ObjectiveThis study aimed to test the effects on sperm viability of transporting cryopreserved semen samples on dry ice.MethodsTwenty normozoospermic semen samples were cryopreserved and divided into five groups. The samples in Group 1 were immersed in liquid nitrogen throughout the experiment in cryogenic storage tanks; the cryopreserved straws in Group 2 were placed in a Styrofoam box containing dry ice and kept under these conditions for 48 hours; the samples in Group 3 were kept for 48 hours on dry ice under the same conditions as the Group 2 samples, and were then moved to a storage tank filled with liquid nitrogen; Group 4 samples were also kept for 48 hours in dry ice storage, and the Styrofoam box containing the samples was shipped by plane to assess the effects of shipping; the samples in Group 5 were shipped together with the Group 4 samples and were placed in a storage tank with liquid nitrogen after spending 48 hours stored on dry ice. After thawing, sperm parameters were analyzed for viability, vitality, and motility; spermatozoa were also tested for mitochondrial activity.ResultsSignificant decreases in motility recovery rates (P=0.01) and vitality (P=0.001) were observed in all groups when compared to the control group. Mitochondrial activity was significantly decreased only in Group 5 (P=0.04), as evidenced by greater numbers of sperm cells not stained by reagent 3,3'-diaminobenzidine.ConclusionsTransportation did not affect the quality of cryopreserved semen samples, but dry ice as a means to preserve the samples during transportation had detrimental effects upon the sperm parameters assessed in this study.

SS is an efficient technique for the recovery of motile spermatozoa from native semen preparations and yielded better results than SF and SL. Routine use for assisted reproduction remains to be evaluated.

ObjectiveTo evaluate storage time effects in cryopreserved human semen samples, kept in the freezer at a controlled temperature of -80°C, on sperm viability after thawing.MethodsWe used 20 semen samples. Each sample was cryopreserved in 10 fingers, which were divided into five groups: one group was kept in cryogenic canisters throughout the experiment(control), and four groups were kept in a VIP Ultra Low MDF-U76V- PE freezer, with the temperature set at -80°C, for 24, 48, 72 and 96 hours, respectively. After the exposure time, the samples were stored in cryogenic canisters after being thawed. The analyzed parameters were: motility, vitality and mitochondrial activity.ResultsAfter thawing, we noticed decreased sperm motility, vitality and mitochondrial activity, when comparing the tested groups with the control group, as well as a progressive reduction in the analyzed parameters between the times evaluated.Conclusions Cryopreservation of semen samples at -80°C is potentially harmful to sperm viability, causing damage when submitted to longer exposure times.

Background: The present study evaluates the effects of energy drinks on the reproductive and biochemical parameters of adult male rats. Methods: A total of 40 male rats (Wistar) were exposed to an energy drink mixed with the drinking water for a period of 120 days. The animals were divided into four groups and exposed to increasing therapeutic doses (DT) of an energy drink, based on allometric extrapolation, resulting in values (mL/day) per animal of 250 g: DT1 2.36 mL, DT3 7.47 mL, and DT6 14.16 mL. The control group (CTRL) consumed water only. During the treatment, the rats were assessed for signs of toxicity. After treatment, the animals were sacrificed and their organs were weighed. Sperm parameters (motility, concentration, and morphology) were evaluated. The biochemical markers alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactic dehydrogenase, urea, creatinine, creatine phosphokinase, and creatine kinase MB fraction were measured, in addition to total cholesterol and testosterone. Results: There was a significant decrease (p < 0.05) in the concentration of sperm in the treated groups (DT1 8.5 ± 0.7; DT3 7.2 ± 0.9; DT6 8.4 ± 0.9) compared to the control group (12.3 ± 1.2). No difference was observed with respect to relative weights of the animals' organs, water consumption, signs of toxicity, behavioral changes, biochemical markers, and sperm motility and morphology. Conclusion: The long-term consumption of energy drinks interferes negatively with sperm concentration, without affecting sperm motility and morphology or altering the hepatic, cardiac, or renal functions.

ObjectiveTo evaluate the efficiency of two vitrification protocols for rat immature testicular tissue and heterotopic transplantation.MethodsTwenty-four pre-pubertal Wistar rats were divided into three groups (n=8). After orchiectomy, testicular fragments (3mm) from Groups 1 and 2 were vitrified with different cryoprotectant concentration solutions, using sterile inoculation loops as support. After warming up, the fragments were submitted to cell viability assessment by Trypan blue and histological evaluation. Vitrified (Groups 1 and 2) and fresh (Group 3) fragments were grafted to the animals periauricular region. After 8 weeks of grafting, the implant site was histologically analyzed.ResultsThe viability recovery rate from Group 1 (72.09%) was higher (p=0.02) than that from Group 2 (59.19%). Histological analysis showed similar tubular integrity between fresh fragments from Groups 1 and 3. Group 2 samples presented lower tubular integrity. We ran histological analyses in the grafts from the Groups. In all groups, it was possible to see the implant site, however, no fragment of testicular tissue or signs of inflammation were histologically found in most samples from Groups 1 and 3. In one sample from Group 2, we found degenerated seminiferous tubules with necrosis and signs of an inflammatory process. In another sample from Group 2, we found seminiferous tubules in the implant site.ConclusionThe vitrification of pre-pubertal testicular tissue of rats showed little damage to cell viability through histological analysis when we used cryoprotectants in a lower concentration. Heterotopic transplantation could not preserve the structural organization of the testicular tissue.

ObjectiveTo evaluate the efficiency of ovarian tissue heterotopic autografting after vitrification in prepubertal rats.MethodsFragments of excised ovaries from prepubertal rats were used after assessing post-warming cellular viability, to determine the best vitrification protocol prior to retroauricular autografting. Pre-pubertal females (N=24) were castrated and divided into three group: Group 1 - fresh ovarian tissue transplantation; Group 2 - vitrified/warmed tissue transplantation; Group 3 - bilateral oophorectomy without transplantation. The ovarian fragments were exposed to solutions from the Ingamed® commercial kit, allocated in bacteriological loops and immersed in liquid nitrogen. Sixty days after transplantation, a vaginal mucus sample was collected for cytology tests, followed by sacrificing the animal, performing a cardiac puncture for collecting a blood sample to determine luteinizing hormone and estradiol levels, and excision of the transplanted fragment for histology tests.ResultsVaginal cytology revealed that 87.5% of females from groups 1 and 2 had estrus while all females in Group 3 remained in diestrus. The mean LH value in groups 1 (0.08 mIU/mL) and 2 (0.34 mIU/mL) were statistically different from that of Group 3 (2.27 mIU/mL). E2 values did not differ between the groups. The histological analysis of Group 1 excised grafts versus those from Group 2 showed a higher percentage of primary follicles (62.5% vs. 12.5%), developing follicles (75% vs. 25%), corpus luteum (37.5% vs. 12.5%) and stromal region (100% vs. 87.5%).ConclusionThis study indicated that pre-pubertal ovarian tissue vitrification can be used to preserve fertility and to restore endocrine function in castrated rats.

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objectives: To evaluate the efficiency of a vitrification protocol of murine blastocysts, varying the exposure time to the equilibrium solution, at different degrees of blastocoel expansion. Methods: Sixty female mice were superovulated with 10 IU of equine chorionic gonadotropin (eCG, Novormon  , Syntex, Argentina) and after 48 hours with 10 IU of human chorionic gonadotropin (hCG, Vetecor  , Calier, Barcelona, Spain). Females were then coupled with males overnight, and the presence of vaginal plug indicated that mating had occurred. Three days later, females were euthanized and the embryos at the morula stage (n = 925) were recovered by flushing the uterine tubes with GV-Hepes medium (Ingámed  , Maringá, Brazil). Embryos were then cultivated until the blastocyst stage and classified, at the time the vitrification process was initiated, into 3 groups (G1, G2, G4) according to the degree of blastocoele expansion. A control group remained in culture until hatching. All embryos, after warming, were put back into culture for measurement of the rehydration (RR) and hatching (HR) rates. Results: For G1 group, the mean RR and HR obtained over a range of exposure times to the equilibration solution of 94.0 and 84.4%, respectively. For G2, these rates were 95.2 and 87.1%, respectively. For G1 and G2, RR and HR obtained after an equilibration time of 9 min were statistically higher than those obtained after 10, 11, 12, 13 and 14 min. For G4, RR and HR were 76.4 and 69.9%, respectively, but in this case, an equilibration time of 15 min presented statistically higher survival rates compared to shorter exposition times. conclusions: The results of this study indicate that the variation in the exposure time of the embryos to the equilibration solution influences significantly the rehydration and hatching of the blastocysts, and the degree of blastocoel expansion is inversely correlated with the survival and development potential after warming.