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.

In this study, sperm from Africanized honey bee drones obtained during the dry and wet seasons of a semiarid region, the Caatinga biome, were characterized. The experiment was conducted during the peaks of the wet (March to June 2018) and dry (October to December 2018) seasons. Significantly higher cumulative rainfall was observed during the wet season (475.2 mm) in comparison to the dry season (27.6 mm). In each season, sperm from 50 Africanized honey bee (Apis mellifera L.) drones were obtained using the manual endophallus eversion technique and were evaluated in terms of the number of sperm per drone, motility, viability, morphology, and morphometry. The number of sperm per drone, the sperm motility, and the viability showed significantly better mean values during the wet season than in the dry season (3.8 ± 8.0 × 10⁶ sperm/drone vs 3.0 ± 6.6 × 10⁶ sperm/drone; 85.6 ± 8.5% vs 52.0 ± 2.9%; 82.8 ± 1.9% vs 70.9 ± 1.8%, respectively). A high incidence of sperm morphological defects (∼60%) was observed in both periods and was significantly more evident during the wet season. All morphometric parameters related to the length of the sperm head, tail, and total sperm were significantly higher during the wet season than during the dry season. In conclusion, honey bee drones present better sperm quality during the wet season than during the dry season in the Caatinga biome.

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.

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 10⁶ spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

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.

The effect of Equex STM^® paste supplementation on the Tris-extender for collared peccaries' semen cryopreservation was assessed. Semen from 12 mature individuals was obtained by electroejaculation and evaluated for morphology, membrane integrity, osmotic response, and sperm kinetic metrics. Samples were diluted in Tris plus 20% egg yolk and divided into three aliquots. The first aliquot was without any supplementation, the second and third contained 0.5 and 1.0% Equex STM, respectively. The samples were added with 3% glycerol, frozen in liquid nitrogen, thawed, and assessed for the same parameters after a thermal resistance test (TRT) for 120 minutes. Similar values were detected for the different treatments immediately after thawing, except for the amplitude lateral head that was reduced in samples containing Equex (p < 0.05). During TRT, samples containing Equex were more efficient in preserving the sperm motility (at 0.5%: 25.5% ± 4.4%; at 1%: 33.3% ± 6.3%) at 30 minutes, in comparison with the control group (16.6% ± 6.0%), in which sperm motility decreased at 15 minutes (p < 0.05). Moreover, Equex, especially at 0.5% concentration, was able to maintain plasma membrane integrity and sperm motility in all the samples after incubation for 60 minutes. In conclusion, we recommend the addition of Equex STM at 0.5% to the Tris-extender to improve post-thawing sperm longevity in collared peccaries.