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

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.

The objective of the study was to evaluate the effects of different cryopreservation techniques including glycerol-based cryoprotectant combinations on the structure and viability of testicular tissues from adult collared peccaries. Tissue biopsies (3.0 mm³) from 5 different individuals were allocated to 10 different groups: fresh control; slow freezing (SF), conventional vitrification (CV), or solid-surface vitrification (SSV); each of them using three different combinations of cryoprotectants [dimethyl sulfoxide (DMSO) + ethylene glycol (EG); DMSO + Glycerol; and EG + Glycerol]. After thawing/warming, samples were evaluated for histomorphology, viability, proliferative capacity potential, and DNA integrity. Most effective preservation of testicular histomorphology was achieved using SF and CV with DMSO + EG. However, the use of glycerol-based cryoprotectant combinations increased the occurrence of tubular cell swelling, tubular cell loss and shrinkage from the basal membrane. Cell viability was comparable among cryopreservation methods and cryoprotectant combinations. Regarding cell proliferative capacity, the use of SF with EG + Glycerol and SSV with DMSO + Glycerol impaired the conservation of spermatogonia proliferative potential compared to other treatments. Moreover, CV with DMSO + EG was better than SF with EG + Glycerol for Sertoli cell proliferation potential. Regarding DNA integrity, less damage occurred when using SF with DMSO + EG while more fragmentations were observed when using CV with EG + Glycerol or DMSO + Glycerol as well as SSV with EG + Glycerol or DMSO + Glycerol. In sum, SF and CV appeared to be the most suitable methods for the cryopreservation of adult peccary testicular tissues. Additionally, the use of glycerol-based cryoprotectant combinations did not improve testicular tissues preservation with DMSO + EG being the most efficient option.

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

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.

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

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

The objective of the study was to understand the influence of climatic variations in a semiarid environment on serum testosterone, testicular morphology and semen quality in collared peccaries (Pecari tajacu). Reproductive metrics (semen quality, testicular morphometry and testosterone serum profiles) of 10 mature males were measured monthly for 18 months. Meteorological data (rainfall, air temperature, relative humidity, wind speed and radiant heat load) also were recorded during the same period. Rainfall regimes were classified in different classes (Class 1: months with no rain; Class 2: months with up to 50 mm of rain; and Class 3: months with50 mm of rain). Among rainfall classes, average air temperature (°C) and relative humidity (%) were different. Climatic changes between rainfall classes did not lead to overall variations of testicular size, testosterone production, and semen metrics. However, relative humidity recorded before semen collection (one day, one week, or over 51-55 days) was positively correlated (P 0.05) with semen motility metrics (total motility, beat cross frequency and straightness) and sperm subpopulations (medium and static sperm), as well as with volume. Negative correlations (P 0.05) were revealed between air temperature and the same semen motility patterns and volume. Additionally, radiant head load measured on the day of semen collection negatively influenced (P 0.05) sperm straightness. This study demonstrates for the first time that no seasonal changes could be detected overt the 18-month period on the serum testosterone, testicular morphology and semen quality of collared peccaries raised in the Caatinga biome; however, it is expected that long term environmental changes will influence the reproductive physiology of species leaving in that habitat.

Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents' germplasm, we verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the sperm parameters of Spix's yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20% concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters, morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided 515.4 × 106 sperm/mL and 561.6 × 106 sperm/mL, presenting >65% motile sperm, respectively. After cryopreservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both extenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender supplemented of 10% egg yolk for cryopreservation of Spix's yellow-toothed cavy epidydimal sperm.

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.

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 (PP

The influence of environmental factors in a semiarid climate on characteristics of fresh and frozen/thawed sperm collected from collared peccaries (Pecari tajacu) was assessed. Semen from 11 male c...

Dasyprocta spp. (agouti) include wild rodents with highlighted ecological and economic importance, and are considered experimental models for endangered hystricognath rodents. Of late, development of techniques to conserve their genetic material as well as the formation of biobanks is increasing. In this context, this review describes the main advances in the knowledge of the reproductive morphophysiological specificities of agouti as well as the development and improvement of assisted reproductive techniques aimed at conservation, multiplication, and exploitation of their reproductive potential under captivity.

In collared peccaries, the development of artificial insemination (AI) is scarce, requiring search for alternative methods for the evaluation of sperm fertilizing ability. Thus, the aims of this study were to estimate the binding capability of collared peccaries sperm, using swine oocytes and the egg perivitelline membrane, and to evaluate the prognostic value of sperm parameters on the in vitro interactions among sperm and heterologous substrates. Eleven ejaculates were collected by eletroejaculation and evaluated for viability and morphology by light microscopy, for functionality by hypo-osmotic swelling test, for plasma membrane integrity by epifluorescence microscopy, and for sperm motility by computerized analysis. Subsequently, for analysis of the in vitro interactions, sperm samples were cultured in an incubation medium with swine oocytes and egg perivitelline membrane for 18 h and 20 min, respectively, at 38.5 °C and humidified atmosphere. The sperm-oocyte interaction rate was 100% with sperm penetrating 19.8+ 5.5% of oocytes. The average values of bound sperm and penetrated sperm per oocyte were 39.4 + 4.6 and 2.5 + 0.7, respectively. Already for perivitelline membrane binding assay, all samples presented sperm bound (100%) with average of 140.6 ± 19.4 bound sperm (range 33.9–308.7). Moreover, positive correlations were observed for the number of sperm bound to swine oocytes and osmotic response (r = 68.5%; P = 0.02), membrane integrity (r = 65.1%; P = 0.03), and straightness (r = 66.5%; (P = 0.03), as weel as for the number of sperm bound to egg perivitelline membrane and sperm viability (r = 74.0%; P = 0.01), total motility (r = 63.6%; P = 0.04), and linearity (r = 70.5%; P = 0.02). Finally, a negative correlation among slow (r = −80.5%; P = 0.01) and static (r = −84.3%; P = 0.01) sperm with the egg perivitelline membrane was observed. In conclusion, swine oocytes and perivitelline membrane can be used as indicators for the functional evaluation of the binding capability of sperm derived from collared peccaries. These tests could be incorporated into the routine of semen technologies.

SummaryThe aim of this study was to evaluate the performance of cavy (Galea spixii) epididymal sperm following addition to TES or TRIS extenders and using a thermal resistance test (TRT), as well as fluorescence analysis as a complementary method to predict the viability of these gametes. Nine testicle–epididymis complexes were used for sperm collection using a flotation method. Epididymis tails were sliced and one was immersed in 3 ml of TRIS buffer, and the other in 3 ml of TES, for 5 min. After sperm recovery, the samples were subjected to a TRT which involved incubation in a water bath at 37°C for 3 h. During incubation, sample parameters were assessed at 0, 15, 30, 60, 90, 120, 150 or 180 min intervals. Results indicated that the TRIS diluent was more efficient than TES (P < 0.05) for the maintenance of sperm parameters in Spix's yellow-toothed cavies over the whole TRT, maintaining sperm longevity for an extended time. In conclusion, we indicate the use of TRIS diluent for recovery and maintenance of longevity of epididymal sperm from cavies (G. spixii).

AIM To evaluate the bacterial composition of collared peccary semen and foreskin mucosa, and to verify the sensitivity of isolates to antimicrobials used in semen conservation and to Aloe vera gel, which is an alternative external cryoprotectant. METHODS AND RESULTS Nine foreskin mucosa and ejaculate samples from adult animals were used. Sperm characteristics and bacterial load were evaluated in fresh semen. The preputial mucosa and semen bacterial isolates were identified and tested against five concentrations of each antimicrobial (streptomycin-penicillin and gentamicin) and A. vera gel. Corynebacterium sp. and Staphylococcus sp. were isolated in greater numbers than others in both semen (64·10 and 20·51%, respectively) and the foreskin mucosa (60·60 and 24·25%, respectively), and ranged from 0·4 to 21 × 105 colony-forming units (CFU) per ml. The average load of Corynebacterium sp. was negatively correlated (P

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.

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

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 ± 7.7%) and membrane functionality (60.5 ± 4.2%) and higher mitochondrial activity (21.5 ± 3.7%) than those preserved in ACP-117c (20.9 ± 5.4% motile sperm; 47.1 ± 2.5% functional membrane; 11.8 ± 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 ± 3.3%) in comparison to samples diluted in ACP-117c (18.6 ± 1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.

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

The aims were to identify the effects of growth differentiation factor 9 (GDF-9) on the in vitro development of ovarian preantral follicles (PAFs) of collared peccaries. Ovarian fragments were in vitro cultured for 1 or 7 days without or with inclusion of GDF-9 in the medium (0, 50, 100, or 200 ng/mL). The non-cultured (control) and cultured fragments were evaluated for PAF viability, activation, and cell proliferation. Although there were no differences in the percentage of morphologically normal follicles, the percentage of growing follicles was greater compared to the control in all treatment groups, especially those cultured with 200 ng/mL GDF-9 for 7 days (P0.05). The inclusion of GDF-9 in the medium did not interfere with PAF viability (P0.05); however, treatment with 200 ng/mL GDF-9 resulted in greater (P0.05) cell proliferation in PAFs cultured for 1 or 7 days (∼2.5 nucleolar organizing regions - NORs) compared to the follicles of the control group (2.0 NORs). In addition, peccary ovarian cortexes were subjected to PCR analysis and there was detection of the mRNA GDF-9 receptor transcripts of the BMPR2 (type I receptor) and ALK-5 (type II receptor) types. In conclusion, GDF-9, especially at a 200 ng/mL inclusion in the culture medium, was actively involved in the in vitro development of collared peccary PAFs.

The aim of the present study was to characterize the epididymal sperm of Spix's yellow-toothed cavy (Galea spixii) through two different recovery methods. Nine sexually mature males were euthanized and the complexes, testes–epididymis, were dissected. For each animal, one epididymis was processed by flotation method and the other was processed by retrograde flushing method, both using a TES-based buffered media. After recovery, we evaluated the sperm for motility, vigour, viability, functional membrane integrity and morphology. Morphometric data from the different sperm regions were evaluated by using an appropriate software. After recovery, both methods provide similar values for all the sperm parameters, aiming the recovery of more than 300 × 106 sperm, presenting >50% motile sperm, with normal morphology and functional membrane. The total sperm length in this sperm was 48.87 ± 0.1, and the sperm head presented 9.4%, on average. A notable characteristic was the prominent acrosome found in the G. spixii sperm. In conclusion, we demonstrate that either flotation or retrograde flushing methods are suitable for the recovery of sperm from cauda epididymis of Spix's yellow-toothed cavies.

As an alternative for the conservation of collared peccary semen, this research aims at evaluating the use of Aloe vera (AV) extract as a cryoprotectant for semen chilling and freezing. Five ejaculates were divided in two aliquots that were diluted in Tris plus egg yolk (EY; 20%) or AV extract (20%) and chilled at 5 °C. In both treatments, an adequate semen conservation was achieved and values closer to 40% motile sperm with viability and osmotic response ranging from 20% to 40%, and normal morphology of 80% were found after 36 hours of storage. Moreover, 12 other ejaculates were diluted in Tris plus EY (20%) or AV extract (5, 10, or 20%) and glycerol (3%). Samples were frozen in liquid nitrogen and thawed after 1 week. After thawing, all the treatments containing EY or AV provided similar values for sperm morphology, viability, osmotic response, membrane integrity, sperm motility, amplitude of lateral head, beat cross frequency, and rapid, low, and static subpopulations, but the highest values for straightness and the lowest values for curvilinear velocity were found using 20% AV (P

Systemic lupus erythematosus (SLE) is an autoimmune disorderwith several clinicalmanifestations. SLE etiology has a strong genetic component, which plays a key role in disease's predisposition, as well as participation of environmental factors, such and UV light exposure. In this regard, we investigated whether polymorphisms in STK17A, a DNA repair related gene, encoding for serine/threonine-protein kinase 17A, are associated with SLE susceptibility. A total of 143 SLE patients and 177 healthy controls from Southern Brazil were genotyped for five STK17A TagSNPs. Our results indicated association of rs7805969 SNP (A and G/A genotype, OR = 1.40 and OR = 1.73, respectively) with SLE predisposition and the following clinical manifestations: arthritis, cutaneous and immunological alterations. When analyzing haplotypes distribution, we found association between TGGTC, TAGTC and AAGAT haplotypes and risk to develop SLE. When considering clinical manifestations, the haplotypes TGGTT and TAGTC were associated with protection against cutaneous alterations and the haplotype TAGTC to hematological alterations. We also observed association between SLE clinical manifestations and ethnicity, with the European-derived patients being more susceptible to cutaneous and hematological alterations.

The aim of this study was to characterize the preantral ovarian follicular population in agoutis (D. leporina) by estimating the number of follicles at each developmental category, and also describe the morphometry and the specific features of the follicle and the oocyte by using light and transmission electron microscopy. The length of each ovary was measured using a caliper rule, longitudinally sectioned into two halves and both were immediately fixed to perform the estimation of follicular population and ultrastructural analysis. The mean (±S.E.M.) population of follicular per pair of ovary was estimated at 4419.8±532.26 and 5397.52±574.91 for right and left ovaries, respectively, but no differences were observed between them. The diameters for follicles, oocyte and nuclei were: 18.62±3.40μm, 12.28±2.37μm and 6.10±0.93μm for primordial, 23.75±5.70μm, 14.22±3.00μm and 6.70±1.24μm for primary and 88.55±17.61μm, 52.85±17.56μm and 22.33±17.61μm for secondary follicles, respectively. The most of the follicles found belonged to the primordial category (86.63%), followed by primary (13.01%) and secondary (0.35%) one. Additionally, polyovular follicles were observed in all the animals and they represented 7.51% of the total follicles counted. The ultrastructural analysis showed that the oocyte presented a central and regular nuclei, displaying a homogenous mass. Among the organelles, the mitochondria were the most abundant and the oocyte Golgi apparatus was rarely observed. In conclusion, this work shows for the first time the characterization of the population of preantral follicles in the ovary of Dasyprocta leporina. Those information will be useful for further development and adaptation of biotechniques such as germplasm cryopreservation and in vitro gametes manipulation. RESUMO: O objetivo deste trabalho foi caracterizar a população folicular ovariana pré-antral em cutias (D. leporina) estimando o número de folículos em cada categoria de desenvolvimento, e também descrever a morfometria e as características específicas do folículo e oócito usando microscopia de luz e eletrônica de transmissão. O comprimento de cada ovário foi medido utilizando um paquímetro, seccionados longitudinalmente em duas metades e ambos foram imediatamente fixados para realizar a estimativa da população folicular e análise ultraestrutural. A média (±S.E.M.) da população folicular por par de ovário foi estimada em 4419,8±532,26 e 5397,52±574,91 nos ovários direito e esquerdo, respectivamente, mas não foram observadas diferenças entre eles. Os diâmetros dos folículos, oócito e núcleos, respectivamente, foram: 18,62±3,40μm, 12,28±2.37μm e 6,10±0,93μm para primordial, 23,75±5,70μm, 14,22±3,00μm e 6,70±1,24μm para primário e 88,55±17,61μm, 52,85±17,56μm e 22,33±17,61μm de folículos secundários. A maioria dos folículos encontrados pertencia à categoria primordial (86,63%), seguido pelo primário (13,01%) e um secundário (0,35%). Adicionalmente, os folículos poliovulares foram observados em todos os animais e representavam 7,51% do total de folículos contados. A análise ultra-estrutural mostrou que o oócito apresentou núcleos centrais e regulares, exibindo uma massa homogênea. Dentre as organelas, as mitocôndrias foram as mais abundantes e o aparelho de Golgi do oócito foi raramente observado. Em conclusão, este trabalho mostra pela primeira vez a caracterização da população de folículos pré-antrais do ovário da Dasyprocta leporina. Essas informações serão úteis para o desenvolvimento e adaptação de biotécnicas, como a criopreservação de germoplasma e manipulação de gametas in vitro.

Foram avaliados os efeitos da centrifugação associada ao uso de dois diluentes na manutenção da viabilidade espermática em tatus-peba (Euphractus sexcinctus) ao longo do teste de termorresistência (TTR). Amostras de sêmen (n=12), oriundas de 04 machos adultos coletados por eletroejaculação, foram divididas em quatro alíquotas, sendo duas imediatamente diluídas em Tris ou água de coco em pó (ACP-119(r)), e as outras duas centrifugadas (800g10min-1) previamente à diluição. As amostras foram incubadas a 34°C por 3h, e os parâmetros seminais avaliados em intervalos de 1h. Em termos gerais, verificou-se uma redução da viscosidade espermática imediata à diluição em ambos os diluentes, independente do uso da centrifugação. Aos 60 minutos, verificou-se uma redução dos parâmetros avaliados (P0,05). Ainda, verificou-se um efeito deletério da centrifugação sobre a qualidade do sêmen de tatus-peba durante todo o teste de termorresistência. Nas condições do presente estudo, conclui-se que o diluente Tris mostrou-se superior ao ACP-119(r) para a manutenção da viabilidade do sêmen de tatus-peba, sendo desnecessária a realização de centrifugação prévia à diluição. The effects of the centrifugation associated to the use of two extenders on the viability of six-banded armadillo's (Euphractus sexcinctus) sperm were evaluated during a thermo resistance test (TRT). Semen samples (n=12) derived from 04 stud males collected by electroejaculation were divided in four aliquots; two of that were immediately diluted in Tris or powdered coconut water (ACP-119(r)); the two others were centrifuged (800g10min-1) prior to the dilution. Samples were incubated at 34ºC per 3h, and the semen parameters were evaluated at each hour. In general, dilution promoted a reduction in semen viscosity in the use of both diluents using centrifugation or not. At 60min, a decrease was verified for all semen parameters (P

In order to establish protocols for gamete recovery from accidentally killed wild animals, or to take advantage of those slaughtered by captive breeders, we assess the influence of two methods on the recovery of epididymal sperm from collared peccaries, and verify the effect of centrifugation on such gametes. Genitalia from nine animals were used. For each animal, one epididymis was processed by flotation and the other was processed by retrograde flushing, both using a buffered media based on Tris. Following recovery, sperm were evaluated for motility, vigor, viability, functional membrane integrity, and morphology. A 1-mL aliquot of each sample was centrifuged, the supernatant removed, and the pellet suspended and evaluated as fresh samples. The sperm characteristics did not differ between the samples collected by flotation or retrograde flushing (P < 0.05). Centrifugation promoted an increase in head and tail defects, thus reducing the percentage of viable sperm (P < 0.05). No other parameter assessed for both methods was affected by centrifugation. In conclusion, epididymal sperm from collared peccaries can be efficiently collected through flotation or retrograde flushing, but not when either is followed by centrifugation.

The aim of this study was to evaluate environmental effects in a semiarid region on collared peccary seminal plasma content and sperm motility. Ejaculates from 12 mature males were obtained during the peak of rainy and dry periods of the Caatinga biome. Samples were evaluated for semen volume, pH, as well as sperm concentration, morphology, osmotic response, membrane integrity, chromatin condensation, and kinetic motility. Seminal plasma was evaluated for ions and organic compounds. The values for chloride, iron, magnesium, phosphorus, citric acid, cholesterol, triglycerides, total proteins, albumin, and fructosamine were similar during the dry and rainy periods; however, concentrations of fructose (849.2 mg/dL compared with 119.4 mg/dL) and calcium (32.3 mg/dL compared with 15.6 mg/dL) were greater during the rainy compared with dry period (P 0.05). There were correlations (P 0.05) among values for semen variables and biochemical contents, particularly between fructose and sperm velocity average pathway (r = 0.65), velocity straight line (r = 0.78), velocity curvilinear (r = 0.57), amplitude lateral head (r = 0.62), linearity (r = 0.41), and subpopulation with a medium velocity (r = -0.75). Furthermore, values for relative humidity were positively correlated with concentrations of fructose (r = 0.49), while air temperature (r = -0.43) and wind velocity values (r = 0.66) were negatively affected by concentration of fructose (P 0.05). There were novel results regarding collared peccary seminal plasma biochemistry indicating there are important correlations with values for semen variables that are affected by the environment in a semiarid climate.

The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide - PI and Hoechst - H342), and mitochondrial activity (CMXRos - Mito Tracker Red®). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol. Resumo: O objetivo deste estudo foi criopreservar o sêmen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelação-descongelação, utilizando marcadores fluorescentes e análise ultraestrutural. As amostras de sêmen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculação foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rápida de congelação. A análise clássica das amostras foi realizada após a diluição, refrigeração e descongelação, seguida por análise de fluorescência, utilizando uma combinação de sondas fluorescentes para avaliar a integridade da membrana (Iodeto de Propídio - PI e Hoechst - H342), e a atividade mitocondrial (CMXRos - Mito Tracker RED®). Foi também utilizada a análise ultraestrutural para verificar possíveis alterações morfológicas causadas pela crioinjúria. Quando comparadas com as amostras a fresco, verificou-se uma queda significativa em todos os parâmetros seminais dos tatus após a descongelação, em que apenas 6,1% de espermatozoides móveis foram encontrados. No entanto, o percentual de espermatozoides que permaneceu com membrana viável (13%) e funcional (24,7%) após a descongelação sugere que algumas células podem estar vivas, mas imóveis. Análises utilizando marcadores fluorescentes revelaram que as mitocôndrias dos espermatozoides de tatus são altamente sensíveis ao protocolo de congelação e os achados através da análise ultraestrutural comprovaram esta afirmação. Além disso, as imagens obtidas por microscopia eletrônica de transmissão revelaram que espermatozoides congelados-descongelados apresentaram membranas plasmáticas danificadas, modificações nucleares como alterações na cromatina, e alterações acrossomais relativas à capacitação espermática. Em conclusão, este estudo é a primeira tentativa de criopreservação de sêmen em uma espécie de tatu, e nos auxiliou a identificar pontos críticos no processo de congelação-descongelação, a fim de melhorar o protocolo.

The study of the seminal plasma help us to understand the mechanisms by which reactive oxygen species (ROS) affect the sperm. The antioxidant enzymes, as the superoxide dismutase - SOD and catalase - CAT, are capable of removing the oxidative agents before they produce injuries. The aim of the current study was to investigate the activity of antioxidant enzymes SOD and CAT in seminal plasma, and their association with sperm quality in collared peccaries. Study was conducted during the dry period (August and September) on a region characterized by a semiarid climate, with an average annual temperature of 27°C and irregular rainfall (Mossoro, RN, Brazil; 5°10´S and 37°10´W). Nine ejaculates were obtained from sexually mature males (1 sample per animal) by electroejaculation. Semen was evaluated for microscopic parameters and the activity of SOD and CAT was measured by spectrophotometry. All ejaculates were white in color. Mean values for concentration were of 207 ± 160 x106 sperm/mL, motility of 83.0 ± 20.9% and viability of 72.5 ± 10.4%. In regards to the enzymatic activity, none was observed for the CAT enzyme. Trace levels of SOD (0.034 ± 0.049 AU/mgP) were detected in the ejaculates of all individuals; however, no correlation was observed between SOD levels and the sperm motility (R = 0.35; P = 0.931), vigor (R = 0.29; P = 0.133), viability (R = 0.16; P = 0.29), functional membrane (R = 0.04; P = 0.617) or morphology (R = 0.03; P = 0.637). In conclusion, we demonstrated the first description of antioxidant enzyme activity in seminal plasma of fresh ejaculates obtained from collared peccaries. SOD antioxidant activity was evident during the dry period of a semi-arid region, but no relationship between SOD and semen parameters was observed.

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

We verify the efficiency of a protocol for estrus synchronization in captive female collared peccaries (Pecaricari tajacu) using the prostaglandin analog D-cloprostenol. Five adult female collared peccaries received an intramuscular administration of 60 µg D-cloprostenol, which procedure was repeated after a 9-day interval. For 10 days after second the D-cloprostenol administration, females were monitored for changes in external genitalia, ovarian ultrasonography, vaginal cytology and reproductive hormonal dosage. As a result, four females synchronized their estrous at 9.5 ± 0.5 days after the second administration of the prostaglandin analog. Such females showed external signs of estrus, including vulvar opening, hyperemic vaginal mucosa, and vaginal mucus, concomitant with an increase in the proportion of superficial cells (52.2 ± 9.9%) verified through vaginal cytology. An estrogen peak of 22.7 ± 3.4 pg/ml was detected by hormonal dosage, and the presence of anechoic follicles measuring 0.29 ± 0.05 × 0.32 ± 0.07 mm were detected in the ovary by ultrasonography. Given these findings, we suggest that D-cloprostenol may be effective for use in estrus synchronization in collared peccaries.

O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune do tecido conectivo, que apresenta diversas manifestações clínicas e sorológicas. Embora sua causa seja desconhecida, sabe se que o LES é uma doença multifatorial, no qual pacientes apresentam uma deficiência no reparo de quebras de dupla fita de DNA (DSBs), causada tanto por agentes endógenos quanto exógenos. O gene STK17A (Serina/Threonina quinase 17A) pode estar envolvido no desencadeamento da doença, uma vez que codifica uma proteína que participa do processo de reparo de DSBs e apoptose. Deficiência nesse processo pode induzir a produção de anticorpos anti ds-DNA e consequente deposição de imunocomplexos, causando inflamação nos tecidos. Neste estudo, foi investigada a associação de cinco polimorfismos de base única (SNPs) no gene STK17A com a susceptibilidade ao LES e as principais manifestações clínicas da doença. O grupo de estudo foi composto por 143 pacientes com LES e 177 indivíduos saudáveis como grupo controle. A genotipagem foi realizada pela metodologia de PCR em tempo real ABI7500HT (Applied Biosystems, Foster City, CA, USA) utilizando Probe Taqman SNP Genotyping Assay. As frequências alélicas e genotípicas foram calculadas através do programa Genotyper Transposer e avaliadas para o equilíbrio de Hardy-Weinberg. As análises estatísticas foram realizadas pelo teste exato de Fisher, juntamente com o programa R, versão 2.1.1 para verificar associação entre os SNPs testados e a susceptibilidade à doença. A distribuição haplotípica foi analisada através do programa SNPstat. A avaliação da associação dos alelos e genótipos dos SNPs com as manifestações clínicas e sorológicas da doença foi realizada pelo EpiInfo (versão 3.5.2) e teste exato de Fisher. Para ajustar os valores de p-values para testes múltiplos, foi aplicada a correção de Bonferroni. Foi observada diferença significativa quando comparados pacientes e controles após a estratificação para o sexo: O genótipo A/A do SNP rs10259269 se mostrou mais frequente nos controles (7.6%) do que nos pacientes (0,7%) conferindo proteção contra o desenvolvimento do LES no sexo feminino (OR = 0,09, p = 0,01). Quando analisada a distribuição dos haplótipos, foi encontrada associação significativa em pacientes com LES entre dois haplótipos: TGGTT e TAGTC (OR = 0,54, p = 0,01 e OR = 0,25, p = 9.04e-08, respectivamente) com efeito protetor contra o desenvolvimento da doença. Interessantemente, após estratificação para etnia (descendentes europeus) e sexo (feminino) o haplótipo TAGTC novamente conferiu proteção ao LES (OR = 0,25, p = 2.30e-06 e OR = 0,41, p = 0,01 respectivamente). Uma associação significativa foi observada entre o genótipo A/G para o SNP rs7802995 com manifestação cutânea em pacientes com LES (OR = 3,44, p = 0,004). Finalmente, depois da estratificação dos pacientes para etnia, foi encontrada uma associação significativa com a alteração sorológica anti ds-DNA na proteção de descendentes de Africanos (OR = 0,11, p = 0,009). Não foi observada associação entre o sexo e as manifestações clínicas/laboratoriais. Em síntese, concluímos que polimorfismos no gene STK17A podem estar envolvidos no desenvolvimento do LES, entretanto, outros estudos de réplica avaliando o efeito funcional desses SNPs na expressão e/ou atividade da proteína são necessários para confirmar o seu papel na susceptibilidade/proteção à doença.

The aim of this research was to study the individual variation with regard to the morphometry of the testes evaluated by ultrasonography and semen characteristics and to verify the existence of relationship between these variables in collared peccaries. In addition, the testes of the animals were evaluated by histology in order to determine the proportion occupied by the seminiferous tubules. A total of 52 ejaculates were obtained from ten adult specimens that had been restrained by anesthesia. The testicular measurements (length, height, and width) were performed by ultrasonography, and the testicular volume was calculated according to Lambert's formula. The scrotal circumference was measured by encircling the thickest portion of the testicle with a graduated nylon tape. The semen was collected by electroejaculation. Testicular fragments were analyzed through classic histology for the determination of the area occupied by the seminiferous tubules. The results show a great amount of individual variation with regard to testicular morphometry and semen characteristics. No significant correlations were obtained between testicular measurements and semen characteristics. The histometric analysis revealed that 67.8% of the testes are occupied by seminiferous tubules. Results show that the measurement of testicular dimensions does not serve as an indicator of the quality of semen obtained by electroejaculation in collared peccaries, as there is no correlation between testicular morphometry and semen characteristics in this species that presents large variations among individuals.