RESUMO
BACKGROUND: Fundamental knowledge of cellular and molecular mechanisms in developing testicular tissues is critical to better understand gonadal biology and responses to non-physiological conditions. The objective of our study was to (1) analyze transcriptome dynamics in developing testis of the domestic cat and (2) characterize age effects on the initial response of the tissue to vitrification. Tissues from adult and juvenile cats were processed for histology, DNA integrity, and RNA sequencing analyses before and after vitrification. RESULTS: Transcriptomic findings enabled to further characterize juvenile period, distinguishing between early and late juvenile tissues. Changes in gene expression and functional pathways were extensive from early to late juvenile to adult development stages. Additionally, tissues from juvenile animals were more resilient to vitrification compared to adult counterparts, with early juvenile sample responding the least to vitrification and late juvenile sample response being closest to adult tissues. CONCLUSIONS: This is the first study reporting comprehensive datasets on transcriptomic dynamic coupled with structural analysis of the cat testis according to the age and exposure to cryopreservation. It provides a comprehensive network of functional terms and pathways that are affected by age in the domestic cat and are either enriched in adult or juvenile testicular tissues.
Assuntos
Testículo , Transcriptoma , Animais , Gatos , Criopreservação , Masculino , VitrificaçãoRESUMO
Rimapenaeus constrictus is a penaeid shrimp widely distributed in the Western Atlantic, frequently captured as bycatch in trawling activities. Here we describe the weight vs. carapace length relationship and the condition factor of the species. Shrimps were sampled in the Ubatuba region, northern littoral of São Paulo State, monthly. We analyzed 4,952 individuals (1,371 males and 3,581 females). We measured the individuals' weight and carapace length, and the condition factor (CF) was calculated for both sexes. Females had a heavier body when compared to males, probably due to their greater maximum body size achieved. Both sexes presented a negative allometric growth in weight, probably due to their reproductive pattern and activities. We found similar mean CF values for males and females. From temporal analysis, the highest CF values for females were observed during the seasons with lower water temperatures. Such a situation may happen because females' CF tend to be influenced by a greater food availability in the environment, induced by the intrusion of the South Atlantic Central Water during the spring and early summer in the Ubatuba. The information presented here could be used as subside in protection actions and management of bycatch species.
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Penaeidae , Exoesqueleto , Animais , Brasil , Feminino , Masculino , Reprodução , Estações do AnoRESUMO
Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n = 8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFßR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p > .05) between SWD (4.00 ± 1.17) and ALD (2.50 ± 0.43) group. The total number of follicles per animal and amounts of small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFßR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.
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Artiodáctilos/fisiologia , Gonadotropina Coriônica/farmacologia , Ovário/efeitos dos fármacos , Animais , Peso Corporal , Gonadotropina Coriônica/administração & dosagem , Feminino , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Receptores da Gonadotropina/genética , Receptores da Gonadotropina/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismoRESUMO
Ex-situ conservation strategies such as the formation of somatic cell banks are valuable tools for the conservation of jaguars, whose population has been declining in recent years. Once properly established, these cells can be successfully leveraged for future applications. We aimed to assess the effects of in vitro culture and cryopreservation on the establishment of fibroblasts derived from jaguars. Initially, we identified five dermal fibroblastic lines using morphology and immunophenotyping assays; these lines were then subjected to two experiments. In the first experiment, the viability, metabolism, and proliferative activity of cells at different passages (first, third, and tenth) were evaluated. In the second experiment, the cells were cryopreserved and the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptosis were evaluated after one, three, and ten passages. Noncryopreserved cells were used as controls. The in vitro culture after first, third, and tenth passages and cryopreservation conditions did not affect the proliferative activity and viability. However, cells cultured until tenth passage and frozen/thawed cells showed reduced metabolism. In addition, cryopreserved cells showed higher levels of intracellular ROS and altered ΔΨm when compared with those of noncryopreserved cells. Finally, frozen/thawed cells cultured after ten passages showed reduced proliferative activity and number of viable cells than did frozen/thawed cells cultured after one and three passages. In summary, we have shown that viable fibroblasts can be established from jaguar skin and that although these cells do not show altered viability and proliferative activity, they do undergo damage during extended culture and cryopreservation.
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Técnicas de Cultura de Células/veterinária , Conservação dos Recursos Naturais , Criopreservação/veterinária , Derme/citologia , Fibroblastos/fisiologia , Panthera , Animais , Proliferação de Células , Sobrevivência CelularRESUMO
In contributing to the conservation of wild rodents, the aim of this study was to evaluate the use of distinct cryoprotectants, separately or in combination, for solid surface vitrification (SSV) of red-rumped agouti ovarian tissue. Ovarian cortex from nine females was recovered and fragmented. Fresh fragments (control) were used to analyse the pre-antral follicle (PF) morphology using a histologic procedure, viability using the Trypan blue test, cell proliferation by counting the argyrophilic nucleolar organizing regions (Ag-NORs technique) and DNA integrity using the TUNEL assay. The remaining fragments were vitrified using SSV method with 3 M or 6 M ethylene glycol (EG) or dimethyl sulfoxide (DMSO), or in combination (3 M EG/3 M DMSO), and further evaluated as reported for the fresh samples. All cryoprotectants were effective at preserving PFs morphology compared to the control group (80.7 ± 5.21%), except 6 M EG and 3 M DMSO that provoked a significant (p < .05) decrease on the values of morphologically normal primary (60.0 ± 19.0%) and primordial (44 ± 4.5%) follicles, respectively. Regarding viability, all cryoprotectants provided values similar to that verified for the control group (79.0%), but a significant decrease (p < .05) was observed with EG/DMSO combination (59%). Using Ag-NORs technique, the highest (p < .05) cell proliferative capacity was detected when using EG at each tested concentration. The TUNEL proved the preservation of DNA integrity regardless of the cryoprotectant. In summary, we suggest the use of 3 M EG for the solid surface vitrification of red-rumped agouti ovarian tissue.
Assuntos
Criopreservação/veterinária , Crioprotetores , Dasyproctidae , Ovário , Animais , Sobrevivência Celular , Criopreservação/métodos , Dano ao DNA , Dimetil Sulfóxido , Etilenoglicol , Feminino , Folículo OvarianoRESUMO
This study investigated the effects of BMP-15 on the in vitro development of preantral follicles of collared peccaries. Ovarian fragments were cultured for 1 or 6 days in Tissue Culture Medium 199 (TCM199+ ) supplemented with BMP-15 at rates of 0, 1, 25 or 50 ng/ml. The fragments were analysed histologically by evaluating follicular morphology, activation and growth as well as the potential for proliferation of granulosa cells. Our results show the addition of 25 ng/ml BMP-15 in the medium provided the greatest percentage of normal follicles (79.67% ± 0.69) when compared to other treatments (p < .05); however, this result is similar to 1 ng/ml BMP-15 (74.00% ± 1.90, p > .05). Moreover, 25 and 50 ng/ml of BMP-15 promoted follicular activation. BMP-15 supplements did not affect oocyte and follicular growth. All concentrations of BMP-15 increased the number of nucleolus organizer regions (NORs) after 1 day of culture when compared to fresh fragments or the control samples (p < .05). However, at the end of the experiment, the number of NORs in follicles cultured in all treatments was higher than that observed in the fresh control (sample taken prior to culturing) (p > .05). In summary, the addition of 25 ng/ml BMP-15 to the culture medium of collared peccary preantral follicles maintained a high number of morphologically healthy follicles and stimulated the activation of primordial follicles after 6 days in culture.
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Artiodáctilos/fisiologia , Proteína Morfogenética Óssea 15/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Proteína Morfogenética Óssea 15/administração & dosagem , Técnicas de Cultura de Células/veterinária , Feminino , Folículo Ovariano/fisiologiaRESUMO
The aim of the present study was to evaluate the development of fresh and vitrified agouti ovarian tissue after xenografting to C57Bl/6 severe combined immunodeficiency (SCID) female mice. Ovaries were obtained from five female agoutis and divided into 16 fragments. Five fragments were transplanted immediately to ovariectomised SCID mice and the others were vitrified, stored for 2 weeks and transplanted only after rewarming. Tissue fragments were transplanted under the kidney capsule in recipients. The return of ovarian activity in recipients was monitored by the observation of external signs of oestrus and vaginal cytology over a period of 40 days after transplantation, after which the grafts were removed and evaluated for morphology, cell proliferation and the occurrence of DNA fragmentation. Ovarian activity returned in four of five mice that received fresh ovarian tissue from agoutis and in one of six mice that had received vitrified tissue a mean (±s.e.m.) 20.6±8.6 days after xenotransplantation. After graft removal, a predominance of primordial and primary follicles was observed in all grafts. Vitrification reduced cell proliferation and increased the occurrence of DNA fragmentation in grafted agouti ovarian tissue. In conclusion, the present study demonstrates that xenografted agouti ovarian tissue, fresh or vitrified, is able to promote the return of ovarian activity in ovariectomised SCID C57B1/6 mice. However, improvements to vitrification protocols for agouti ovarian tissue are necessary.
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Criopreservação , Preservação da Fertilidade/métodos , Ovariectomia , Ovário/transplante , Animais , Proliferação de Células , Fragmentação do DNA , Ciclo Estral , Feminino , Sobrevivência de Enxerto , Xenoenxertos , Camundongos Endogâmicos C57BL , Camundongos SCID , Ovário/metabolismo , Ovário/patologia , Gravidez , Recuperação de Função Fisiológica , Fatores de Tempo , VitrificaçãoRESUMO
SummaryThe aim of this study was to establish a functional freezing-thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen-thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.
Assuntos
Cocos/química , Criopreservação/veterinária , Crioprotetores/farmacologia , Epididimo/fisiologia , Extratos Vegetais/farmacologia , Espermatozoides/fisiologia , Trometamina/farmacologia , Animais , Artiodáctilos , Criopreservação/métodos , Epididimo/efeitos dos fármacos , Masculino , Análise do Sêmen , Espermatozoides/efeitos dos fármacosRESUMO
The aim of this study was to assess a vitrification protocol for asinine ovarian tissue, to preserve preantral follicles using different cryoprotectant solutions, composed of various concentrations (EG 3 M or 6 M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3 M + EG 3 M). Ten pairs of ovaries from Brazilian north-eastern breed jennies were obtained through videolaparoscopy, and cortical fragments were submitted to a solid-surface vitrification (SSV) using each cryoprotectant solution. The ovarian tissue was evaluated for follicular morphology and viability, DNA integrity (TUNEL technique) and the presence of nucleolar organizing regions in granulosa cells (AgNOR technique). After thawing, the percentage of normal preantral follicles was significantly reduced in the vitrified ovarian tissue fragments compared to the fresh control (p < 0.05). When comparing treatments, the use of DMSO 3 M (81.7 ± 37.5%), EG 3 M (83.7 ± 27.4%) and the combination of both DMSO 3 M + EG 3 M (81.8 ± 46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6 M (69.8 ± 16.5%) and EG 6 M (72.3 ± 18.0%; p < 0.05). When vitrified using the DMSO + EG combination, a higher percentage (62.5 ± 29.1%) of viable follicles (trypan blue) was observed in relation to the other vitrification treatments (p < 0.05). The TUNEL technique identified that all treatments tested showed DNA fragmentation in the follicular cells, except in the case of the DMSO 3 M + EG 3 M treatment. When evaluating the presence of NORs, no significant differences were observed in the amount of NORs between the fresh and vitrified groups using DMSO 3 M + EG 3 M (p > 0.05). We concluded that the combination DMSO 3 M + EG was more efficient for the vitrification of ovarian tissue taken from Equus asinus, allowing adequate preservation of PAFs morphology, viability, DNA integrity and cell proliferative capacity.
Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Equidae , Folículo Ovariano/fisiologia , Animais , Brasil , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Feminino , Folículo Ovariano/citologia , VitrificaçãoRESUMO
The aim of the present study was to characterise the ovarian preantral follicle (PF) population and to establish a solid surface vitrification (SSV) process using dimethyl sulfoxide (DMSO) as a cryoprotectant for preservation of ovarian tissue from yellow-toothed cavies (Galea spixii). Ovaries were fixed for PF population analysis or were subjected to the SSV process. The mean (± s.e.m.) PF population per ovarian pair was estimated to be 416.0±342.8. There were 140.0±56.0 (63.4%) and 125.0±58.0 (64.0%) primary follicles on the right and left ovaries, respectively. The proportion of this follicle category was significantly greater than that of other follicle categories (P<0.05). The diameter of follicles (123.7±18.3µm), oocytes (50.1±5.0µm) and nuclei (14.27±2.01µm) was larger for secondary ones when compared with other PFs categories. Most PFs were morphologically normal (94.6%), with light microscopy identifying only a few atretic follicles (5.4%). After SSV, there was a reduction in the proportion of morphologically normal PFs compared with the non-vitrified group (69.5% vs 91.2%, respectively). Transmission electron microscopy revealed preservation of oocytes and granulosa cell membranes and the morphological aspect of follicles; the primary change observed in some vitrified PFs was the presence of vacuoles in the oocytes and granulosa cells cytoplasm and turgid mitochondria. In conclusion, the present study provides an estimative and characterization for the PF population in ovaries of G. spixii. Moreover, we report its PFs cryopreservation using an SSV process.
Assuntos
Criopreservação , Folículo Ovariano/anatomia & histologia , Ovário/anatomia & histologia , Vitrificação , Animais , Feminino , Microscopia Eletrônica de Transmissão , Folículo Ovariano/ultraestrutura , Ovário/ultraestrutura , RoedoresRESUMO
BACKGROUND: Alpha adrenergic drugs are usually used in the treatment of erectile and ejaculatory dysfunction in humans. The influence of such drugs on the seminal characteristics of wild animals has not been verified; whereas their impact on the seminal characteristics and erectile and ejaculatory functions of collared peccaries (Tayassu tajacu) has already been determined. This study aimed at investigating and comparing the effects of medetomidine and atipamezole on the seminal variables of collared peccaries undergoing electroejaculation as well as at determining whether these drugs affected the erectile and ejaculatory functions of this species. RESULTS: A statistically significant difference in sperm concentration was observed between AP (100.0 ± 26.0 × 106 sperm/ml) and MP (220.2 ± 49.8 × 106 sperm/ml); however, both these treatments did not differ from P treatment (180.0 ± 50.7 × 106 sperm/ml). No statistically significant difference was observed among all treatments with regard to erectile function. With regard to ejaculation time, no significant difference was observed between the MP and AP treatments; however, when compared with the P treatment, AP exhibited a significantly higher difference. CONCLUSIONS: When collared peccaries were anesthetized with propofol, neither medetomidine nor atipamezole significantly affected the characteristics of the semen or the erectile function, despite the fact that the AP treatment increased ejaculation time. Therefore, the data indicate that using propofol alone is an effective anesthetic protocol for collecting semen in collared peccaries. Other non-injectable anesthetic drugs, such as inhaled anesthetics, may be used in future research to collect semen from peccaries.
Assuntos
Artiodáctilos/fisiologia , Ejaculação/efeitos dos fármacos , Imidazóis/farmacologia , Medetomidina/farmacologia , Ereção Peniana/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Estudos Cross-Over , Estimulação Elétrica , Masculino , Análise do Sêmen/veterinária , Contagem de Espermatozoides/veterináriaRESUMO
To assist in the conservation of collared peccary, it is important to strengthen semen processing protocols. The aim of this study was to compare the effects of different commercial extenders (BTS; NUTRIXcell+ and PRIMXcell Ultra) and TRIS + egg yolk on the functional and morphological aspects of collared peccary semen stored at 17 °C for 48â¯hours. Ten ejaculates obtained by electroejaculation were divided into 4 aliquots and diluted in the respective extenders, then stored in a biological incubator at 17 °C for 12, 24, 36, and 48â¯hours. The samples were evaluated for kinetic parameters, membrane functionality, membrane integrity, mitochondrial activity, morphology, and sperm-binding capacity. At the end of storage (48â¯h), promising results were found for motility parameters, with TRIS + egg yolk (71.0 ± 4.6%) being more efficient than NUTRIXcell+ (38.9 ± 10.9%) (P < 0.05) and similar to BTS (42.9 ± 11.9%) and PRIMXcell Ultra (46.8 ± 10.8%). The results for membrane integrity and mitochondrial activity were around â¼30-50%, with TRIS being the only extender to preserve both parameters (58.9 ± 5.3 and 59.2 ± 5.6%) for up to 48â¯hours, respectively (P < 0.05). Finally, the extenders could guarantee 60% membrane functionality and â¼ 60-70% normal sperm morphology, as well as similar binding capacity among the groups. In conclusion, TRIS + egg yolk is effective in preserving the sperm parameters of collared peccary semen at 17 °C for 48â¯hours, while PRIMXcell Ultra and BTS are viable alternatives for this purpose.
Assuntos
Gema de Ovo , Preservação do Sêmen , Animais , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Masculino , Gema de Ovo/química , Crioprotetores/farmacologia , Análise do Sêmen/veterinária , Artiodáctilos/fisiologia , Trometamina/farmacologia , Trometamina/química , Refrigeração/veterinária , Espermatozoides/fisiologia , SêmenRESUMO
We verified the possibility of cooling peccary semen for 4, 24, and 48 h before cryopreservation, using different dilution media (TRIS + egg yolk (20%) and PRIMXcell Ultra). Ten ejaculates were divided equally into six aliquots and then diluted. Two aliquots were stored in a biological incubator (4 h), and the remaining aliquots were stored in a commercial container, the Botutainer® (24 and 48 h), both at 5 °C. The samples were cryopreserved and then evaluated for kinetic parameters, functionality, integrity, mitochondrial activity, morphology, and sperm binding capacity. After thawing, samples diluted in TRIS showed total motility of 43.4 ± 6.8%, 48.4 ± 6.2%, and 38.6 ± 5.0% after cooling for 4, 24, and 48 h before cryopreservation, respectively. Such results are significantly greater than those achieved with the use of PRIMXcell diluent for 4 (8.3 ± 2.8%), 24 (4.7 ± 1.4%), and 48 h (4.8 ± 2.9%) storage (p < 0.05). Furthermore, TRIS provided better preservation of sperm membrane integrity when samples were cooled for 24 h (44.5 ± 4.7%) before cryopreservation compared to those samples diluted in PRIMXcell Ultra stored for 24 (25.7 ± 4.0%) and 48 h (25.2 ± 4.0%) before freezing (p < 0.05). In summary, we suggest TRIS diluent + egg yolk (20%) as an effective option to allow semen to cool for 24 or 48 h in a transport container before cryopreservation.
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Knowledge on male reproductive physiology is essential for the development of effective conservation strategies. This study investigated the influence of environmental variables on certain reproductive metrics in white-lipped peccaries (Tayassu pecari) raised in the Atlantic Forest. After anesthetization, testicular and cauda epididymis biometry were evaluated in nine adult male individuals subjected to electroejaculation. Semen was evaluated for volume, pH, concentration, total number of sperm, sperm morphology, membrane integrity, and kinematic parameters. Concurrently, environmental variables were collected from the day before, for the previous 14 days (estimated for sperm maturation in epididymis), and the period of 51-55 days (corresponding to the spermatogenic cycle) before semen collection. Overall, it was observed that rainfall is the most important environmental variable influencing the reproductive parameters of white-lipped peccaries, being positively correlated with the amplitude of lateral sperm head displacement (ρ = 0.62, P < 0.05) and the appearance of proximal cytoplasmic droplets in sperm (ρ = 0.62, P < 0.05). In addition, the testicular biometry of the species is influenced by the set of environmental variables of air temperature, rainfall, and relative humidity (ρ ≥ 0.60, P < 0.05). On the other hand, epididymal biometric data showed numerous correlations between cauda epididymis metrics and sperm parameters (ρ = 0.68, P < 0.05). This information will be useful to improving conservation strategies for these animals, contributing to their management in captivity and to reintroduction programs, especially in the Atlantic Forest where the species is declining.
Assuntos
Artiodáctilos , Benchmarking , Animais , Masculino , Brasil , Sêmen , Artiodáctilos/fisiologia , Espermatozoides , FlorestasRESUMO
Methods for seminal plasma (SP) removal and the selection of collared peccary sperm for fertilization were compared. The experiments evaluated the following: the (I) impact of centrifugation for SP removal before swim-up for sperm selection and (II) a comparison of different Percoll® gradient densities (PG 45-90% and PG 35-70%). Non-selected sperm served as the control. Sperm quality was assessed based on motility patterns, morphology, membrane functional integrity, viability, reactive oxygen species (ROS), glutathione (GSH), and DNA integrity. Subsequently, the most successful group in the previous experiment and washing by centrifugation (WC) were compared for motility patterns and fertilization using pig oocytes. Swim-up decreased motility and enhanced ROS compared to the control. Centrifugation before swim-up harmed integrity and viability compared to the control. PG 45-90% (96.8 vs. 69.7 vs. 40.7 µm/s) allowed for a better velocity average pathway (VAP), a better velocity straight line, and better linearity (LIN) than those of the control and PG 35-70% (88.4 vs. 56.0 vs. 27.3 µm/s). Thus, PG 45-90% was used for fertilization. PG 45-90% obtained a higher VAP, a higher amplitude of the lateral head, straightness, and higher LIN than those of the control and WC. Cleavage (25.2-26.3%) and morula (8.1-10.5%) rates did not differ between the groups. Therefore, PG 45-90% and WC were efficient in isolating collared peccary sperm capable of fertilizing pig oocytes.
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We evaluated the effects of detergents based on sodium dodecyl sulfoxide (SDS) on the functional parameters of collared peccary frozen-thawed sperm. Semen aliquots from ten individuals were diluted in a Tris-egg yolk-glycerol extender alone or with 0.5% Equex STM® paste or SDS (at 0.1%, 0.3% or 0.5% (v/v) concentration). Samples were fast frozen in liquid nitrogen with a post-thaw evaluation of motility, membrane functionality and integrity, mitochondrial activity, sperm binding ability and thermal resistance. The treatments without SDS (41.8 ± 3.5%) and those containing Equex (41.8 ± 4.4%) or 0.1% SDS (41.2 ± 5.5%) provided greater sperm motility (p < 0.05) than those containing SDS 0.3% (30.5 ± 4.7%) and 0.5% (31.2 ± 6.3%). Immediately after thawing, only treatments containing 0.1% SDS effectively preserved sperm straightness (STR) when compared to the negative control. All treatments preserved the amplitude of lateral head (ALH) and straightness (STR) during a thermal resistance test (p > 0.05), but SDS 0.5% impaired the membrane functionality and mitochondrial activity after thawing (p < 0.05). All treatments provided a similar recovery of sperm binding ability after thawing (p < 0.05). Our results showed that the addition of 0.1% SDS to the Tris-yolk-glycerol extender optimized the freeze-thaw recovery of peccary semen.
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The objective was to characterize morphological, morphometric, and ultrastructural changes in rhea spermatozoa between the epididymis and the vas deferens. Sperm samples were collected from the reproductive tracts of seven adult individuals and evaluated for sperm characteristics using brightfield microscopy as well as ultrastructural features using scanning electron microscopy (SM). Mean sperm count tended to increase in the vas deferens (378.0 ± 135.0 × 106) compared to the epididymis (201.0 ± 77.4 × 106). Percentages of motile sperm grew from 37.0 ± 4.9% in the epididymis to 58.5 ± 7.7% in the vas deferens. The proportion of normal spermatozoa was 75.6 ± 1.8% and most common defects were bent tails (9.7 ± 0.9%). However, these proportions were not different between epididymis and vas deferens. SM analysis revealed further features of rhea spermatozoa. Normal rhea spermatozoa were threadlike with an acrosome (0.95 ± 0.0 µm), head (7.53 ± 0.01 µm), midpiece (2.08 ± 0.01 µm), and tail (30.7 ± 0.06 µm). Lengths of sperm acrosome, head, midpiece, and tail were longer in the vas deferens compared to the epididymis. Our findings suggest that rhea spermatozoa undergo a maturation process during the passage from the epididymis to the vas deferens.
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The aim of the study was to perform the first in-depth analysis of miRNAs in ovarian and testicular tissues of the domestic cat, a critical biomedical model. Specifically, potential miRNA involvement was explored in gonadal function, testis development, and cellular stress response to preservation protocols. We performed miRNA-sequencing on 20 ovarian and 20 testicular samples from 15 cats, including different ages and tissue treatments. Using fresh tissues (n = 15), we confirmed gonadal expression of 183 miRNA precursors and discovered additional 52 novel feline candidate precursors. We integrated the mRNA data from our previous study on the same age and treatment groups to create in-silico miRNA-mRNA networks and their functional enrichment, which allows comprehensive exploration into possible miRNA functions in cat gonads. Clusters of miRNAs united by shared differentially expressed mRNA targets are potentially involved in testicular development and spermatogenesis. MicroRNAs could play a significant role in ovarian tissue response to stress from microwave-assisted dehydration, with smaller roles in cellular response to vitrification in both ovary and testis. This new list of miRNAs with potential function in cat gonads is a major step towards understanding the gonadal biology, as well as optimizing fertility preservation protocols.
RESUMO
BACKGROUND: Polyarthritis has been associated with canine visceral leishmaniasis (CanVL), and co-infection with Ehrlichia canis is common and may alter clinical manifestations. METHODS: A total of 89 dogs presenting CanVL were subdivided into two groups: (1) G1, consisting of 46 dogs seronegative to Ehrlichia spp., and (ii) G2, consisting of 43 dogs seropositive to Ehrlichia spp. Eight joints (carpal, tarsal, stifles and elbows) from each dog were evaluated by radiography and synovial fluid (SF) cytologic analysis. RESULTS: Overall, 74 of the 89 (83.1%) dogs presented joint abnormalities suggestive of osteoarthritis by radiography (G1: 40/46 [86.9%]; G2: 34/43 [79.0%]), with no statistically significant between-group difference. All dogs with abnormal joint X-ray images presented radiographic lesions bilaterally, independent of the characteristics of the lesion. Soft tissue swelling around the joint and joint space narrowing were more commonly observed in G1 than in G2 dogs. There was no significant between-group difference in terms of other radiographic abnormalities suggestive of osteoarthritis (evident trabecular pattern, subchondral bone sclerosis, osteolysis, osteolytic-proliferative lesions or bone proliferation). SF from 174/315 (55.2%) and 152/307 (49.5%) joints from G1 and G2 dogs, respectively, presented an inflammatory infiltrate, but there was no significant association between the presence of inflammatory infiltrate and group. There was also no statistical difference between groups in either of the evaluated joints in terms of the percentage of neutrophils or mononuclear cells. Leishmania spp. amastigotes were found in 69/315 (21.9%) joints from G1 dogs and in 100/307 (32.5%) joints from G2 dogs (Fisher's exact test, P = 0.002, odds ratio = 0.5, 95% confidence interval = 0.4-0.8). The neutrophilic infiltrate was significantly higher in joints with amastigote forms in both G1 (Mann-Whitney U-test, U(18) = 817, Z = -3.76, P = 0.0001) and G2 dogs (Mann-Whitney U-test, U(18) = 6543, Z = - 5.06, P < 0.0001). CONCLUSIONS: A high prevalence of arthritis in dogs with CanVL was found, and all dogs presented involvement in multiple joints. Although no difference was observed between groups in terms of the number of dogs with polyarthritis and the presence of an inflammatory infiltrate in SF, Leishmania spp. amastigotes were found more frequently in joints from G2 dogs. Further studies evaluating SF in dogs co-infected with L. infantum and E. canis should be performed to evaluate this finding.
Assuntos
Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Osteoartrite , Animais , Doenças do Cão/epidemiologia , Cães , Leishmaniose Visceral/complicações , Leishmaniose Visceral/diagnóstico por imagem , Leishmaniose Visceral/veterinária , Osteoartrite/complicações , Osteoartrite/diagnóstico por imagem , Osteoartrite/veterinária , Líquido SinovialRESUMO
The objective was to investigate the effects of semen freezing extender supplementation with antibiotics on bacterial load of semen samples, sperm functional and morphological metrics in the collared peccary. Fresh ejaculates from 10 males were extended in Tris-egg yolk-glycerol supplemented or not (control) with gentamicin (70 µg/mL) streptomycin-penicillin (SP; 1 mg/mL-1000 IU/mL) or and cryopreserved in liquid nitrogen. Bacterial load, sperm motility patterns, morphology, membrane functionality and integrity, mitochondrial activity, chromatin integrity and sperm-binding ability were evaluated in fresh and frozen-thawed samples. Regardless of the use of antibiotics, the sole cryopreservation provoked a significant decrease (P < 0.05) in bacterial load compared to fresh samples (from average values > 1 x 106 CFU/mL to <0.4 × 106 CFU/mL). Post-thawing sperm kinetic parameters were not affected by the absence or presence of different antibiotics, except for beat cross frequency that was significantly (P < 0.05) impaired by SP supplementation compared to the group without antibiotics. After thawing, sperm morphology, membrane functionality and integrity, and mitochondrial activity were also not affected by the presence or absence of antibiotics; however, a significant decrease was observed in the group without antibiotics (P < 0.05) in comparison to fresh samples. Regarding sperm-binding ability, there were no differences among the different groups. While collared peccary semen could be efficiently cryopreserved in the absence of antibiotics in the extender, the use of both gentamicin or the streptomycin-penicillin combination is recommended as effective antibiotic supplementation for a further control of bacterial loads without affecting sperm parameters.