€¯4.2%) and higher mitochondrial activity (21.5 ±â€¯3.7%) than those preserved in ACP-117c (20.9 ±â€¯5.4% motile sperm; 47.1 ±â€¯2.5% functional membrane; 11.8 ±â€¯1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ±â€¯3.3%) in comparison to samples diluted in ACP-117c (18.6 ±â€¯1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.(AU)

Conservação da capacidade ligante de espermatozoides caninos refrigerados em diluente tris-gema / Conservation of the binding ability of chilled canine sperm in Tris-egg yolk extender

The aim was to evaluate the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for up to 48 h. The semen of four dogs was collected by digital manipulation and evaluated. The sperm fractions were diluted in Tris-egg yolk and stored at 4 °C. The sperm parameters of motility, vigor, morphology and osmotic response were evaluated in fresh semen, immediately after dilution (0h), at 24h and at48h. The sperm binding test using the chicken egg's perivitelline membrane was performed on fresh and 48 h-chilled samples. As a result, fresh semen showed motility of 99.2+/-0.8%, vigor 5+/-0,1 with 84.2+/-1.8% morphologically normal sperm, 95.0+/-0.8% osmotic response and 302.3+/-27.0 membrane-bound sperm. After refrigeration for 48 h, a significant reduction (p<0.05) in the sperm parameters was observed however, values were within the ideal range for the use of semen, such as 90.8+/-1.5% mobile sperm, with vigor 4.3+/-0.2, normal morphology of 71.5+/-1.9%, osmotic response of 77.0+/-4.1%, and 205.5+/-27.7 bound sperm. In conclusion, the hen egg perivitelline membrane binding test proved the efficiency of the Tris-egg yolk extender in conserving the binding capability of canine sperm chilled for 48h.

Combination of intracellular cryoprotectants preserves the structure and the cells proliferative capacity potential of adult collared peccary testicular tissue subjected to solid surface vitrification.

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.

Antioxidant effects of the essential oil of Syzygium aromaticum on bovine epididymal spermatozoa.

Focusing on its application in reproductive biotechnology, we evaluated the effects of the essential oil of Syzygium aromaticum (EOSA) on bovine epididymal sperm quality variables, including morphology, membrane functional integrity, membrane structural integrity, mitochondrial activity, metabolic activity, motility and oxidative stress by reactive oxygen species (ROS) levels. Bovine spermatozoa from eight males were incubated into the following groups: EOSA0 (without EOSA), EOSA10 (10 µg/ml of EOSA), EOSA15 (15 µg/ml of EOSA) and EOSA20 (20 µg/ml of EOSA); the incubation time with and without the EOSA was 1 or 6 hr. None of the sperm quality variables presented difference among the EOSA concentrations. However, the incubation time had a significant effect on the membrane functional integrity, membrane structural integrity, mitochondrial activity, progressive motility and some kinetic parameters. The effect of interaction among EOSA and incubation time was significant only on ROS levels. Spermatozoa incubated in the presence of 15 µg/ml of the EOSA for 1 hr had significantly reduced ROS levels compared with all other groups in the same time. In conclusion, the EOSA at a concentration of 15 µg/ml has antioxidant effects and protects bovine epididymal spermatozoa; hence, the EOSA may potentially be used in the field of reproductive biotechnology.

Vitrification of collared peccary ovarian tissue using open or closed systems and different intracellular cryoprotectants.

This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions - NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ±â€¯8.6%); however, the SSV was only efficient with DMSO alone (63.9 ±â€¯7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ±â€¯2.9% viable cells with 2.0 ±â€¯0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.

Establishment of a protocol for the isolation of ovarian preantral follicles derived from collared peccaries (Pecari tajacu).

We compare the efficiency of mechanical or enzymatic methods, and their combination, for the isolation of ovarian preantral follicles (PFs) from collared peccaries. The ovaries from six females were subjected to the different methods investigated here. For the enzymatic method, ovary fragments were exposed to collagenase type IV in TCM-HEPES medium; the mechanical procedure was based on ovarian cortex dissociation by using a scalpel blade. The residual solution obtained after the mechanical isolation was subjected to the enzymatic procedure. The number of isolated PFs was quantified and classified as primordial, primary, or secondary; their viability was assessed using trypan blue dye assay. To confirm the results, PFs derived from the most efficient method were evaluated for integrity using scanning electron microscopy (SEM) and subjected to a 24 h in vitro culture for subsequent evaluation of viability by using fluorescent probes. A higher number of PFs (P < 0.05) was obtained from the enzymatic method (961.7 ± 132.9) in comparison with the mechanical method (434.3 ± 88.9), but no difference was observed between the two methods and their combination (743.2 ± 92.8). The trypan blue assay showed that the enzymatic method (98.7 ± 0.6%) provided the highest percentage of viable follicles (P < 0.05). Furthermore, SEM confirmed the ultrastructural integrity of the surface architecture of peccary PFs isolated by the enzymatic procedure; epifluorescence microscopy was used to confirm their viability (86.0%). In conclusion, we suggest that the enzymatic method investigated here is useful for the isolation of viable ovarian PFs from collared peccaries.