Cryo letters最新文献

Backgrund: Goldfish Carassius auratus is a popular ornamental fish extensively cultured worldwide. Sperm cryopreservation is a common fish breeding method that ensures sperm availability around the year. Studies on cryopreservation of goldfish sperm, especially on the suitability of cryoprotectant types and pre-freezing time, are scarcely available.

Objective: To determine the most suitable type of cryoprotectant and pre-freezing for the successful cryopreservation of goldfish sperm.

Materials and methods: A completely randomized design with two factors was utilized in this study. The first factor is the type of cryoprotectants, which included methanol, ethanol, ethylene glycol, glycerol, and DMSO. The second is pre-freezing times of 10, 20, 30, and 40 min at each of the pre-freezing temperatures of 4 degree C, -10 degree C, and -79 degree C, meaning that the total times for the ramping down of temperature were 30, 60, 90 and 120 min, respectively. The Ringer solution and 10% egg yolk were used as extender and extracellular cryoprotectant. The sperm was stored at -179 degree C for 7 days.

Results: The ANOVA test showed that cryoprotectants and pre-freezing significantly affected the motility, viability, and fertility of goldfish sperm after freezing in liquid nitrogen for 7 days (P<0.05). Furthermore, 10% DMSO combined with 15% egg yolk with an pre-freezing time of 20 min can maintain sperm motility, viability, and fertility higher than other treatments, by 79%, 80%, and 33%, respectively. The agarose gel electrophoresis showed no DNA fragmentation in all samples, including fresh sperm.

Conclusion: We conclude that 10% DMSO combined with 15% egg yolk and 20 min pre-freezing is the best treatment for goldfish sperm cryopreservation. DOI: 10.54680/fr23310110412.

Background: Indian Wild Orange (Citrus indica Tanaka) is an endangered and endemic species from northeast India for which effective ex situ conservation strategies, including embryo cryopreservation, are urgently needed.

Materials and methods: Desiccation tolerance and cryopreservation ability for embryonic axes of Citrus indica was determined using three techniques (air desiccation-freezing, PVS2 vitrification-freezing and encapsulation-dehydration-freezing). Success was assessed as survival and recovery in vitro.

Results: Successful cryopreservation of embryonic axes was achieved using all three methods, with the highest survival achieved when using air desiccation-freezing (90%) followed by encapsulation-dehydration (85%) and PVS2 vitrification cryopreservation (80%). Regeneration levels were lower than survival levels for all three proceedures. Post-cryo regeneration success was: encapsulation-dehydration (64%) > air desiccation-freezing (55%) > PVS2 vitrification (52%).

Conclusion: Although there was relatively high post-cryopreservation recovery growth obtained using all the three techniques, the air desiccation-freezing technique is preferred, as it is a simple, practical and reproducible technique for the long-term cryobanking of this important wild species. Doi: 10.54680/fr23310110512.

Background: Vitrification of embryos has become the basic means of assisted reproductive technology (ART) therapy in recent years. Concerns have also been raised about the safety of vitrification and the effect of cryopreservation time. Most of the previous studies were on the data within 6 years of cryopreservation.

Objective: In this study, we aimed to evaluate the impact of long-term cryopreservation (>6 years) on pregnancy and neonatal outcomes.

Materials and methods: This research was a single-center, retrospective analysis, including 426 frozen-thawed embryo transfer (FET) cycles. Patients who participated in IVF-FET cycles between January 2013 to December 2020 were analyzed. Preferentially matched participants were divided into three groups according to storage time: group A (>72 months), group B (0-3 months, propensity score matching [PSM] according to the age of oocyte retrieval), and group C (0-3 months, PSM according to the age of embryo transfer).

Results: Our results revealed that there were no significant differences in human chorionic gonadotropin [HCG] positive rate, clinical pregnancy rate, miscarriage rate, live birth rate, and neonatal outcomes when the embryo storage duration >72 months. But the proportion of high birth weight was higher in group A (>72 months) when matched according to age at embryo transfer.

Conclusion: The results of our study showed that long-term cryopreservation had no effect on the pregnancy and neonatal outcomes of vitrification. The results offer evidence for the safety of using long-term cryopreservation embryos after vitrification. DOI: 10.54680/fr23310110612.

Background: The recovery of spermatozoa from the cauda epididymis may be the only option to obtain genetic material from elite stallions that had undergone castration or sudden death due to colic or severe injury.

Objective: To evaluate two different protocols for retrieval of stallion epididymal spermatozoa and to evaluate different cryoprotectants on the freezability of the epididymal spermatozoa.

Materials and methods: Six epididymides from three stallions were collected immediately after routine castration under general anesthesia. In the first experiment, each epididymis (of two testes) of the same stallion were processed using different methods for retrieval of the epididymal spermatozoa and were pooled and cryopreserved either using 5% glycerol or 5% dimethyl formamide (DMF) as cryoprotectant. The semen quality parameters viz., progressive motility, HOST, viability and acrosome integrity were evaluated at the fresh, pre-freeze and post-thaw stages.

Results: Retrograde method of flushing of epididymis yielded significantly (p < 0.05) higher concentration of the stallion sperm than that of the floating method. The qualitative semen parameters i.e., viability, plasma membrane integrity and acrosome integrity were found to be significantly restored using 5% DMF as cryoprotectant in comparison to when 5% glycerol was used.

Conclusion: Retrograde flushing method of epididymis yielded significantly higher sperm concentration to that of the floating method, and 5% DMF as cryoprotectant provided acceptable freezability of stallion epididymal spermatozoa. DOI: 10.54680/fr23310110312.

Several clinical trials have proved the efficacy and safety of T-cells chimeric antigen receptor (CAR-T cells) in treatment of malignant lymphoma and the first products were registered in the European Union in 2018. The shelf-life of CAR-T cell products in the liquid state is short, so cryopreservation offers a significant benefit for logistics in manufacturing and patient management. Direct shipment of the cryopreserved CAR-T cell therapy products to the clinical department is feasible, nevertheless, intermediate storage in the hospital cryostorage facility gives significant advantage in planning of their administration to patients. Moreover, some manufacturers prefer transport of the starting material cryopreserved at the collection site. The cryopreservation protocol used for starting material by the authors is based on combining dimethyl sulphoxide (DMSO) with hydroxyethyl starch (HES) and slow controlled cooling in cryobags housed in metal cassettes. This achieves the mononuclear cell post-thaw viability of 98.8 ± 0.5 % and recovery of 72.8, ± 10.2 %. Transport of the starting material to the manufactures and return transport of the CAR-T therapy product is performed by authorized courier companies. Intermediate cryostorage of the final CAR-T cell therapy product is performed in a separate dry-storage liquid nitrogen container. On the day of infusion, the cryopreserved products are transported to the clinical department in a dry shipper. On the wards the product is removed from the cassette, inserted into a sterile plastic bag, thawed in a 37 degree C water bath followed by immediate intravenous administration. The authors discuss the adherence of the used technology to good manufacturing practice (GMP) principles and genetic safety assurance rules. Doi: 10.54680/fr23310110112.

Background: Semen cryopreservation is a biotechnology used frequently in animal production; however, there are some obstacles, such as those caused by high levels of reactive oxygen species (ROS). Moringa oleifera (MO) is known as a potent source of antioxidants and might be an important adjuvant.

Objective: The objective of this study was to determine the effect of different concentrations of MO extract supplementation on goat semen cryopreservation efficiency.

Materials and methods: Ejaculates (n=6) from four goat breeders were pooled and diluted in skimmed milk (SM) or Tris-egg yolk (TEY)-based extenders and supplemented with different concentrations of MO extract (0, 1, 2 and 5 mg/mL). After the freeze-thaw cycle, sperm kinetics and viability were assessed.

Results: With the SM extender, straightness, wobble and plasma membrane integrity were lower than in the control group (P < 0.05). With the TEY extender, wobble was lower in with 5 mg/mL MO extract than in the control group (P < 0.05). As regards sperm ultrastructure, evaluated by SEM, the MO extract, regardless of the diluent used, damaged the membrane of sperm cells in a dose-dependent manner.

Conclusion: The addition of aqueous extract of MO leaves in both diluents at all concentrations tested affects the parameters of sperm progressivity and damages the plasma membrane in a dose-dependent manner. DOI: 10.54680/fr23310110712.