Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes).

Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks' Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.

Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Investigation of the protective effects of different forms of selenium in freezing dog semen: Comparison of nanoparticle selenium and sodium selenite.

This study aimed to investigate the protective effects of nanoparticle selenium (SeNP) and sodium selenite (SS) on preventing oxidative stress during the freezing process of dog semen. A total of six dogs were used in the study. The ejaculate was collected from dogs three times at different times by massage method. A total of 18 ejaculates were used and each ejaculate was divided in five experimental groups. The experimental groups were designed to tris extender containing no antioxidants control, 1 µg/mL SeNP1, 2 µg/mL SeNP2, and 1 µg/mL SS1 and 2 µg/mL SS2. Extended semen were equilibrated for 1 h at 4°C, then frozen in liquid nitrogen vapour and stored in liquid nitrogen (~-196°C). After thawing, semen samples were evaluated in terms of CASA motility and kinematic parameters, spermatozoa plasma membrane integrity and viability (HE Test), spermatozoa morphology (SpermBlue) and DNA fragmentation (GoldCyto). Antioxidant enzyme activity (glutathione peroxidase; GPX, superoxide dismutase; SOD, catalase; CAT) and lipid peroxidation (malondialdehyde; MDA) were evaluated in frozen-thawed dog sperm. When the results were evaluated statistically, the progressive motility, VCL, and VAP kinematic parameters in the SeNP1 group were significantly higher than the control group after thawing (p < .05). The highest ratio of plasma membrane integrity and viable spermatozoa was observed in the SeNP1 group, but there was no statistical difference found between the groups (p > .05). Although the ratio of total morphological abnormality was observed to be lower in all groups to which different selenium forms were added, compared to the control group, no statistical difference was found. Spermatozoa tail abnormality was significantly lower in the SeNP1 group than in the control and SS2 group (p < .05). The lowest ratio of fragmented DNA was observed in the SeNP1 group, but there was no statistical difference was found between the groups (p > .05). Although there was no statistical difference between the groups in the evaluation of sperm antioxidant profile, the highest GPX, SOD and CAT values and the lowest lipid peroxidation values were obtained in the SeNP1 group. As a result, it was determined that 1 µg/mL dose of SeNP added to the tris-based extender in dog semen was beneficial on spermatological parameters, especially sperm kinematic properties and sperm morphology, and therefore nanoparticle selenium, a nanotechnology product, made a significant contribution to the freezing of dog semen.

Effect of autologous platelet-rich plasma on the fertility and quality of cryopreserved buffalo bull semen: a comparative study using OptiXcell® and tris egg yolk extenders.

BACKGROUND: Buffalo spermatozoa have a distinct membrane structure that makes them more vulnerable to cryopreservation, resulting in lower-quality post-thawed sperm. This decreases the success rate of artificial insemination in buffaloes. Understanding and addressing these specific vulnerabilities are essential for improving reproductive techniques in buffalo populations. The properties of cryopreserved buffalo bull semen were examined in this study regarding the impact of adding autologous platelet-rich plasma (PRP) to OptiXcell® or Tris egg yolk-based extenders. Ten buffalo bulls were used to collect semen. Each bull's ejaculate was separated into two main equal amounts, each of which was then diluted with either OptiXcell® or Tris egg yolk-based extender, supplemented with various PRP concentrations (5%, 10%, and 15%), and the control (0%), before being cryopreserved according to established protocols. Following equilibration and thawing, the quality and functionality of the sperm were evaluated, along with the antioxidant enzyme activities (GSH and TAC), malondialdehyde (MDA) content, and in vivo fertilization rate of the thawed semen. RESULTS: All PRP concentrations in both extenders, particularly 10% PRP, improved the quality and functionality of the sperm in both equilibrated and frozen-thawed semen. Additionally, the antioxidant enzyme activities in both extenders were higher in the PRP-supplemented groups compared to the control group in thawed semen (P < 0.05). All post-thaw sperm quality, antioxidant enzyme activities, and functionality aside from DNA integrity were higher (P < 0.05) in the PRP-supplemented OptiXcell® than in the PRP-supplemented Tris egg yolk-based extender. The fertility of cryopreserved semen in the extenders supplemented with 10% and 15% PRP increased (P < 0.05) significantly more than that of the control extenders, with 10% PRP being the optimum concentration in OptiXcell® (80%) compared to that of Tris egg yolk-based extender (66.67%) and control of two extenders (53.33% and 46.67%, respectively). CONCLUSIONS: Even though autologous PRP-supplemented extenders have a protective impact on equilibrated and cryopreserved semen, 10% PRP-supplemented OptiXcell® extenders are more effective at preserving post-thaw semen quality, functionality, and antioxidant capacity, which increases the in vivo fertility of buffalo bulls.

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure.

BACKGROUND: The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. RESULTS: Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. CONCLUSIONS: The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

The effects of MitoQ as a mitochondrial-targeted antioxidant in a plant-based extender on buck sperm quality parameters during cryopreservation.

Sperm cryopreservation plays an important role in the artificial insemination (AI) industry of small ruminants. It, however the use of frozen-thawed goat semen is limited due to the insufficient number of sperm with good biological functions. Mitochondria are the most sensitive organelles to cryopreservation damage in sperm. This study was conducted to determine the effects of MitoQ, the mitochondrial-targeted antioxidant, in a plant-based extender on the quality parameters of Markhoz goat sperm after the freezing and thawing process. Semen samples were collected and diluted in the extender, divided into five equal aliquots and supplemented with 0, 1, 10, 100 and 1000 nM MitoQ and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, lipid peroxidation (LPO), DNA fragmentation, reactive oxygen species (ROS) concentration, viability and apoptotic-like changes were measured. The use of 10 and 100 nM MitoQ resulted in higher (P≤0.05) total motility (TM), progressive motility (PM), viability, membrane functionality, mitochondrial activity, and acrosome integrity compared to the other groups. On the other hand, LPO, apoptotic-like changes, DNA fragmentation and ROS concentration were lower (P≤0.05) in MQ10 and MQ100 groups compared to the other groups. MitoQ has no effect (P>0.05) on sperm abnormal morphology and velocity parameters. In conclusion, MitoQ can reduce oxidative stress by regulating mitochondrial function during the cryopreservation process of buck sperm and could be an effective additive in the cryopreservation media to protect sperm quality.

FBS-based cryoprotective compositions for effective cryopreservation of gut microbiota and key intestinal microorganisms.

OBJECTIVE: The need for innovative techniques to preserve microbiota for extended periods, while maintaining the species composition and quantitative balance of the bacterial community, is becoming increasingly important. To address this need, we propose an efficient approach to cryopreserve human gut microbiota using a two-component cryoprotective composition comprising fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). Fetal serum is a commonly utilized component in the freezing media for eukaryotic cells, however, its effects on prokaryotic cells have not been extensively researched. RESULTS: In our study, we demonstrated the high efficiency of using a two-component cryoprotective medium, FBS + 5% DMSO, for cryopreservation of human gut microbiota using three different methods. According to the obtained results, the intact donor microbiota was preserved at a level of 85 ± 4% of the initial composition based on fluorescent analysis using the LIVE/DEAD test. No differences in survival were observed when comparing with pure DMSO and FBS media. The photometric measurement method for growth of aerobic bacteria (A. johnsoni), facultative anaerobes (E. coli, E. faecalis), microaerophilic (L. plantarum), and obligate anaerobic bacterial cultures (E. barkeri, B. breve) also demonstrated high viability rates of 94-98% in the two-component protective medium, reaching intact control levels. However, for anaerobic microflora representatives, serum proved to be a more suitable cryoprotectant. Also, we demonstrated that using cryoprotective media with 50-75% FBS content is enough to preserve a significant level of bacterial cell viability, from an economic standpoint.

VCA supercooling in a swine partial hindlimb model.

Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia-reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at - 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.

Structure and slow dynamics of protein hydration water with cryopreserving DMSO and trehalose upon cooling.

We study, through molecular dynamics simulations, three aqueous solutions with one lysozyme protein and three different concentrations of trehalose and dimethyl sulfoxide (DMSO). We analyze the structural and dynamical properties of the protein hydration water upon cooling. We find that trehalose plays a major role in modifying the structure of the network of HBs between water molecules in the hydration layer of the protein. The dynamics of hydration water presents, in addition to the α-relaxation, typical of glass formers, a slower long-time relaxation process, which greatly slows down the dynamics of water, particularly in the systems with trehalose, where it becomes dominant at low temperatures. In all the solutions, we observe, from the behavior of the α-relaxation times, a shift of the Mode Coupling Theory crossover temperature and the fragile-to-strong crossover temperature toward higher values with respect to bulk water. We also observe a strong-to-strong crossover from the temperature behavior of the long-relaxation times. In the aqueous solution with only DMSO, the transition shifts to a lower temperature than in the case with only lysozyme reported in the literature. We observe that the addition of trehalose to the mixture has the opposite effect of restoring the original location of the strong-to-strong crossover. In all the solutions analyzed in this work, the observed temperature of the protein dynamical transition is slightly shifted at lower temperatures than that of the strong-to-strong crossover, but their relative order is the same, showing a correlation between the motion of the protein and that of the hydration water.

Effects of hydroxytyrosol on post-thaw quality of rooster sperm.

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 µg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 µg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 µg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.

Impact of cryoprotective agents on human gut microbes and in vitro stabilized artificial gut microbiota communities.

The availability of microbial biobanks for the storage of individual gut microbiota members or their derived and artificially assembled consortia has become fundamental for in vitro investigation of the molecular mechanisms behind microbe-microbe and/or microbe-host interactions. However, to preserve bacterial viability, adequate storage and processing technologies are required. In this study, the effects on cell viability of seven different combinations of cryoprotective agents were evaluated by flow cytometry for 53 bacterial species representing key members of the human gut microbiota after one and 3 months of cryopreservation at -80°C. The obtained results highlighted that no universal cryoprotectant was identified capable of guaranteeing effective recovery of intact cells after cryopreservation for all tested bacteria. However, the presence of inulin or skimmed milk provided high levels of viability protection during cryoexposure. These results were further corroborated by cryopreserving 10 artificial gut microbiota produced through in vitro continuous fermentation system technology. Indeed, in this case, the inclusion of inulin or skimmed milk resulted in a high recovery of viable cells, while also allowing consistent and reliable preservation of the artificial gut microbiota biodiversity. Overall, these results suggest that, although the efficacy of various cryoprotective agents is species-specific, some cryoprotectants based on glycerol and the addition of inulin or skimmed milk are preferable to retain viability and biodiversity for both single bacterial species and artificial gut microbiota.

A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender.

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Limits of pressure-based ice detection during isochoric vitrification.

Vitrification under isochoric (constant-volume or volumetrically confined) conditions has emerged as an intriguing new cryopreservation modality, but the physical complexities of the process confound straight-forward interpretation of experimental results. In particular, the signature pressure-based ice detection used in many isochoric techniques becomes paradoxical during vitrification, wherein the emergence of a sharp increase in pressure reliably indicates the presence of ice, but avoidance of this increase does not necessarily indicate its absence. This phenomenon arises from the rich interplay between thermochemical and thermovolumetric effects in isochoric systems, and muddies efforts to confirm the degree to which a sample has vitrified. In this work, we seek to aid interpretation of isochoric vitrification experiments by calculating thermodynamic limits on the maximum amount of ice that may form without being detected by pressure, and by clarifying the myriad physical processes at play. Neglecting kinetic effects, we develop a simplified thermodynamic model accounting for thermal contraction, cavity formation, ice growth, solute ripening, and glass formation, we evaluate it for a range of chamber materials and solution compositions, and we validate against the acutely limited data available. Our results provide both counter-intuitive insights- lower-concentration solutions may contract less while producing more pressure-undetectable ice growth for example- and a general phenomenological framework by which to evaluate the process of vitrification in isochoric systems. We anticipate that the model herein will enable design of future isochoric protocols with minimized risk of pressure-undetectable ice formation, and provide a thermodynamic foundation from which to build an increasingly rigorous multi-physics understanding of isochoric vitrification.

Theory-based cryopreservation mode of mesenchymal stromal cell spheroids.

Cryopreservation of spheroids requires development of new improved methods. The plasma membranes permeability coefficients for water and cryoprotectants determine time characteristics of mass transfer through the cell membranes, and therefore the optimal modes of cells cryopreservation. Here we proposed an approach to cryopreservation of multicellular spheroids which considers their generalized characteristics as analogues of the membranes' permeability coefficients of the individual cells. We have determined such integral characteristics of spheroids from mesenchymal stromal cells (MSCs) as osmotically inactive volume; permeability coefficients for water and Me2SO molecules and the activation energy of their penetration. Based on these characteristics, we calculated the osmotic behavior of multicellular spheroids under cooling conditions to select the optimal cooling rate. We also determined the optimal cooling rate of spheroids using the probabilistic model developed based on the two-factor theory of cryodamage. From the calculation it follows that the optimal cooling rate of the MSC-based spheroids is 0.75°Ð¡/min. To verify the obtained theoretical estimates, we conducted experiments on freezing MSC-based spheroids under different modes. The obtained results of primary viability screening indicate that freezing at a constant linear cooling rate of 0.75-1.0°Ð¡/min gives a good result. Theoretical prediction of the spheroid osmotic behavior during cooling provided the basis for experimental verification of varying the temperature to which slow cooling should be carried out before immersion in liquid nitrogen. Slow freezing of spheroids to -40 °C followed by immersion in liquid nitrogen was shown to preserve cells better than slow freezing to -80 °C. Obtained data allow more effective use of MSC-based spheroids in drug screening and regenerative medicine.

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves.

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation. Sperm data were analysed using an ANOVA or Friedman test (results mean ± SD). Paired comparison between the two freezing curves was analysed using the Wilcoxon test. Total and progressive motility were significantly higher (P<0.05) in the EY and CE-3 samples using the slow curve, whereas for the fast curve, total and progressive motility were significantly higher (P<0.05) in the EY samples compared to all the extenders and the samples frozen in CE-3 were significantly higher than the remaining extenders (P<0.05). The percentages of live acrosome intact sperm and of live non-peroxidized sperm were significantly higher (P<0.05) in the EY extender when using either of the freezing curves and in turn, were significantly higher (P<0.05) in samples frozen in CE-3 compared to the remaining extenders. Intact DNA was significantly lower (P<0.05) in the BIO extender, using the rapid curve. To conclude, the commercial equine extender with 3% dimethylformamide, without egg-yolk, could be a suitable alternative for extenders with egg-yolk.

Melatonin application during cryopreservation improves the development and clinical outcomes of human vitrified-warmed oocytes.

In this clinical study, we investigated the potential of melatonin (MT) supplementation in the freeze-thaw medium used for cryopreserved human oocytes. In total, 152 patients who underwent in vitro fertilization between January 2020 and December 2022 were included and categorized into different groups as follows: the donor group, comprising 108 patients who donated their oocytes, with 34 patients using a vitrification and warming medium supplemented with MT (D-MT subgroup) and 74 patients using conventional medium without MT (D-0 subgroup); and the autologous group, comprising 38 patients who used their own oocytes, with 19 patients using medium supplemented with MT (A-MT subgroup) and 19 patients using medium without MT (A-0 subgroup). After thawing, the surviving oocytes in the D-MT and A-MT subgroups and D-0 and A-0 subgroups were cultured in a fertilization media with and without 10-9 MMT for 2.5 h, respectively, followed by intracytoplasmic sperm injection insemination, embryo culture, and transfer. The survival, cleavage, high-quality embryo, clinical pregnancy, ongoing pregnancy, and implantation rates were significantly higher in the D-MT subgroup than in the D-0 subgroup (all P < 0.05). Similarly, the survival, fertilization, high-quality embryo, and high-quality blastocyst rates were significantly higher in the A-MT subgroup than in the A-0 subgroup (all P < 0.05). These findings indicate that MT addition during cryopreservation can enhance the development of vitrified-warmed human oocytes and improve clinical outcomes.

Use of coenzyme Q-10 to improve the pregnancy rate in sheep.

One of the factors responsible for less pregnancy rates is the use of frozen semen in sheep due to the oxidative stress created by the process. The aim of this experiment was to test the effects of adding coenzyme Q-10 (CoQ10) to the seminal extender on sperm quality and the pregnancy rate of sheep. In this study, ejaculates from eight Dorper rams of reproductive age were used and tested in four treatments: Control (pure BotuBov®), C1 (175 µM of CoQ10), C3 (350 µM of CoQ10), and C7 (700 µM of CoQ10). Samples were collected in triplicate from each animal, and sperm analysis was performed by CASA after thawing at 0 h and 2 h. The samples were also analyzed by flow cytometry for plasma and acrosomal membrane integrity, stability, lipid peroxidation, mitochondrial potential, and superoxide anion production. In total, 198 ewes were inseminated by laparoscopy and divided into two groups: control (n=98) and C7 (n=100). Pregnancy diagnosis was performed at 30 days. Coenzyme Q10 proved to be safe for semen cryopreservation, not altering sperm kinetic values between the groups post-thawing. In flow cytometry, the C1 and C7 groups achieved a better index of plasma membrane integrity and membrane stability (P<0.05). A increased pregnancy rate was observed in C7 (52 %) compared to the control (38 %). In conclusion, coenzyme Q10 assists in the cryopreservation process, protecting the sperm cell and improving pregnancy rates in ewes.

Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm.

The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN2) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN2. Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, PI-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress.

Sesame oil as a partial substitute for egg yolk in goat semen extenders.

This study aimed to evaluate the effects of replacing egg yolk extender with sesame oil on the quality of sperm in goats following incubation at 37°C, chilling at 4°C, and freezing. Semen samples were collected from four intact male goats. The individual semen sample was divided into six groups consisting of a control group and five treatment groups with different egg yolk-to-sesame oil ratios. Seminal plasma was removed, and the sperm pellet was diluted with experimental semen extenders. The control group contained an extender of 10 % egg yolk (SO0), and the experimental extenders were composed of 8.75 % egg yolk and 1.25 % sesame oil (SO1.25); 7.5 % egg yolk and 2.5 % sesame oil (SO2.5); 5 % egg yolk and 5 % sesame oil (SO5); 2.5 % egg yolk and 7.5 % sesame oil (SO7.5); and 10 % sesame oil (SO10). Each group of semen was divided into three groups, incubated at 37°C for 1 h, chilled at 4°C for 4 h, or frozen for 24 h. Five replicates were performed. Sperm quality was evaluated, including motility, viability, and functional membrane integrity. The SO1.25 group achieved the highest sperm quality rate among the treatment groups, and the extender did not have a negative effect compared to the control. However, the total replacement of egg yolk with sesame oil in an extender resulted in the lowest sperm quality. In conclusion, the ratios of egg yolk and sesame oil that were acceptable for goat semen cryopreservation were 8.75 % and 1.25 %, respectively.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.