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.

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue.

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α-MEM+ alone or supplemented with melatonin (250, 500, 1,000 or 2,000 pM) for a period of six days. Non-cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p < .05. The results showed that the presence of 1,000 and 2,000 pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500 pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000 pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000 pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250 pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000 pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.

Ultrastructural description of fresh and frozen/thawed sperm derived from collared peccaries (Pecari tajacu Linnaeus, 1,758).

The aim was to describe, through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the ultrastructure of peccaries' fresh and frozen-thawed sperm. For that, semen derived from three mature males was obtained by electroejaculation and evaluated for motility, membrane integrity, membrane functionality, chromatin integrity, and morphology through light microscopy. Samples were frozen using a Tris extender plus egg yolk (20%) and glycerol (6%). Then, fresh and frozen-thawed semen samples were mixed in different sperm pools that were processed for SEM and TEM. Sperm motility, membrane integrity, and functionality were impaired (p < .05) by freezing-thawing procedures, but sperm morphology, and chromatin integrity evaluated by light microscopy were not significantly affected. The SEM revealed that peccaries' sperm presents a flattened head in a paddle format, measuring 6.07 µm in length and 3.84 µm in width, with a vastus acrosome (4.46 µm). Normal tails measure 38.11 µm, being formed by an extensive midpiece with 15.52 µm in length. In frozen-thawed samples, both SEM and TEM provide us information about damage undetected through light microscopy as the presence of vesicles in the acrosome, loose plasma membrane, vacuolized mitochondria, dense fibers disorganized, and decondensed chromatin. In conclusion, we provide the first description of the sperm ultrasctruture in collared peccaries. Moreover, SEM and TEM help us to identify some nanometric damage provoked by freezing-thawing procedures, thus providing valuable information for the improvement of such important protocols used for biobanking formation.

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.

Sperm characteristics following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins in the collared peccary (Pecari tajacu).

The aim of the current study was to compare sperm quality characteristics of the collared peccary (Pecari tajacu) following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins (LDL). Semen from 11 adult males was obtained by electroejaculation and evaluated for sperm motility, vigor, morphology as well as membrane integrity analyzed by the hypo-osmotic swelling (HOS) test and a fluorescent staining. Moreover, the semen was diluted in a Tris-based extender containing 20% egg yolk (control group) or 5, 10 or 20% LDL (treatment groups). The semen samples were frozen in liquid nitrogen and thawed in a water bath for 60s at 37°C. The treatments did not affect (p>0.05) sperm vigor, morphology or membrane integrity analyzed by the HOS test. However, post-thaw sperm motility was significantly higher (p<0.05) in the extender supplemented with 20% LDL (36.4 ± 5.3%) compared with the egg yolk extender and extender supplemented with 10% LDL. Furthermore, the percentage of membrane-intact frozen-thawed spermatozoa analyzed by the fluorescent staining was significantly higher (p<0.05) in the extender supplemented with 20% LDL (27.4 ± 6.5%) than in the other groups. In conclusion, 20% LDL can be used to substitute the whole egg yolk as a cryoprotective additive for freezing semen of the collared peccary.