Extenders with vitamins C and E applied to Rhamdia quelen sperm cryopreservation / Extensores com vitaminas C e E aplicados à criopreservação de esperma de Rhamdia quelen

We performed this experiment to evaluate the effects of adding vitamins C and E on extenders for sperm cryopreservation of Rhamdia quelen over spermatic mobility after thawing. At cryopreservation, sperm samples were diluted in a proportion of 1:3 (v/v), following pre-freezing in nitrogen steam and subsequent immersion in liquid nitrogen. The diluents were composed by 5% milk powder, 5% glucose, 10% methanol and different levels of vitamin. Three sperm cryopreservation tests were carried out with (1) diluent containing 0.0; 4.0; 6.5; 9.0 and 11.5 mg of vitamin C mL-1, (2) diluent containing 0.0; 2.0; 4.0; 6.0 and 8.0 mg of vitamin E mL-1; (3) diluent containing 0.0; 4.0 + 2.0; 6.5 + 4.0; 9.0 + 6.0 and 11.5 + 8.0 mg of vitamin C mL-1 plus vitamin E mL-1, respectively. The spermatic motility rate, spermatic curvilinear velocity, average path and straight line velocities were measured in thawed semen by CASA. Data were submitted to ANOVA and Duncan´s test at 5% of significance. After thawing the effect (P0.05) of vitamin C was observed only for sperm motility, with higher values (38.2±20.7%) on solution containing 4.0 mg of vitamin C mL-1. The concomitant addition of both vitamins influenced (P0.05) only the curvilinear velocity, reducing the velocity at any concentration. In conclusion, diluents with 4.0 mg vitamin C mL-1 to cryopreservation of the silver catfish semen improve the sperm quality after thawing, and the use of diluents with vitamin E or both vitamins are not recommended because do not ensure the cells protection.

Effect of Sperm Cryopreservation on miRNA Expression and Early Embryonic Development

Although sperm preservation is a common means of personal fertility preservation, its effects on embryonic development potential need further investigation. The purpose of this study was to identify key microRNA (miRNA) in cryopreserved sperm and determine the changes of these miRNAs and their target genes during embryonic development using cryopreserved sperm. Moreover, the embryonic development potential of cryopreserved sperm was estimated in assisted reproductive technology (ART), where key miRNAs and target genes were validated in sperm and subsequent embryos. Clinical data of embryonic development from cryopreserved sperm indicated a significant decrease in fertilization rate in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cases, as well as a reduction in blastocyst formation rate in ICSI cases. Meanwhile there was a significant increase in blocked embryo ratio of Day1, Day2, and Day3.5 embryos when frozen-thawed mouse sperm was used, compared with fresh mouse sperm, suggesting a potential negative effect of sperm cryopreservation on embryonic development. From frozen-thawed and fresh sperm in humans and mice, respectively, 21 and 95 differentially expressed miRNAs (DEmiRs) were detected. miR-148b-3p were downregulated in both human and mouse frozen-thawed sperm and were also decreased in embryos after fertilization using cryopreserved sperm. Target genes of miR-148b-3p, Pten, was identified in mouse embryos using quantitative real-time PCR (qRT-PCR) and Western blot (WB). In addition, common characters of cryopreservation of mouse oocytes compared with sperm were also detected; downregulation of miR-148b-3p was also confirmed in cryopreserved oocytes. In summary, our study suggested that cryopreservation of sperm could change the expression of miRNAs, especially the miR-148b-3p across humans and mice, and may further affect fertilization and embryo development by increasing the expression of Pten. Moreover, downregulation of miR-148b-3p induced by cryopreservation was conserved in mouse gametes.

Oxidative Stress Measurement in Frozen/Thawed Human Sperm: The Protective Role of an In Vitro Treatment with Myo-Inositol

Despite its widespread use, sperm cryopreservation induces serious detrimental alterations in sperm function; indeed, it is commonly associated with decreased sperm viability and motility, and DNA fragmentation. Mechanisms of human sperm cryodamage are thought to be multifactorial, but oxidative stress seems to have a prominent role. A huge amount of data supported the cryoprotective effect of different antioxidants able to minimize the detrimental effects of reactive oxygen species (ROS) and improve the quality of spermatozoa. Among others, myo-inositol is one of the most powerful and has been reported to be effective in improving sperm quality and motility when used both in vivo and in vitro. This study aimed to determine the in vitro impact of myo-inositol in ameliorating sperm oxidative status during sperm cryopreservation. In particular, we demonstrated a significant improvement of sperm parameters (vitality and motility) when myo-inositol was added after sperm thawing (p < 0.05). Moreover, we showed that myo-inositol induces a significant increase in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production. Finally, by means of 2D-electrophoresis, we demonstrated a significant decrease in the level of carbonyl groups, the main structural changes occurring in conditions of oxidative stress (p < 0.05). In conclusion, the sperm cryopreservation procedure we developed, assuring the reduction of ROS-induced sperm modifications, may improve the in vitro procedure currently used in ART laboratory for sperm cryostorage.