RESUMO

For the development of efficient management and conservation strategies for wild rodent species, understanding the aspects related to their reproduction, including the interaction of this physiological function with the environment, is essential. This study aimed to evaluate the impacts of weather changes derived from a semiarid region's dry and rainy seasons on the epididymal sperm characteristics of red-rumped agouti (Dasyprocta leporina), a histricognath rodent, still little studied, which inhabits the Brazilian Caatinga. The sperm from the epididymal cauda of 14 agoutis were collected, seven individuals per season (dry and rainy). Samples were evaluated for kinetic parameters, membrane structural and functional integrity, mitochondrial activity, morphology, and morphometry. The environmental variables were measured: maximum air temperature, relative humidity, wind speed, solar radiation, and the total rainfall for dry and rainy seasons were, respectively, 36.2 and 34.1 °C, 66.8 and 80.1%, 4.0 and 1.9 m/s, 527.3 and 441.8 W/m2, and 0.2 and 517.7 mm. There were strong correlations between some sperm parameters and environmental variables, mainly those related to the acquisition of sperm mobility. Sperm concentration and the number of sperm collected were higher in the dry (1028.7 sperm/mL × 106 and 1361.2 × 106 sperm) than in the rainy season (758.9 sperm × 106/mL and 714.6 sperm × 106). During the rainy season, there were fewer sperm defects, higher sperm metrics, and higher membrane structural integrity with mitochondrial activity. Regarding motility patterns, the increases during the rainy season stand out in total and progressive motility, VAP, VSL, VCL, and subpopulations of rapid sperm. In summary, our results suggest that the adverse climatic conditions of the dry season in the semiarid region, mainly high solar radiation and temperature, considerably impair the epididymal sperm quality of red-rumped agoutis. On the contrary, the largest amount of sperm was obtained during this season, probably due to compensatory and adaptive mechanisms of the species to enable its reproduction throughout the year.

Assuntos

RESUMO

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.

Assuntos

RESUMO

Establishing new somatic cell cultures has raised significant attention as an effective and convenient way to preserve genetic samples for different applications. Although many lines have been established in model animals, none derived from six-banded armadillo species is currently available. We report the successful isolation and characterization of fibroblasts from six-banded armadillos, evaluating the cell quality after extended culture and cryopreservation. Initially, we collected ear skin from five captive adult individuals and identified fibroblast lines by morphology, karyotyping, and immunophenotyping assays. The isolated fibroblasts were evaluated after several passages (fourth, seventh, and tenth passages) and cryopreservation by slow freezing. Cell morphology, viability, metabolism, proliferative activity, mitochondrial membrane potential, and apoptosis levels were analyzed. The skin explants had great adhesion, and cell outgrowth could be seen after 3-6 d. The cells were verified as fibroblasts at the fourth passage by vimentin expression and normal karyotype (2n = 58). The viability remained high (> 87%) and constant from the fourth to the tenth passage (p > 0.05). The passages did not change the cell morphology and metabolic and growth rates. Moreover, cryopreservation did not affect most evaluated parameters; post-thawed cells maintained their viability, growth, metabolism, and apoptosis levels. Nevertheless, cryopreservation increased mitochondrial membrane permeability and cell population doubling time compared to non-cryopreserved cells (p < 0.05). In summary, viable fibroblasts can be obtained from six-banded armadillo skin while conserving their quality as the number of passages increases and featuring few changes after cryopreservation.

Assuntos

RESUMO

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.

Assuntos

RESUMO

Semen preservation is a significant biotechnology used to safeguard the genetic material of birds, especially those with declining populations, through biobanking. However, there are limited reports on the successful chilling or cryopreservation of wild bird semen. In general, these techniques are not yet well-established for several species of wild birds and pose several challenges such as the need for bird handling and training, contamination of semen samples, low volume of semen collected, and inefficient preservation protocols. To address these challenges and improve post-thawing outcomes, new possibilities are being investigated, including alternative collection methods to traditional digital massage, the use of antioxidants and enzymes in the medium for chilling or freezing, storage methods using different straws from the usual pellet, and slower freezing rates. This review aims to discuss the various aspects of applying semen preservation in wild birds to create germplasm banks, highlighting the primary results obtained and the challenges that need to be addressed.

RESUMO

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.

RESUMO

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.

Assuntos

RESUMO

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.

RESUMO

The search for assisted reproduction techniques applied to the conservation and even the genetic improvement of wild species is becoming increasingly common. Regarding conservation of male gametes from wild animals, although current advances are focused on cryopreservation, the development of protocols for sperm refrigeration seems to be underrated, despite its various advantages and applications. Therefore, this review aims to highlight the importance of short-term conservation of sperm from wild mammals, report the development of state-of-the-art refrigeration protocols for both ejaculated and epididymal sperm, and evaluate the challenges and prospects of their application.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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)

Assuntos

RESUMO

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)

Assuntos

RESUMO

O tecido testicular contém células germinativas, que possuem potencial para se desenvolver em espermatozoides viáveis por meio do cultivo in vitro ou xenotransplante, sendo uma alternativa interessante a ser utilizada na formação de biobancos. Esta revisão compila as atualizações, desafios e perspectivas relacionadas às técnicas de criopreservação e cultivo de tecido testicular como estratégia para a conservação de espécies mamíferas. O tecido testicular pode ser obtido tanto de indivíduos adultos como pré púberes, seja após orquiectomia ou até mesmo após a sua morte. O tecido fragmentado pode ser criopreservado por congelação lenta ou rápida e por métodos de vitrificação. Os crioprotetores são indispensáveis durante a criopreservação e podem variar o tipo e concentração de acordo com a espécie. Com os avanços da criopreservação deste material, espermatozoides podem ser obtidos por transplante de fragmentos testiculares ou células germinativas isoladas em camundongos imunodeficientes. No entanto, a obtenção de espermatozoides no cultivo in vitro ainda é um desafio.(AU)

The testicular tissue contains germ cells, which have the potential to develop into viable spermatozoa through in vitro culture or xenotransplantation, being an interesting alternative to be used in the formation of biobanks. This review compiles updates, challenges and perspectives related to cryopreservation techniques and testicular tissue culture as a strategy for the conservation of mammalian species. Testicular tissue can be obtained from both adult and pre-pubertal individuals, either after orchiectomy or even after their death. Fragmented tissue can be cryopreserved by slow or fast freezing and by vitrification methods. Cryoprotectants are indispensable during cryopreservation and may vary in type and concentration according to the species. With advances in cryopreservation of this material, spermatozoa can be obtained by transplanting testicular fragments or isolated germ cells into immunodeficient mice. However, obtaining spermatozoa in in vitro culture is still a challenge.(AU)

Assuntos

RESUMO

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)

Assuntos

RESUMO

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.

RESUMO

We studied the sperm membrane functionality through the epididymal transit by comparing different hypoosmotic solutions and verifying possible associations among osmotic response and functional parameters of sperm in red-rumped agouti (Dasyprocta leporina). For this purpose, epididymal sperm from six sexually mature male agoutis were collected via flotation. Then, analyses of sperm parameters and hypoosmotic swelling test using different hypoosmotic solutions (0, 50 and 200 mOsm/L) in different regions of the epididymis (caput, corpus and cauda) were performed. There was an increase (p < .05) in the values for sperm concentration, the total number of sperm recovered, total and progressive motility, average path velocity, straight-line velocity, curvilinear velocity, and rapid and medium subpopulations following the caput-corpus-cauda direction. Regardless of the hypoosmotic solution, the agouti sperm membrane presented similar functional integrity in all the epididymal regions. Moreover, the highest (p < .05) osmotic responses were reached with the use of 50 mOsm/L solution in comparison to 0 and 200 mOsm/L for all the regions. Significant correlations among osmotic response and some sperm kinetic parameters were observed, especially in epididymal caput, while no correlations were found in the region of the cauda. In summary, red-rumped agouti sperm present similar membrane functionality during epididymal transit, but there are evident correlations among such functionality and sperm kinetic parameters, especially in the caput region. Moreover, we indicate the use of a 50 mOsm/L hypoosmotic solution for the analysis of this parameter through the hypoosmotic swelling test.

Assuntos

RESUMO

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

RESUMO

Biological Resource Banks (BRB) or Genetic Resource Banks (GRB) are critical tools for the conservation of animal biodiversity. According to the International Union for Conservation of Nature, more than 38,500 species are threatened with extinction, out of a total of 138,300 surveyed species. These banks are repositories of biological samples and data recovered and preserved for the long term by zoos, universities, research centers and other conservation organizations. In recent years, BRB have increasingly included ovarian and testicular tissues as additional options to rescue and propagate wild species, especially those at risk of extinction. After in vitro culture or grafting, gonadal tissues are potential sources of matured gametes that can be used for Assisted Reproduction Technologies while informing about gametogenesis or mechanisms involved in infertility. It therefore is crucial to properly recover, cryopreserve, and culture these tissues using species-specific protocols. Developing BRBs is currently one of the strategies to preserve species from the Caatinga biome - an exclusively Brazilian biome with a rich wild fauna that suffers from anthropogenic activities. Among wild species from this biome, studies have been primarily conducted in collared peccaries, agoutis, cavies, and armadillos to preserve their ovarian and testicular tissues. Additionally, domestic species such as the domestic cat and donkeys have been proposed as models for wild species that are phylogenetically close. This review addresses the main technical aspects involved in obtaining BRB derived from gonadal tissues in some wild species of the Caatinga biome. It reports recent advances and perspectives to use these biological materials for wildlife conservation.

RESUMO

This review was designed to summarize the most important information around seminal plasma composition and discuss its impact on the freezability of wild mammal semen samples. Seminal plasma is made up of various biochemical constituents, including ions, lipids, proteins, enzymes, and sugars, which vary between species in response to the presence and size of any relevant accessory glands. The biochemical constituents of seminal plasma may change as a result of age, individual variability, and seasonality. These constituents are responsible for supporting different functions in sperm cells, contributing to motility, acrosomal reaction, and fertilization events. A detailed understanding of seminal plasma biochemistry may help to optimize semen freezing protocols, enabling the dynamic alteration in diluents to allow for increased sperm viability rates after thawing.

Assuntos

RESUMO

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.(AU)

Assuntos