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2.
Biopreserv Biobank ; 18(3): 235-243, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32282240

RESUMO

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.

3.
PLoS One ; 15(2): e0221843, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045413

RESUMO

GPS collars have revolutionized the field of animal ecology, providing detailed information on animal movement and the habitats necessary for species survival. GPS collars also have the potential to cause adverse effects ranging from mild irritation to severe tissue damage, reduced fitness, and death. The impact of GPS collars on the behavior, stress, or activity, however, have rarely been tested on study species prior to release. The objective of our study was to provide a comprehensive assessment of the short-term effects of GPS collars fitted on scimitar-horned oryx (Oryx dammah), an extinct-in-the-wild antelope once widely distributed across Sahelian grasslands in North Africa. We conducted behavioral observations, assessed fecal glucocorticoid metabolites (FGM), and evaluated high-resolution data from tri-axial accelerometers. Using a series of datasets and methodologies, we illustrate clear but short-term effects to animals fitted with GPS collars from two separate manufacturers (Advanced Telemetry Systems-G2110E; Vectronic Aerospace-Vertex Plus). Behavioral observations highlighted a significant increase in the amount of headshaking from pre-treatment levels, returning below baseline levels during the post-treatment period (>3 days post-collaring). Similarly, FGM concentrations increased after GPS collars were fitted on animals but returned to pre-collaring levels within 5 days of collaring. Lastly, tri-axial accelerometers, collecting data at eight positions per second, indicated a > 480 percent increase in the amount of hourly headshaking immediately after collaring. This post-collaring increase in headshaking was estimated to decline in magnitude within 4 hours after GPS collar fitting. These effects constitute a handling and/or habituation response (model dependent), with animals showing short-term responses in activity, behavior, and stress that dissipated within several hours to several days of being fitted with GPS collars. Importantly, none of our analyses indicated any long-term effects that would have more pressing animal welfare concerns.


Assuntos
Antílopes , Sistemas de Informação Geográfica , Dispositivos Eletrônicos Vestíveis/efeitos adversos , África do Norte , Animais , Animais Selvagens , Comportamento Animal , Espécies em Perigo de Extinção , Cabeça , Movimento , Estresse Psicológico , Fatores de Tempo
4.
Theriogenology ; 150: 360-373, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32102745

RESUMO

Conservation strategies in natural habitats as well as in breeding centers are necessary for maintaining and reinforcing viable populations of wild felids. Among the fundamental knowledge that is required for conservation breeding, a solid understanding of reproductive biology is critical for improving natural breeding and enhance genetic diversity. Additionally, it offers the opportunity to develop assisted reproductive technologies (ARTs) in threatened and endangered species. Conservation breeding and reproductive biotechnologies of wild felids have advanced in the past decade. It has been clearly shown that female felids have species and individual patterns of reproductive cycles and respond differently to exogenous hormones. In males, several species still have poor semen quality often due to the loss of genetic diversity in small populations. To overcome the challenges of natural breeding (incompatibility between individuals or suboptimal environment) and mitigate inbreeding, artificial insemination, embryo production and embryo transfer have been further developed in 24 wild cat species. Major factors limiting ART success are inconsistent responses to ovarian stimulation, variable quality of gametes and embryos, and preparation of recipient females. Additional approaches including stem cell technologies have been explored for future medical applications. However, there still is a critical need for better knowledge of feline reproductive biology and improvement of ARTs efficiency to increase the genetic diversity and create sustainable populations of wild felids.

5.
Cryobiology ; 92: 53-61, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704199

RESUMO

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.

6.
PLoS One ; 14(12): e0225440, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800613

RESUMO

Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues.


Assuntos
Dessecação/métodos , Ovário/citologia , Preservação de Tecido/métodos , Animais , Gatos , Sobrevivência Celular , DNA/química , DNA/normas , Feminino , Micro-Ondas , Ovário/metabolismo , RNA Mensageiro/química , RNA Mensageiro/normas , Temperatura
7.
Cryobiology ; 91: 53-60, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31678072

RESUMO

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.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Testículo/citologia , Animais , Artiodáctilos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/química , Feminino , Masculino , Vitrificação
8.
Mol Reprod Dev ; 86(12): 1822-1831, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31549479

RESUMO

Understanding cellular and molecular damages in oocytes during exposure to extreme conditions is essential to optimize long-term fertility preservation approaches. Using the domestic cat (Felis catus) model, we are developing drying techniques for oocytes' germinal vesicles (GVs) as a more economical alternative to cryopreservation. The objective of the study was to characterize the influence of desiccation on nuclear envelope conformation, chromatin configuration, and the relative fluorescent intensities of histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 9 (H3K9me3) compared to vitrification. Results showed that higher proportions of dried/rehydrated GVs maintained normal nuclear envelope conformation and chromatin configuration than vitrified/warmed counterparts. Both preservation methods had a similar influence on epigenetic patterns, lowering H3K4me3 intensity to under 40% while maintaining H3K9me3 levels. Further analysis revealed that the decrease of H3K4me3 intensity mainly occurred during microwave dehydration and subsequent rehydration, whereas sample processing (permeabilization and trehalose exposure) or storage did not significantly affect the epigenetic marker. Moreover, rehydration either directly or stepwise with trehalose solutions did not influence the outcome. This is the first report demonstrating that the incidence of GV damages is lower after desiccation/rehydration than vitrification/warming.

9.
PeerJ ; 7: e7540, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31497402

RESUMO

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies.

10.
Adv Exp Med Biol ; 1200: 1-10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471792

RESUMO

The previous edition of this book mainly provided a snapshot of the state of the art in terms of species-specific reproductive biology and emerging technologies. The influence of environmental changes on reproductive fitness was introduced but not fully explored. The objectives of this second edition were to (1) emphasize the need for holistic and global efforts to understand and sustain reproduction in a constantly changing environment and (2) provide more knowledge in the reproductive physiology of different taxa. The first section of the book is dedicated to survival and adaptation of species in a changing environment (including chapters on environmental impacts in different taxa, as well as the role of microbiomes). The second section focuses on progress in understanding, assisting or even suppressing reproduction in wild species, keeping in mind the influence of environmental factors as well. It contains chapters from the previous edition that were updated (reproduction in elephants, koalas, marsupials, amphibians, and corals), new chapters on species such as sharks and rays, and contributions about the increasing role of reproductive manipulations, such as assisted reproduction and contraception. While the present book emphasizes the overarching issue of environmental impacts on reproduction (resulting in infertility, subfecundity, or fitness), it also highlights the challenges of maintaining wild species in captivity, including those associated with ensuring good welfare. Captive environments can influence reproduction in a multitude of ways, some unexpected, such as the selection of unwanted genetic traits, an essential dimension to be considered to ensure the success of conservation breeding programs. Lastly, new approaches, such as the use of allostatic load indexes and reproductive microbiome analyses also will be closely examined for the first time in rare and endangered species to address conservation issues.


Assuntos
Conservação dos Recursos Naturais , Espécies em Perigo de Extinção , Reprodução , Animais , Cruzamento
11.
Adv Exp Med Biol ; 1200: 225-240, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471799

RESUMO

Communities of microbes have coevolved in animal organisms and are found in almost every part of the body. Compositions of those communities (microbiota) as well as their genomes and genes (microbiomes) are critical for functional regulations of the body organ systems-the digestive or 'gut' microbiome being the most described so far. Based on extensive research in humans, microbiomes in the reproductive tract may play a role in reproductive functions and pregnancy. However, in wild animal species, those microbiomes have been poorly studied, and as a result, little is known about their involvement in fertility or parental/offspring health. This emerging research area is highly relevant to conservation biology from captive breeding management to successful reintroduction or maintenance of wild populations. The objective of this chapter is to review current knowledge about reproductive microbiomes in healthy wild animal species. While recognizing the current technical limits of microbial identification in all animal species, we also explore the link between microbial communities (within female or male reproductive systems) and fertility, from conception to birth outcome. In addition, it is critical to understanding how reproductive microbiomes are affected by environmental factors (including captivity, contact with other individuals, or changes in the ecosystem) to optimize conservation efforts. Thus, reproductive microbiomes represent a novel dimension in conservation biology that will likely gain importance in the future.


Assuntos
Animais Selvagens , Conservação dos Recursos Naturais , Microbiota , Reprodução , Animais , Feminino , Masculino , Gravidez
12.
Adv Exp Med Biol ; 1200: 545-550, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471809

RESUMO

While many of the traditional scientific disciplines have developed over centuries, animal conservation is a relative newcomer. It relies on multiple specialties with different levels of expertise that, eventually, generate vast amounts of data. More specifically, conservation physiology is an emerging area that can be defined as 'an integrative scientific discipline applying physiological concepts, tools, and knowledge to characterizing biological diversity and its ecological implications; understanding and predicting how organisms, populations, and ecosystems respond to environmental change and stressors; and solving conservation problems across the broad range of taxa, including microbes, plants, and animals' (Cooke et al. 2013). Reproductive biology is more focused, given that it mainly deals with the physiology underlying the production of gametes, embryos, and offspring, and the many associated processes that control these events. However, it is integrated into the different components of conservation physiology. In bringing together the various contributors for this book, the editors' purpose was to provide readers with a new perspective about the complexity behind reproduction and the role it plays in species conservation. Chapters highlight the diversity of reproductive mechanisms across taxa, and provide insight into how they may have evolved, and likely will continue to evolve in a changing environment. To conservation physiologists, the hope is that this information will be applied to sustain populations in both natural habitats and managed facilities. Ultimately, a major goal is to forecast and mitigate negative impacts of environmental change or anthropogenic pressures on animal fitness, which will only follow once we have acquired a solid understanding of reproductive processes.


Assuntos
Biodiversidade , Conservação dos Recursos Naturais , Ecossistema , Reprodução , Animais
13.
Anim Reprod Sci ; 208: 106112, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31405481

RESUMO

The establishment of protocols for the control of the ovarian function of collared peccaries is recommended for the development of assisted reproductive techniques. The goals were to (1) compare a gonadotropin combination with prostaglandin analogue to synchronize timing of onset of estrus among animals, and (2) elucidate the effects of the most desirable protocol for performing an artificial insemination study and macroscopic evaluation of the ovaries. Three of five females treated with a double administration of 120 µg prostaglandin (cloprostenol) at a 9-day interval expressed symptoms of estrus 9 days after the second injection. One female presented estrus after 6 days, whereas other did not respond to the treatment. All females (5/5) treated with a single dose containing 400 IU eCG and 200 IU hCG manifested estrus 6 days after the hormone injection. In a second experiment, ten females that were estrous synchronized using eCG/hCG, were artificially inseminated with fresh semen and monitored for pregnancy every 30 days. Although there was no detection of fetuses by ultrasonic examination, seven females (7/10) had greater than basal progesterone values for 60 days after the treatments were imposed. Ovaries from two females treated with eCG/hCG were collected 6 days post-injection. There was confirmation of an ovarian stimulation as a result of the presence of 88 and 25 antral follicles, as well as three and eight hemorrhagic structures in ovaries of each female, respectively. It, therefore, is proposed that eCG/hCG can be used as an effective treatment for estrous synchronization in collared peccaries.


Assuntos
Artiodáctilos/fisiologia , Gonadotropina Coriônica/farmacologia , Sincronização do Estro/métodos , Animais , Gonadotropina Coriônica/administração & dosagem , Cloprostenol/farmacologia , Relação Dose-Resposta a Droga , Feminino
14.
Mol Reprod Dev ; 86(11): 1671-1681, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31429169

RESUMO

Incomplete transgene-silencing remains a challenge in the generation of induced pluripotent stem cells (iPSC) in felids-a critical family in biomedical and biodiversity conservation science. In this study doxycycline-inducible transgenes (NANOG, POU5F1, SOX2, KLF4, and cMYC) were used to reprogram cat fetal fibroblasts with the objective of obtaining iPSC with fully silenced transgenes. Colony formation was slower (14 vs. 8 days) and at lower efficiency than mouse embryonic fibroblasts (0.002% vs. 0.02% of seeded cells). Alkaline-phosphatase positive colonies were grown on feeder cells plus LIF and GSK3, MEK, and ROCK inhibitors. Cells could be passaged singly and transgene expression was silenced at passage 3 (P3) after doxycycline removal at P2. NANOG, POU5F1, and SOX2 were expressed at P3, P6, and P10, although at lower immunostaining intensities than in cat inner cell masses (ICM). Transcripts related to pluripotency (NANOG, POU5F1, SOX2, KLF4, cMYC, and REX1) and differentiation (FGF5, TBXT, GATA6, SOX17, FOXF1, PAX6, and SOX1) were assessed by a reverse transcription-quantitative polymerase chain reaction in iPSC and embryoid bodies. The immunostaining patterns, relatively low levels of NANOG and REX1 in comparison to ICM along with the expression of TBXT (mesoderm) suggested that cells were a mix of reprogrammed pluripotent and differentiating cells.

15.
Biol Reprod ; 101(5): 906-915, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31359037

RESUMO

Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial-blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

16.
Theriogenology ; 138: 39-46, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31284220

RESUMO

Control of ovarian function in cheetahs is sub-optimal, which currently limits the integration of assisted reproductive techniques into the genetic management of that endangered species. The objective of this study was to determine the effect of preemptive progestin treatment on the quality of ovarian responses after exogenous gonadotropin stimulation in cheetahs. Adult females received either 1) 200 IU equine chorionic gonadotropin (eCG) followed with 3,000 IU porcine luteinizing hormone (pLH) (intramuscular route) (n = 5; control group) or 2) similar eCG/pLH administration preceded by a 7-day treatment with oral progestin (0.1 mg/kg altrenogest; ALT group; n = 7). At 42 h post-pLH administration, a series of metrics was assessed via laparoscopy (number of follicles ≥ 2 mm, number of corpora lutea, oviduct and uterine cornua diameter and overall vascularization). Concentrations of fecal estradiol, progesterone and glucocorticoid metabolites (FEM, FPM, and FGM, respectively) were measured by enzyme immunoassay for 3 wk before ALT treatment (Period 1), 7 d during ovarian suppression period (Period 2), throughout eCG/LH treatment and laparoscopy (Period 3), and 6 wk following laparoscopy (Period 4). Overall, nine out of 12 cheetahs (4/5 in control and 5/7 ALT group) had freshly-formed corpora lutea at the time of laparoscopy. Mean follicle and corpora lutea numbers in the control versus ALT group were not different (P > 0.05). Overall measurements and vascularization scores also did not differ (P > 0.05) among groups. FEM average concentrations increased (P ≤ 0.05) in response to eCG for the ALT-treated females between Periods 2 and 3 and were sustained during Period 4. However, FEM average concentrations did not vary (P > 0.05) for control females throughout Periods 1-4. Post-ovulatory FPM average concentrations (Period 4) did not differ (P > 0.05) between the ALT-treated females and controls. FPM average concentration from both groups increased in Period 4 compared to Periods 1-3 (P ≤ 0.05). Females receiving the ALT treatment also had lower (P ≤ 0.05) FGM metabolite average concentrations than control females during ovarian suppression (suggesting adrenal suppression). Collective results suggest that ovarian response to gonadotropin treatment in the cheetah was improved following oral progestin administration due to the normative increase in estradiol following stimulation for these females compared with control. This treatment should lead to more effective timed assisted reproduction procedures for this species.


Assuntos
Acinonyx/fisiologia , Sincronização do Estro/métodos , Gonadotropinas Equinas/farmacologia , Ovário/efeitos dos fármacos , Indução da Ovulação , Progestinas/administração & dosagem , Administração Oral , Animais , Gonadotropina Coriônica/farmacologia , Estradiol/análise , Estradiol/metabolismo , Fezes/química , Feminino , Glucocorticoides/análise , Glucocorticoides/metabolismo , Hormônio Luteinizante/farmacologia , Ovário/fisiologia , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Progesterona/análise , Progesterona/metabolismo , Progestinas/farmacologia , Resultado do Tratamento , Acetato de Trembolona/administração & dosagem , Acetato de Trembolona/análogos & derivados , Acetato de Trembolona/farmacologia
18.
PLoS One ; 14(5): e0217365, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31136609

RESUMO

Plastic polymers can be combined with additives that modify physical properties and stability of the material. However, the biocompatibility of those additives is not well known. The objective of the study was to characterize the impact of zinc stearate-a common additive-through the development of a novel three-dimensional (3-D) in vitro platform with endometrial cells from domestic cats. Epithelial and stromal cells from adult uteri were isolated and cultured in medium supplemented with 3% Matrigel for two weeks in plastic tissue culture dishes that had been identified as polystyrene with and without zinc stearate by Raman, FTIR, and X-ray fluorescence spectroscopies. Three-dimensional cell structures that were obtained were measured and categorized by shape. Cell viability, proliferation, differentiation, organization, and apoptosis then were assessed by immuno-staining. Results indicated that zinc stearate did not affect 3-D endometrial cell structure morphology, viability, or cellular composition. This first study of a new in vitro platform will be useful for studies testing the influence of other additives, drugs, or exogenous hormones.


Assuntos
Técnicas de Cultura de Células/métodos , Endométrio/citologia , Plásticos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Gatos , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Organoides/citologia , Organoides/efeitos dos fármacos , Poliestirenos/toxicidade , Ácidos Esteáricos/toxicidade , Células Estromais/citologia , Células Estromais/efeitos dos fármacos
19.
J Assist Reprod Genet ; 36(7): 1401-1412, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31079268

RESUMO

PURPOSE: Increasing intracellular energy storage by chemically activating adenosine monophosphate-activated protein kinase (AMPKα) prior to sperm cryopreservation may improve post-thawed sperm function. Using the domestic cat as a biomedical model, the objectives were to (1) confirm the expression of AMPKα and its regulatory kinases in epididymal spermatozoa and (2) assess the influence of AMPK activator, 5'-aminoimidasole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) on epididymal sperm function before and after cryopreservation. METHODS: In study I, sperm samples of different qualities were obtained from cauda epididymides of domestic cats and evaluated for AMPKα expression. In study II, epididymal spermatozoa were equilibrated for either 30 or 60 min in the presence of 0 (control), 0.5, 2.0, and 5.0 mM AICAR and sperm functions were assessed before and after cryopreservation. In study III, epididymal spermatozoa were treated as in study II and evaluated for AMPKα signaling protein expressions (phospho-AMPKα Thr172 and GLUT1) as well as ATP levels. RESULTS: AMPKα protein expression was higher in high-motility vs poor-motility samples. Thirty-minute equilibration with 0.5 mM AICAR improved motion characteristics and fertilizing ability of cryopreserved sperm to the control. Increased expressions of phospho-AMPKα Thr172 and GLUT1 as well as intracellular ATP level were confirmed in sperm samples equilibrated with 0.5 or 2.0 mM AICAR for 30 min. CONCLUSIONS: Presence and role of AMPKα protein in cat regulating sperm function were demonstrated before and after cryopreservation. Findings could be used to potentially enhance cryopreserved sperm function in sub-fertile men.


Assuntos
Criopreservação , Metabolismo Energético/genética , Proteínas Quinases/genética , Espermatozoides/crescimento & desenvolvimento , Animais , Gatos , Feminino , Fertilização/genética , Fertilização/fisiologia , Humanos , Masculino , Preservação do Sêmen/métodos , Motilidade Espermática/genética , Motilidade Espermática/fisiologia , Espermatozoides/metabolismo
20.
Reprod Domest Anim ; 54(8): 1121-1130, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145489

RESUMO

The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α-MEM+ supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α-MEM++ supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri-cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis.


Assuntos
Adhatoda/química , Folículo Ovariano/crescimento & desenvolvimento , Extratos Vegetais/farmacologia , Ovinos , Animais , Meios de Cultura/química , Feminino , Extratos Vegetais/química , Técnicas de Cultura de Tecidos , Trealose/química , Trealose/farmacologia
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