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

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

Influence of antibiotics on bacterial load and sperm parameters during short-term preservation of collared peccary semen.

Studies on semen and sperm cells are critical to develop assisted reproductive technologies for the conservation of the collared peccary. The objective of the study was to compare the effect of different antibiotics on the bacterial load and sperm quality during short-term storage of peccary semen. Fresh semen samples from 10 males were extended in Tris-egg yolk or Tris-Aloe vera supplemented with streptomycin-penicillin (SP; 1 mg/mL - 1000 IU/mL or 2 mg/mL - 2000 IU/mL) or gentamicin (30 µg/mL or 70 µg/mL) before storage at 5°C. Bacterial load and sperm motility, membrane integrity and function, mitochondrial activity, and morphology, were evaluated at different time points for 36 h. The SP and gentamicin treatments concentration inhibited (p < 0.05) bacterial growth for 36 h regardless of the extender. Compared to the other treatments, Tris-egg yolk plus 70 µg/mL gentamicin maintained the sperm parameters for longer, including total motility (41.9 ± 6.1%) at 24 h, and membrane integrity (58.3 ± 2.1%) at 36 h. In contrast, the highest SP concentration in both extenders impaired sperm membrane integrity at 36 h (p < 0.05). For the liquid storage of collared peccary semen, it therefore is recommended to use Tris extender supplemented with egg yolk and gentamicin (70 µg/mL).

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

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

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

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

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

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

Comparison of different extenders on the recovery and longevity of epididymal sperm from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831).

The 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).

Estimating the binding ability of collared peccary (Pecari tajacu Linnaeus, 1758) sperm using heterologous substrates.

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.

Estrous synchronization in captive collared peccaries (Pecari tajacu) using a prostaglandin F2α analog.

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.