Ana Glória Pereira

The cryopreservation of male gonadal tissue is critical to conserve genetic material and use it later via assisted reproduction. This study aimed to evaluate cryopreservation methods (slow freezing, SF; solid surface vitrification, SSV) as well as the optimal concentrations of intracellular cryoprotectants during the SSV of testicular tissue from prepubertal collared peccaries. Five pairs of testes were dissected on different days into small fragments (3 mm³) and allocated to a non-cryopreserved, a control group or one of three treatment groups: SF; SSV 3 M (1.5 M dimethyl sulfoxide [DMSO] plus 1.5 M ethylene glycol [EG]); or SSV 6 M (3 M DMSO plus 3 M EG). After one week of storage in liquid nitrogen, tissue samples were warmed and evaluated in terms of histology, viability, proliferative capacity potential, and DNA integrity. The scores for histological integrity and cellular damage for SF (2.08 ± 0.05 and 2.33 ± 0.07, respectively) were similar to the results found in SSV 6 M (1.93 ± 0.04 and 2.30 ± 0.07; p > 0.05). However, these scores were better when compared to SSV 3 M (1.87 ± 0.05 and 2.08 ± 0.06; p < 0.05). The percentage of cellular viability was around 57% after all preservation treatments (p > 0.05), which was lower than in the control group (88.8 ± 1.9%; p < 0.05). The SSV 6 M treatment was better than the other treatments regarding the proliferative capacity potential of spermatogonia cells (3.52 ± 0.03) (p < 0.05), although it was lower than in the control group (4.00 ± 0.12) (p < 0.05). Additionally, SSV 6 M led to the same DNA integrity (97.0 ± 0.7%) as in the control group (99.4 ± 0.3%). These collective findings suggest that the combination of SSV with 6 M cryoprotectants is the most efficient for the cryopreservation of testes from prepubertal collared peccaries.

This study aims to advance knowledge of the reproductive aspects of the red-rumped agouti (Dasyprocta leporina). The objective was to describe the organic and inorganic constituents present in the seminal plasma of agouti specimens analyzed under different environmental conditions in a semiarid region. Seminal plasma was collected from six adult males via electroejaculation during both the dry and rainy seasons. Organic components (total proteins, albumin, cholesterol, triglycerides, fructose, and glucose) and inorganic components (phosphorus, magnesium, calcium, iron, chloride, sodium, and potassium) were analyzed. Significantly higher concentrations of glucose (88.24 vs. 26.27 mg/dL), phosphorus (66.40 vs. 3.67 mg/dL), and potassium (92.67 vs. 19.68 mmol/L) were detected during the dry season compared to the rainy season (p < 0.05). Conversely, chloride concentrations (43.04 vs. 201.40 mEq/L) were significantly higher during the rainy season (p < 0.05). This study provides the first description of the biochemical composition of seminal plasma in this species, revealing important seasonal variations in these constituents within the Caatinga biome.

For the development of efficient management and conservation strategies for wild rodent species, understanding the aspects related to their reproduction, including the interaction of this physiological function with the environment, is essential. This study aimed to evaluate the impacts of weather changes derived from a semiarid region’s dry and rainy seasons on the epididymal sperm characteristics of red-rumped agouti (Dasyprocta leporina), a histricognath rodent, still little studied, which inhabits the Brazilian Caatinga. The sperm from the epididymal cauda of 14 agoutis were collected, seven individuals per season (dry and rainy). Samples were evaluated for kinetic parameters, membrane structural and functional integrity, mitochondrial activity, morphology, and morphometry. The environmental variables were measured: maximum air temperature, relative humidity, wind speed, solar radiation, and the total rainfall for dry and rainy seasons were, respectively, 36.2 and 34.1 °C, 66.8 and 80.1%, 4.0 and 1.9 m/s, 527.3 and 441.8 W/m², and 0.2 and 517.7 mm. There were strong correlations between some sperm parameters and environmental variables, mainly those related to the acquisition of sperm mobility. Sperm concentration and the number of sperm collected were higher in the dry (1028.7 sperm/mL × 10⁶ and 1361.2 × 10⁶ sperm) than in the rainy season (758.9 sperm × 10⁶/mL and 714.6 sperm × 10⁶). During the rainy season, there were fewer sperm defects, higher sperm metrics, and higher membrane structural integrity with mitochondrial activity. Regarding motility patterns, the increases during the rainy season stand out in total and progressive motility, VAP, VSL, VCL, and subpopulations of rapid sperm. In summary, our results suggest that the adverse climatic conditions of the dry season in the semiarid region, mainly high solar radiation and temperature, considerably impair the epididymal sperm quality of red-rumped agoutis. On the contrary, the largest amount of sperm was obtained during this season, probably due to compensatory and adaptive mechanisms of the species to enable its reproduction throughout the year.

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.

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.

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.

The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean ± SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 ± 2.5%. The bromophenol blue staining technique revealed that 64.6 ± 5.2% sperm were viable, while that proportion was 72.1 ± 2.5% using eosin-nigrosine. An average of 77.6 ± 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 ± 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.

This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm³) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface vitrification (SSV), and conventional vitrification (CV). Each method used dimethyl sulfoxide (DMSO), ethylene glycol (EG), or a DMSO + EG combination. Morphology, viability, mitochondrial activity, and proliferative potential were assessed in fresh and frozen tissue samples. Testicular morphology was better using SSV with a combination of DMSO and EG. Across the different cryopreservation approaches, as well as cryoprotectant combinations, cell viability was comparable. Regarding mitochondrial activity, DMSO + EG/SSV or CV, and DMSO + EG/CV were similar to the EG/SF group, which was the best group that provided values similar to fresh control groups. Adequate preservation of the proliferative potential of spermatogonia, Leydig cells, and Sertoli cells was obtained using SSV with DMSO + EG. Overall, the use of SSV with DMSO + EG was the best protocol for the preservation of testicular tissues from adult red-rumped agoutis.