Cold storage and cryopreservation methods for spermatozoa of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus.

BACKGROUND: Sea urchins have contributed greatly to knowledge of fertilization, embryogenesis, and cell biology. However, until now, they have not been genetic model organisms because of their long generation times and lack of tools for husbandry and gene manipulation. We recently established the sea urchin Lytechinus pictus, as a multigenerational model Echinoderm, because of its relatively short generation time of 4-6 months and ease of laboratory culture. To take full advantage of this new multigenerational species, methods are needed to biobank and share genetically modified L. pictus sperm. RESULTS: Here, we describe a method, based on sperm ion physiology that maintains L. pictus and Strongylocentrotus purpuratus sperm fertilizable for at least 5-10 weeks when stored at 0°C. We also describe a new method to cryopreserve sperm of both species. Sperm of both species can be frozen and thawed at least twice and still give rise to larvae that undergo metamorphosis. CONCLUSIONS: The simple methods we describe work well for both species, achieving >90% embryo development and producing larvae that undergo metamorphosis to juvenile adults. We hope that these methods will be useful to others working on marine invertebrate sperm.

Assessment of reproductive outcomes of fresh versus cryopreserved ejaculated sperm samples-a retrospective analysis of 44 423 oocyte donation ICSI cycles.

STUDY QUESTION: Does the use of frozen sperm affect live birth rate (LBR) and cumulative LBR (CLBR) compared to fresh sperm samples in oocyte donation ICSI cycles? SUMMARY ANSWER: Although there were slight decreases in pregnancy rates (PRs) and LBR, as well as CLBR per embryo replaced and per embryo transfer (ET), when frozen sperm samples were used compared to fresh ejaculates, their clinical impact was limited. WHAT IS KNOWN ALREADY: Sperm cryopreservation is part of the daily routine in reproduction clinics worldwide because of its many advantages in cycle planning. Nonetheless, there is a lack of agreement in terms of its impact on the outcomes of ICSI cycles. Previous studies showed conflicting conclusions and focused on different populations, which makes reaching consensus on the impact of sperm freezing-thawing complicated. Moreover, classical parameters are used to assess cycle success: pregnancy, live birth and miscarriage rates per ET. This study reports those measurements plus CLBR, which more accurately reflects the impact of the technique on the likelihood of achieving a newborn. STUDY DESIGN, SIZE, DURATION: A retrospective multicenter observational cohort study, including data from 37 041 couples and 44 423 ICSI procedures from January 2008 to June 2022, was carried out. The group using frozen sperm included 23 852 transferred embryos and 108 661 inseminated oocytes, whereas the fresh sample group comprised 73 953 embryos replaced and 381 509 injected oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Outcomes measured per first ET and per ET were compared between groups using Fisher's exact test and Chi-squared test, as appropriate. Binary-logistics regression models were used to adjust the analyses according to clinically relevant co-variables. Kaplan-Meier curves plotted the CLBR per oocyte inseminated, per embryo replaced and per ET, and compared between groups using the Mantel-Cox test. Cox regressions were employed for the multivariate analyses of CLBR. MAIN RESULTS AND THE ROLE OF CHANCE: The frozen sperm group showed a slightly lower biochemical (3.55% and 2.56%), clinical (3.68% and 3.54%) and ongoing (3.63% and 3.15%) PR compared to the cycles using fresh sperm, respectively, both per first ET and per ET. LBR was 4.57% lower per first ET and 3.95% lower per ET in the frozen sperm group than the fresh sperm group. There was also a subtle increase of 2.66% in biochemical miscarriage rate per ET when using frozen versus fresh sperm. All these differences remained statistically significant after the multivariate analysis (adjusted P ≤ 0.001). There were statistically significant differences in CLBR per embryo replaced and per ET but not per oocyte used (adjusted P = 0.071). Despite the statistical significance of the differences between the groups, those using frozen sperm required only 0.54 more oocytes injected, 0.45 more embryos transferred and 0.41 more ET procedures, on average, to achieve a live birth compared to the fresh samples. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study subjects the data to biases or potential errors during annotation on the source clinical and cycle records. This study uses multivariate analyses to control biases as much as possible. Using the oocyte donation model also contributes to reducing heterogeneity in the oocyte quality factor. WIDER IMPLICATIONS OF THE FINDINGS: The large sample sizes included in this study allowed for the detection of small changes in cycle success rates between groups. Although statistically significant, the decrease in PRs, LBR, and CLBR when using frozen sperm can be clinically overlooked in favor of the many benefits of sperm cryopreservation. STUDY FUNDING/COMPETING INTEREST(S): None declared. TRIAL REGISTRATION NUMBER: Not applicable.

Semen cryopreservation for an oncological reason: a retrospective study.

RESEARCH QUESTION: How do cancer type and treatment affect semen quality before and after treatment, and what effect does it have in their clinical management of infertility? Also, what is the rate of patients using cryopreserved semen samples after treatment? DESIGN: Patients who cryopreserved spermatozoa for oncological reasons between 2000 and 2022 in IVI clinics in Spain were retrospectively reviewed. Semen parameters were analysed before and after treatment, and utilization and destruction rates were calculated. Total motile sperm count (TMSC) was used for assisted reproductive technology (ART) counselling. RESULTS: A total of 724 patients cryopreserved their semen during the study period. The semen parameters of the cancer patients' semen before and after treatment were heterogeneous, with significant differences between cancer type and semen parameters. The utilization rate was relatively low (0.4%), whereas the destruction rate was 23.1%. CONCLUSION: Cancer and antineoplastic treatment affect everyone differently. Therefore, sperm cryopreservation should be offered to all patients before starting treatment to ensure their reproductive future. Furthermore, in addition to considering the semen parameters defined by the World Health Organization, it is important to use TMSC in the diagnosis of men to choose appropriate ART according to type of cancer.

Post-treatment sperm cryopreservation practices in childhood and young adult cancer survivors.

RESEARCH QUESTION: What are current practices of post-treatment fertility preservation in male childhood cancer survivors (CCS) who have not benefitted from pre-therapeutic fertility preservation in France and other European countries? DESIGN: A survey was conducted of all fertility preservation centres in France (n = 30) and European fertility specialists (n = 9) in five European countries. Eight clinical cases and 40 questions were included to assess the effect of age at diagnosis, type of treatment (alkylating-agents, orchidectomy, testicular radiotherapy) and sperm parameters on the probability of a post-treatment fertility preservation proposal. Demographic characteristics of the responding practitioner were also collected. RESULTS: Post-treatment sperm cryopreservation was proposed by 100% of fertility specialists in cases of severe oligoasthenoteratozoospermia, 77-88% in cases of moderate oligoasthenoteratozoospermia and in 65-77% in cases of sperm motility and vitality impairment. In cases of normal sperm parameters, 27-54% of fertility specialists would propose post-treatment sperm cryopreservation. These results did not differ significantly according to the type of treatment received or to responder-related factors. Practices of European specialists were also guided by sperm parameter results; 44-67% of specialists responding that they would propose sperm cryopreservation in cases of moderate to severe sperm parameter alterations. CONCLUSION: Post-treatment semen analysis could be widely proposed to CCS who have not benefitted from pre-therapeutic fertility preservation. Post-treatment fertility preservation could be proposed in cases of persistent moderate to severe sperm parameter alterations. Guidelines would be important to homogenize practices and to encourage oncologists to refer CCS for fertility assessments.

Development of sperm cryopreservation protocol for patin buah, Pangasius nasutus.

The development of sperm cryopreservation for Pangasius nasutus is necessary in order to serve the growing demand of this species through artificial fertilization and the preservation of valuable strains of male broodstocks. In the present study, the basic protocol of sperm cryopreservation for P. nasutus was established by identifying the optimal conditions for optimum cryoprotectant, toxicity of cryoprotectants, extenders, freezing condition and dilution ratio. Methanol (MeOH) at 10% concentration had the best post-thaw motility (26.3 ± 0.9%) and curvilinear velocity (VCL) compared to dimethyl acetamide and dimethyl sulfoxide. MeOH was the least toxic cryoprotectant; sperm suspended in 5 and 10% MeOH maintained motility up to 50 min. No significant differences were detected between the three types of extenders tested (0.9% sodium chloride, Calcium-free Hanks' Balance salt solution and ringer solution). P. nasutus sperm had a narrow range of optimal cooling rate. Significantly higher post-thaw motility was identified when cooling at 9.23 °C min-1, obtained by freezing at height of 14 cm above liquid nitrogen vapor for 7 min, showing lower cooling rate is suitable for this species. However, when cooling below and above the optimal cooling rate, post-thaw motility dropped drastically. There were no significant differences among the dilution ratios investigated, indicating the volume of cryodiluent at all tested ratios (1:9, 1:19 and 1:49) was sufficient for the protection of cells during the cryopreservation process. The development of the protocol for cryopreserved P. nasutus sperm will assist artificial seed production and provide an important tool for genetic and breeding research.

Applying a heat transfer mathematical model for the cryopreservation of rainbow trout (Oncorhynchus mykiss) sperm: How straw location over liquid nitrogen level affects freezing rate and fertilization yield.

Cryopreservation of rainbow trout semen under field conditions was analyzed. Straw location over liquid nitrogen level is a crucial variable that affects freezing rate and fertilization yield due to changes in nitrogen vapor external temperature. The objectives were: to analyze cryopreservation protocols by experimentally measuring the cooling rates and fertilization yield of 0.5 ml plastic straws located in nitrogen vapor at different heights corresponding to different external temperatures; to numerically simulate the freezing process, by solving the heat transfer partial differential equations with the corresponding thermo-physical properties of the biological system and the plastic straw; to evaluate and analyze the surface heat transfer coefficient (h) during the freezing process of the straws; to introduce a new variable, the characteristic freezing time (tc), that enables comparison between protocols; this variable was defined as the elapsed period between the initial freezing temperature and a final reference temperature of -40 °C (temperature in which more than 80 % of the water is in a frozen state). The mathematical model predicted the temperature distribution inside the straw, showing a low effect of straw plastic materials (polyethylene-terephthalate glycol, polyvinyl-chloride, and polypropylene) on freezing rates. The average h value obtained from numerical simulations was 25.5 W/m2 K, close to that obtained from the analytical Nusselt correlation for natural convection. An improvement on fertilization trials was observed when the average external nitrogen temperature was -129.6 °C (temperature range: -94 to -171 °C) with an average tc of 56.8 s (ranging between 47 and 72 s). These results corresponded to a height above the level of liquid nitrogen of 2 cm. Comparison with literature reported data showed satisfactory results. Applying mathematical models in the cryobiology field achieved results that are relevant for cryopreservation activities.

Safety evaluation of single-sperm cryopreservation technique applied in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is a technique that directly injects a single sperm into the cytoplasm of mature oocytes. Here, we explored the safety of single-sperm cryopreservation applied in ICSI. This retrospective study enrolled 186 couples undergoing ICSI-assisted pregnancy. Subjects were allocated to the fresh sperm (group A)/single-sperm cryopreservation (group B) groups based on sperm type, with their clinical baseline/pathological data documented. We used ICSI-compliant sperm for subsequent in vitro fertilization and followed up on all subjects. The recovery rate/cryosurvival rate/sperm motility of both groups, the pregnancy/outcome of women receiving embryo transfer, and the delivery mode/neonatal-related information of women with successful deliveries were recorded. The clinical pregnancy rate, cumulative clinical pregnancy rate, abortion rate, ectopic pregnancy rate, premature delivery rate, live birth delivery rate, neonatal birth defect rate, and average birth weight were analyzed. The two groups showed no significant differences in age, body mass index, ovulation induction regimen, sex hormone [anti-Müllerian hormone (AMH)/follicle-stimulating hormone (FSH)/luteinizing hormone (LH)] levels, or oocyte retrieval cycles. The sperm recovery rate (51.72%-100.00%) and resuscitation rate (62.09% ± 16.67%) in group B were higher; the sperm motility in the two groups demonstrated no significant difference and met the ICSI requirements. Group B exhibited an increased fertilization rate, decreased abortion rate, and increased safety versus group A. Compared with fresh sperm, the application of single-sperm cryopreservation in ICSI sensibly improved the fertilization rate and reduced the abortion rate, showing higher safety.

Cryopreserved testicular spermatozoa among patients with azoospermia.

PURPOSE: To investigate cryopreserved testicular spermatozoa among patients with azoospermia. METHODS: In this retrospective study spanning from October 1993 to December 2021, we examined men diagnosed with azoospermia who underwent testicular spermatozoa cryopreservation. Data from medical records included utilization and disposal of sperm samples, age at initial cryopreservation. We analyzed the data over 20 years using Kaplan-Meier curves, compared age with the log-rank test, and assessed hazard ratios (HR) with 95% confidence intervals (CI) using Cox regression analysis. RESULTS: A total of 356 patients with a mean age of 32.1 ± 6 were included. Of these, 225 patients utilized thawed testicular sperm for fertility treatments, with 118 patients using all their frozen straws and 107 patients partially using their stored straws. Additionally, 29 patients opted for disposal (six patients partially used their testicular spermatozoa before disposal), resulting in 108 patients who neither used nor disposed of their straws. From a laboratory standpoint, nearly 90% of patients contributed a single testicular sample, which was subsequently divided and cryopreserved as straws, with a median of 4 straws per sample. Notably, in the older age group (> 35 years old), there were a significantly lower usage rate and a higher disposal rate compared to the younger age groups (p < 0.05 for both), corroborated by univariable Cox analysis. CONCLUSIONS: This extensive study unveils unique patterns in the preservation and disposal of testicular spermatozoa among azoospermic patients. Most patients utilize a significant portion of their stored samples, while older patients tend to use their testicular spermatozoa less frequently.

Fertility preservation in males with cancer of trends, region development, and efficacy in mainland China from 16 regions Chinese sperm banks.

PURPOSE: Male cancer survivors experience confusion about fertility following cancer treatment. The aims of this study were to evaluate survivors' semen quality in different tumor type groups in China and to analyze the current situation and challenges of male cancer patients with sperm cryopreservation. METHODS: This was a multicenter retrospective study of male patients with cancer who underwent sperm cryopreservation in 16 regions of the national sperm banks over an 11-year period from 2010 to 2020. RESULTS: The number of male cancer patients with sperm cryopreservation showed an overall upward trend. The development of male cancer fertility preservation (FP) in the eastern, central, and western regions of Chinese displayed imbalance. There are seven tumor types for sperm preservation in the top incidence ten tumor types, including lymphoma, leukemia, nasopharyngeal carcinoma, sarcoma, thyroid cancer, and brain tumor. Moreover, nasopharyngeal carcinoma is a high incidence rate in China, which is related to high sperm preservation rate, different from other countries. The most percentage of males receiving sperm cryopreservation in the testicular cancers (15-39 years old) of China in 2020 was 5.55%, 1.29% in the lymphoma, and 0.39% in the leukemia. According to the type of cancer, a statistically significant lower pre-sperm density, total sperm output, and post-sperm density was observed in testicular cancers. It is worth noting that the prevalence of azoospermia 22.2% in leukemia patients attribute to urgent treatment before sperm cryopreservation. Disposition of cryopreserved sperm categories included continued storage (47.2%), discarded (9%), death (0.9%), and use (3.7%). CONCLUSION: This study provides the first comprehensive national statistical census and review of fertility preservation in male cancer patients with respect to trends, prevalence, and cancer types. The development of male cancer fertility preservation in China is imbalanced and percentage of males receiving sperm cryopreservation in the adolescent and young adult cancers was low. Sixteen human sperm banks from China analyze current problems and challenges, and then prioritize steps toward the achievement of the FP strategy framework for Healthy China 2030.

Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

A Pilot Analysis of Whole Transcriptome of Human Cryopreserved Sperm.

Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for sperm donation programs. The purpose of the study was to assess the impact of cryopreservation on human sperm transcriptome. Semen samples were collected from 13 normospermic men. Each sample was divided into two aliquots. The total RNA was immediately extracted from one aliquot. The second aliquot was frozen and total RNA was extracted after a week of storage in liquid nitrogen. The RNA samples were randomized in four pools, each of six donors, and analyzed by microarrays. The paired Significance Analysis of Microarray was performed. We found 219 lower abundant transcripts and 28 higher abundant transcripts in cryopreserved sperm than fresh sperm. The gene ontology analysis disclosed that cryopreservation alters transcripts of pathways important for fertility (i.e., spermatogenesis, sperm motility, mitochondria function, fertilization, calcium homeostasis, cell differentiation, and early embryo development), although the increase of some transcripts involved in immune response can compensate for the harmful effects of freezing.

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Sperm cryopreservation does not affect live birth rate in normozoospermic men: analysis of 7969 oocyte donation cycles.

STUDY QUESTION: Does sperm cryopreservation influence the reproductive outcomes of normozoospermic patients in oocyte donation cycles? SUMMARY ANSWER: After controlling for confounders, the use of cryopreserved semen from normozoospermic patients does not affect pregnancy and live birth rates after elective ICSI. WHAT IS KNOWN ALREADY: Sperm cryopreservation by slow freezing is a common practice in ART. While frozen-thawed semen typically presents reduced motility and vitality, its use for ICSI is generally considered adequate in terms of reproductive outcomes. Nevertheless, most studies comparing reproductive outcomes between fresh and cryopreserved sperm include patients with severe male factor (testicular sperm, oligo-, and/or asthenozoospermia) or women of advanced maternal age, where the altered quality of the gametes can partially mask the full effect of freezing/thawing. STUDY DESIGN, SIZE, DURATION: The study included a retrospective cohort of 7969 couples undergoing their first oocyte donation cycle between January 2013 and December 2019 in one large clinic, using normozoospermic semen from the male partner. All cycles involved elective ICSI, fresh oocytes, and a fresh embryo transfer, either at cleavage or blastocyst stage. Two study groups were established based on the sperm status: fresh (n = 2865) and cryopreserved (n = 5104). PARTICIPANTS/MATERIALS, SETTING, METHODS: A slow freezing protocol was used for all sperm cryopreservation. Sperm washing, capacitation, and selection prior to ICSI were performed identically for fresh and frozen-thawed samples, using pellet swim-up. Fertilization rate (FR), pregnancy (biochemical and ongoing), and live birth rates were compared between study groups using univariate and multivariate regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Male and female age, sperm concentration and motility after ejaculation, and number of oocytes inseminated were similar between cycles using fresh or cryopreserved sperm. Analysis by Student's t-test did not indicate a significant difference in FR between fresh and cryopreserved sperm (P = 0.0591); however, after adjusting for confounders, this difference reached statistical significance: 74.65% FR for fresh (CI 95%: 73.92-75.38) versus 73.66% for cryopreserved sperm (CI 95%: 73.11-74.20), P = 0.0334. The adjusted regression analysis revealed higher odds of biochemical pregnancy when using fresh sperm (odds ratio (OR): 1.143, P = 0.0175), but no significant effects of sperm cryopreservation were observed for ongoing pregnancy (OR: 1.101, P = 0.0983) and live birth (OR: 1.082, P = 0.1805). LIMITATIONS, REASONS FOR CAUTION: Caution should be exerted when extrapolating these results to different protocols for sperm cryopreservation and selection, or to IVM, advanced maternal age and classical IVF cycles, which were excluded from analysis. Owing to the retrospective nature of the study, some uncontrolled for variables may affect the results. WIDER IMPLICATIONS OF THE FINDINGS: Sperm cryopreservation does not affect pregnancy and live birth rates in normozoospermic patients, and although it may lower FR s slightly, this would not be clinically relevant. In line with previous studies that included patients with an apparent male or female factor, sperm cryopreservation is a safe and convenient technique. STUDY FUNDING/COMPETING INTEREST(S): The study received no external funding and all authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Optimization of sperm cryopreservation protocol for Basa catfish (Pangasius bocourti).

The aim of this study was to optimize a sperm cryopreservation protocol for Basa catfish (Pangasius bocourti). Key factors for the efficiency of cryopreservation, including cryoprotectants, thawing conditions, equilibration times, dilution ratios and freezing methods, were investigated. The suitable time for post-thaw storage as well as pre-freezing cool storage was also examined. Five cryoprotectants (dimethyl sulfoxide, Me2SO; dimethylformamide, DMF; ethylene glycol, EG; propylene glycol, PG; N-methylacetamide, MA) at different final concentrations (5, 10 and 15%) were tested, 10% PG provided the best cryoprotective effect. Thawing temperature at 30-40 °C yielded significantly higher post-thaw motility than 20, 25, 50 or 60 °C. No obvious effect on sperm motility was detected either in equilibrated or thawed samples during a 2-h equilibration. Regarding dilution ratio (semen/cryomedium, v/v), percentage of motile spermatozoa was significantly higher at ratios of 2:1, 1:1 and 1:3 than those with higher ratios (1:5, 1:7 and 1:9). Thawed sperm was sensitive to post-thaw storage, but no reduction in motility was detected within 30 min. Further evaluation of the effective pre-freezing storage time indicated that sperm in diluted form had more advantage in maintaining its freezability, which could be chilled for 24 h before freezing without compromising post-thaw sperm motility. P. bocourti sperm could be successfully cryopreserved with both a programmable freezer and the floating frame technique (frozen 5 or 7 cm above the surface of liquid nitrogen). Cryopreserved sperm (77.5 ± 5.1%) fertilization was not significantly different from fresh sperm (80.9 ± 4.7%) at the ratio of 2 × 105 spermatozoa per egg. Our results provided more detailed suitable conditions for P. bocourti sperm cryopreservation than previous studies. Standardizing the cryopreservation protocol and storage time would be helpful in facilitating artificial reproduction in this species.

Equilibration time improves the sperm variables of wild ruminant ejaculated and epididymal sperm cryopreserved by ultra-rapid freezing.

This work examines the effect of equilibration time with extender on ultra-rapidly frozen-thawed wild ruminant epididymal (origin: Iberian ibex) and ejaculated (origin: mouflon) sperm variables. Sperm samples were prepared either without prior equilibration, or equilibrated for 30 min before freezing. Higher quality (p < 0.05) frozen-thawed spermatozoa were obtained when equilibration was allowed, for ejaculated sperm in terms of sperm motility, acrosome apical ridge integrity, sperm viability, and percentage of normal cells, and for epididymal sperm in terms of linearity and straightness of sperm movement. The sperm head area, head perimeter, head length and head width were smaller (p < 0.01) in the equilibrated than non-equilibrated frozen-thawed epididymal sperm; no such dimensional changes were recorded for ejaculated sperm. In conclusion, equilibration prior to ultra-rapid freezing improves the cryoresistance of sperm cells, although viable sperm cells can be obtained without equilibration. The epididymal sperm showed greater cryoresistance, supporting the idea that it is more resistant to freeze-thawing than ejaculated sperm.

Effects of cryopreservation in the presence of Natural Deep Eutectic Solvents (NADESs) on sperm parameters.

Natural Deep Eutectic Solvents (NADESs) are being considered as a potential alternative to traditional cryoprotective agents (CPAs) in sperm freezing. The study aimed to assess the effects of NADESs as a CPA on human sperm parameters. A total of 32 normozoospermic semen samples were collected from the Alzahra infertility treatment center (Iran) between July 2021 and September 2022. The samples were categorized into eight different groups: 1) a control (nonfrozen), and groups frozen with 2) SpermFreeze Solution, 3) ChX (Choline chloride and Xylitol), 4) ChS (Choline chloride and D-sorbitol), 5) ChG (Choline chloride and Glucose), 6) ChU (Choline chloride and Urea), 7) EtP (Ethylene glycol and l-proline), and 8) GlyP (Glycerol and l-proline). The study also analyzed the quality of sperm parameters, such as chromatin condensation and integrity, acrosome integrity, and survival, along with the expression of some genes that affect sperm fertility (TRPV1, TRPV4, SPACA3, and OGG1). The study found there were notable variations in sperm parameters (such as viability, chromatin condensation and integrity, and acrosome integrity) among frozen groups with some NADESs compared to the SpermFreeze Solution and control groups (P < 0.05). Analysis of gene expression demonstrated that the levels of TRPV1, TRPV4, SPACA3, and OGG1 genes were superior in the GlyP group compared to the other groups (P < 0.05). Additionally, the ChS and ChU groups exhibited preserved expression of these genes compared with the SpermFreeze Solution group. The use of NADESs led to the discovery of a more appropriate CPA that has low toxicity and is highly effective in maintaining the fertility potential of sperm.

Clinical and neonatal outcomes of using a modified micro cryotube for cryopreservation of small numbers of spermatozoa for TESA-ICSI cycles.

Cryopreservation of a small number of human spermatozoa is still a major challenge for embryologists. The aim of this study was to evaluate the clinical pregnancy and neonatal outcomes of intracytoplasmic sperm injection (ICSI) using a modified micro cryotube as freezing carrier for freezing small numbers of human spermatozoa collected by testicular sperm aspiration (TESA). We conducted a retrospective study to analyses the ICSI outcomes of using frozen-thawed few testicular spermatozoa in males with obstructive azoospermia (OA) from June 2017 to June 2021. Of 155 ICSI treatment cycles, 79 cycles were allocated to frozen sperm group and a modified micro cryotube was used for freezing testicular sperm, 76 cycles were allocated as fresh sperm group. No significant differences were observed in fertilization rate, good quality embryo rate, and blastocyst rate between the frozen sperm group and fresh sperm group (P > 0.05). Similarly, in the fresh embryo transfer cycles plus the first frozen-thawed embryo transfer cycles, the total clinical pregnancy rate (54.43% vs 57.89%), implantation rate (46.08% vs 49.47%), miscarriage rate (13.95% vs 13.64%) and live birth rate (45.57% vs 48.68%) were not statistically different between the frozen and fresh sperm groups (P > 0.05). In addition, there was no statistical differences in the mean gestational age (38.33weeks ± 1.74 vs 37.89weeks ± 1.87), preterm delivery rate (5.56% vs 10.81%), mean birth weight at delivery (3026.50 g ± 577.64 vs 2977.56 g ± 528.93), and low birth weight (12.50% vs 19.51%) between the two groups (P > 0.05 in all cases). Modified micro cryotube for cryopreservation of rare testicula rretrieved spermatozoa did not negatively affect the pregnancy and neonatal outcomes in TESA-ICSI cycles. The presented method may be a useful alternative for cryopreservation of small numbers of human spermatozoa in clinical setting.

The investigation of post-thaw chilled storage and the applicability of large-scale cryopreservation in chub (Squalius cephalus) sperm.

Chub (reophillic cyprinids) is one of the most sensitive bioindicator fish of environmental changes following anthropogenic activities. The improvement of different biotechnological procedures could help support its conservation and strengthen the natural populations. The aim of this study was to compare the effects of two different hormonal agents (carp pituitary extract and Ovopel™) on various motility parameters (pMOT-%, DAP-µm, VCL µm s-1, VSL-µm s-1, LIN-%, ALH-µm, BCF-Hz) of fresh and cryopreserved/thawed sperm (stored at 4 °C for 6 h). Additionally, we sought to develop a novel, large-scale cryopreservation method for chub sperm, assessing freezing methods (Styrofoam box and a controlled-rate freezer) and different containers (0.5, 5 mL straw and 4 mL cryotube) for sperm cryopreservation. The results of this study indicated no difference between the carp pituitary extract and Ovopel treated groups in either the fresh or frozen/thawed sperm (at 0, 3, 6, hour post thawing, P = 0.4351). In contrast, the quality of the thawed chub sperm was negatively affected after 3 h chilled storage in both hormonal treatments (P = 0.0036, P < 0.0001). When assessing the motility parameters of the sperm between the 5 mL straw and 4 mL cryotube groups cryopreserved in a Styrofoam Box, no difference was observed (P = 0.103). Additionally, sperm loaded in 4 mL cryotubes showed no difference in motility when cryopreserved with either the Styrofoam box or controlled-rate freezer methods (P = 0.109). A similar hatching rate was observed in sperm preserved using the Styrofoam box (35 ± 7 %) and controlled rate freezer (25 ± 9 %) methods (P = 0.300). In a second fertilization trial, hatching rate was similar between control (72 ± 19 %) and cryopreserved (4 mL cryotube and Styrofoam box, 61 ± 5 %) groups. (P = 0.257). Based on our findings and its standard features (less species specific, precise dose calculation), Ovopel can be a good candidate for the stimulation of spermiation in chub sperm prior to cryopreservation. Furthermore, our study presents a novel and applicable method for the large-scale cryopreservation of chub sperm.

Cryopreservation Induces Acetylation of Metabolism-Related Proteins in Boar Sperm.

Cryodamage affects the normal physiological functions and survivability of boar sperm during cryopreservation. Lysine acetylation is thought to be an important regulatory mechanism in sperm functions. However, little is known about protein acetylation and its effects on cryotolerance or cryodamage in boar sperm. In this study, the characterization and protein acetylation dynamics of boar sperm during cryopreservation were determined using liquid chromatography-mass spectrometry (LC-MS). A total of 1440 proteins were identified out of 4705 modified proteins, and 2764 quantifiable sites were elucidated. Among the differentially modified sites, 1252 were found to be upregulated compared to 172 downregulated sites in fresh and frozen sperms. Gene ontology indicated that these differentially modified proteins are involved in metabolic processes and catalytic and antioxidant activities, which are involved in pyruvate metabolism, phosphorylation and lysine degradation. In addition, the present study demonstrated that the mRNA and protein expressions of SIRT5, IDH2, MDH2 and LDHC, associated with sperm quality parameters, are downregulated after cryopreservation. In conclusion, cryopreservation induces the acetylation and deacetylation of energy metabolism-related proteins, which may contribute to the post-thawed boar sperm quality parameters.

Optimization of Sperm Cryopreservation Formulation in Portunus trituberculatus.

Portunus trituberculatus is a very important marine economic species, and its aquaculture industry has been developing rapidly. However, the phenomenon of marine wild capture of P. trituberculatus and germplasm degradation has become increasingly serious. It is necessary to develop the artificial farming industry and carry out germplasm resource protection, for which sperm cryopreservation technology is an effective method. This research compared three methods (mesh-rubbing, trypsin digestion, and mechanical grinding) for acquiring free sperm, and the best method was mesh-rubbing. Then, the optimal cryopreservation conditions were selected, and the optimal formulation was sterile calcium-free artificial seawater, the optimal cryoprotectant was 20% glycerol, and the best equilibrium time was 15 min at 4 °C. The optimal cooling program was suspending the straws at 3.5 cm on the liquid nitrogen surface for 5 min and then storing them in liquid nitrogen. Finally, the sperm were thawed at 42 °C. However, the expression of sperm-related genes and the total enzymatic activities of frozen sperm were significantly decreased (p < 0.05), which showed that sperm cryopreservation damaged the sperm. Our study improves the sperm cryopreservation technology and the yield of aquaculture in P. trituberculatus. Additionally, the study provides a certain technical basis for the establishment of a sperm cryopreservation library of crustaceans.