Freezing Medium Research Articles

Dipeptide <i>L</i>-сarnosine (β-alanyl-<i>L</i>-histidine) — nervous tissue cryoprotector non-hybernate animals

In this work, the cryoprotective properties of dipeptide L-carnosine (β-alanyl-L-histidine) were studied on slices of the olfactory cortex of the brain of rats. Changes in the activity of N-methyl-D-aspartate receptors were analyzed as the most vulnerable to the effect of cryopreservation (CP), for this purpose, extracellular NMDA potentials were recorded. Slices were incubated with L-carnosine (20 mM) in the medium and frozen at a slow rate (0.1°C/min) down to –10°C and after CS (30 days) they were heated at the same rate (0.1°C/min) to +37°C. The effectiveness of cryoprotection of L-carnosine was determined by changes in the amplitudes of NMDA potentials after CP compared to before CP. The dipeptide restored the pH of the freezing medium 6.9 (without L-carnosine) to the optimum pH 7.3—7.4, promoted dehydration of free water from slices after CP, inhibited the development of glutamate excitotoxicity in slices. The data obtained prove that L-carnosine exhibits the properties of a non-toxic effective cryoprotector in the nervous tissue of warm-blooded non-hibernating animals.

Investigating the effects of vitamin B12 and Myo-inositol on human sperm parameters during the freeze-thaw process

Background and aims: While sperm freezing has proven to be beneficial for addressing clinical needs, it can also have negative impacts on sperm function. However, it has been observed that additives with antioxidant properties can help alleviate the damage caused by reactive oxygen species (ROS) and cold shock. The objective of this research was to examine the effects of incorporating vitamin B12 and Myo-inositol into the freezing medium on human sperm and how they influence sperm parameters. Methods: The semen samples of 20 people with normal fertility were divided into four equal parts after preparation, including the control group, vitamin B12, Myo-inositol, and both vitamin B12 and Myo-inositol. Sperm parameters were examined after melting and washing the samples. The obtained data were analyzed with SPSS software (version 23) and analysis of variance (P<0.05). Results: The results revealed that the groups receiving antioxidants experienced a significant improvement in sperm motility, viability, morphology, and total antioxidant capacity (TAC) levels compared to the control group. Based on the obtained data, the average sperm motility in the control group was 40.65±4, which was less than that of the B12 group’s average (49.5±5.92), the Myo-inositol group (68.70±15.42), and the vitamin B12+Myo-inositol group (71.5±8.63), and this difference was significant (P<.001). Conclusion: According to the findings, the quality of sperm and the rate of recovery of sperm parameters increase with an increase in the TAC concentration and a decrease in the malondialdehyde (MDA) concentration.

Liposome-based Freezing Medium Improves the Outcome of Mouse Prepubertal Testicular Tissue Cryopreservation

AbstractCryopreservation of testicular tissue holds an important role in the field of fertility preservation, particularly for prepubertal boys diagnosed with cancer. However, prepubertal testicular tissue cryopreservation is still considered to be in the experimental stage necessitating the refinement of cryopreservation protocol. Considering the fact that loss of membrane lipids is the primary cause of freeze–thaw-induced loss of testicular cell functions, in this study, we explored the beneficial properties of exogenous supplementation of membrane lipids in the form of liposomes in enhancing the cryosurvival of prepubertal testicular tissue. The freezing medium supplemented with liposomes (prepared from soy lecithin, phosphatidylethanolamine, phosphatidylserine, and cholesterol) was used for the experiments. Prepubertal testicular tissues from Swiss albino mice were cryopreserved in a liposome-containing freezing medium (LFM) composed of 0.25 mg/mL liposomes, 5% DMSO, and 30% FCS in the DMEM/F12 medium using a slow freezing protocol. The tissues were thawed and assessed for various testicular cell functions. Freezing in LFM mitigated the loss of viability, decreased malondialdehyde level (p < 0.05), and reduced apoptosis (p < 0.05) in the testicular cells compared to the testicular tissue cryopreserved in the control freezing medium (CFM). Further, DMSO (5%) appears to be the ideal penetrating cryoprotectant for prepubertal testicular tissue cryopreservation with liposome-based freezing medium. Similar enhancement in cryosurvival of cells was observed in adult human testicular tissue frozen with LFM. These findings highlight the translational value of liposome-based freezing medium in the cryopreservation of testicular tissue of prepubertal boys undergoing chemotherapy.

Cryopreservation of goat spermatozoa in the Mekong Delta region of Vietnam: evaluating cryoprotectant effectiveness

Conserving and improving the quality of breeds is crucial for enhancing small ruminant production in Vietnam. To support this purpose, cryopreservation was offered as a promising method for semen preservation and maintaining genetic diversity. Moreover, many small-scale farmers in Mekong Delta rely on goats for both income and nutrition, making them an important part of farming activities. This study aimed to develop a sperm cryopreservation protocol and identify the optimal cryoprotectant for goat sperm in the Mekong Delta region of Vietnam. Semen samples were diluted with TCG-EggYolk freezing medium containing cryoprotectant agent (CPA) in a 1:1 ratio. The samples were then placed in 0.5 mL French straws, cooled to 15 °C and 5 °C, and subjected to nitrogen vapor before being immersed in liquid nitrogen at -196 °C. Experiment 1 utilized Glycerol as the CPA in concentrations of 0 %, 5 %, 8 %, and 11 %. Experiment 2 employed dimethyl sulfoxide (DMSO) as the CPA with the same concentrations. Thawed samples were evaluated for sperm quality. Results indicated that in Experiment 1, increasing the Glycerol concentration improved the overall motility, viability, and membrane integrity of sperm. The best outcomes were achieved with 8 % Glycerol, resulting in progressive motility of 57.5 % (P<0.05). In Experiment 2, the extender containing 8 % DMSO demonstrated significantly better performance in terms of overall motility, viability, and membrane integrity compared to 5 % or 11 % DMSO. Progressive motility was 53.5 % (P<0.05). In conclusion, the study determined that using 8 % Glycerol or 8 % DMSO is the optimal choice for freezing goat sperm in the Mekong Delta region of Vietnam.

Post-cooling sperm processing can rescue sperm quality of cooled-stored stallion semen

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen.

Conserving goat sperm post-thawed gene expression and cellular characteristics using the antioxidant coenzyme Q10 supplementation.

The use of frozen goat semen for artificial insemination frequently results in a decline in sperm quality following thawing, which can be attributed to cold shock from cryopreservation, reduced motility, and possible DNA damage. Freezing may compromise mRNA stability due to the presence of free radicals. Despite strong post-thaw motility and no visible DNA fragmentation, sperm can still exhibit altered gene expression patterns. To reduce the damaging impact of free radicals during cryopreservation, antioxidants are typically added to the freezing medium. This study assessed the impact of adding coenzyme Q10 (CoQ10) to frozen sperm diluent on the ATP5F1A and CPT2 gene expression, sperm motility, and viability post-thawing. CoQ10 was added to sperm at six different concentrations: 0 mg/dL (P0), 6.25 mg/dL (P1), 12.5 mg/dL (P2), 25 mg/dL (P3), 50 mg/dL (P4), and 100 mg/dL (P5). The Statistical Package for the Social Sciences (SPSS) software version 22 was used to conduct comparative tests using one-way analysis of variance followed by Duncan's test for motility and viability and Kruskal-Wallis test followed by pairwise comparison test for membrane integrity and gene expression. The addition of CoQ10 to semen diluent has a notable impact on the post-thawed quality of sperm. The most significant outcomes were observed with a 25 mg/dL dosage (P3) for cell viability, membrane integrity, and ATP5F1A gene expression, and with a 50 mg/dL dosage (P4) for sperm motility, membrane integrity, and CPT2 gene expression. Incorporating CoQ10 into frozen semen diluent improves gene expression and prevents deterioration of the cell quality of thawed goat spermatozoa. While the study demonstrates the benefits of CoQ10, the precise molecular mechanisms through which CoQ10 enhances gene expression and cell quality were not fully elucidated. Further investigation is needed to understand these mechanisms in detail. Comparative studies with other antioxidants and cryoprotectants can help establish the relative efficacy of CoQ10 and potentially develop more effective combinations.

Unravelling spermatogenesis in spotted wolffish: Insights from the ultrastructure of juvenile male testes to the cryopreservation of broodstock sperm

The aim of this study was to deepen our understanding of the reproductive biology of male spotted wolffish (Anarhichas minor) using two different experimental approaches involving juvenile and mature broodstock fish. The first approach consisted of a detailed histological examination of the testes to identify the onset of gonadal maturation and characterise the spermatogenic stages in two- and three-year-old juvenile specimens. Light microscopy analysis revealed clear differences between the age groups. Two-year-old fish displayed well-defined interstitial tissue, Sertoli cells and cysts housing spermatogonia stem cells in which meiosis had not yet begun. In contrast, three-year-old fish exhibited cysts containing spermatocytes, spermatids and abundant spermatozoa, indicating the initiation of the spermatogenic cycle, albeit with asynchronous puberty. Histochemical staining revealed a significant presence of smooth myoid cells in the interstitial tissue of sexually mature fish, while electron microscopy further revealed synaptonemal complexes indicating the onset of meiosis and centriolar structures that gave rise to flagella. The second approach focused on optimising semen freezing and cryopreservation procedures in mature broodstock individuals over the age of 10 years. Seven freezing extenders (KT, TS-2, OP, MT, MH, HBSS, or SR), with seawater (SW) as a control, were assessed along with two cryoprotectants dimethylsulfoxide (DMSO) or methanol to evaluate their impact on pre- and post-thaw semen quality. Results showed that the MT and HBSS extenders were superior in total sperm kinetics at 1:3 dilution, and that DMSO showed optimal results in sperm motility and velocity variants. Moreover, the MT and HBSS groups demonstrated consistent sperm viability after cryopreservation, with values similar to fresh samples. Based on the viability results of the SYBR-green-14/PI assay comparing fresh and cryopreserved sperm using MT and HBSS, the MT extender emerged as the most effective freezing medium for cryopreservation of spotted wolffish broodstock sperm. In conclusion, this study provides a comprehensive understanding of the reproductive dynamics of male spotted wolffish, offering valuable insights for both scientific research and aquaculture management.

Effect of microbubbles on immersion freezing of grape tomato

Microbubbles (MBs) were incorporated into calcium chloride solution as a novel freezing medium for immersion freezing of grape tomato. The effects of MB size (39, 43, 48 μm mean diameter), entrapped gas (air, N2, CO2) and freezing temperature (−10, −15, −20 °C) on the freezing behavior and quality attributes of tomato were investigated. MBs increased the nucleation temperature from −7.4 to −3.5 °C and reduced the onset time of nucleation from 5.8 to 2.9 min at freezing temperature of −20 °C, which facilitated the formation of small ice crystals within tomato. MB-assisted freezing reduced the drip loss by 13.7–17.0% and improved the firmness of tomato, particularly when MB size and freezing temperature decreased. Freezing tomato with air-MBs did not compromise its nutritional quality, using N2- and CO2-MBs even increased its lycopene content, by 31% and 23%, respectively. The results proved the preservation effect of MBs on fruit during immersion freezing. This study can benefit the fruit and vegetable industry by providing an efficient freezing technology for producing frozen products with high sensory and nutritional quality.

Effects of different freezing methods on muscle qualities and myofibrillar protein properties of red drum (Sciaenops ocellatus) during storage

The freezing of aquatic products without causing significant changes in quality is recently attracting much attention. In this study, the effects of air freezing (AF, -20 °C), blast freezing (BF, -35 °C), liquid nitrogen freezing (LNF), and immersion freezing (IF, -35 °C) by 20 % ethanol, 20 % propylene glycol, 10 % glycerol, and 10 % sodium chloride aqueous solutions, respectively, on muscle qualities and myofibrillar protein properties of the red drum were investigated. The results indicated that the freezing rate of IF was 2.66 cm/h, which was 6.33 times higher than that in AF groups. Compared with AF and BF, the ice crystals were smaller and more uniformly shaped, with properties of lower water migration and less texture softening after cryogenic liquid freezing of IF and LNF group aquatic products. Immersion freezing showed the same superiority in maintaining the freshness of fish fillets and retaining the protein structure as liquid nitrogen storage. Overall, IF has similar effects to LNF in maintaining muscle qualities and myofibrillar protein properties. This study describes a new freezing method that uses 20 % ethanol, 20 % propylene glycol, 10 % glycerol, and 10 % sodium chloride aqueous solutions as immersion freezing medium. The multi-compound freezing medium has the potential to be used for the freezing of aquatic products.

The evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation.

The aim of this study is the evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation. Semen samples of 50 Asthenoteratozoospermia men (random) were collected. Sperm parameters were analyzed based on World Health Organization (WHO, 2010) criteria (2021) and each sample was divided into 5 groups (E1-E5). E1 (control group): the sperm was cryopreserved without nanoliposome, and Mito-Tempo. E2: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.1 mM) + freezing medium. E3: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.2 mM) + freezing medium. E4: in this group, the cryopreservation sperm with Mito-Tempo 0.3 mM + freezing medium. E5: the cryopreservation sperm with Mito-Tempo 0.2 mM + freezing medium. The result of this study indicated that sperm parameters and total antioxidant capacity (TAC) significantly increase in E3 and E4 groups, compared to E1, E2, and E5 groups respectively (P < 0.05). The percentage of abnormal morphology, DNA fragmentation index (DFI), malondialdehyde (MDA), and the levels of ROS significantly decrease in E3 and E4 groups, compared to E1, E2, and E5 groups (P < 0.05). In addition, the sperm parameters and stress oxidative factors significantly improve in E3 group compared to other groups (P < 0.05). In conclusion, the combination of Mito-Tempo with nanoliposome due to its ability to cooperate with lipid layers may lead to significant performance in reducing oxidative stress damage and increasing the quality of sperm parameters.

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.

Study on the Effect of Post-Freezing Mechanical Properties of Polypropylene Fibre Concrete Based on BAS-BPNN

An indoor accelerated freezing and thawing test of polypropylene fibre-reinforced concrete in chloride and sulphate environments was conducted using the “fast-freezing method” with the objective of investigating the damage law of the post-freezing mechanical properties of hydraulic concrete structures and studying the effects of different mixing amounts of polypropylene fibres on the mechanical properties of concrete. Furthermore, in order to reduce the cost of concrete tests and shorten the time required for conducting concrete tests, a backpropagation neural network based on a Beetle Antenna Search algorithm (BAS-BPNN) was established to simulate and predict the mechanical properties of polypropylene fibre-reinforced concrete. The accuracy of the model was verified. The results indicate that the order of improvement in the macro-physical properties of concrete due to fibre doping is as follows: PPF1.2 exhibited the greatest improvement in macro-physical properties of concrete, followed by PPF0.9, PPF1.5, PPF0.6, and PC. When the freezing and thawing medium and the number of cycles are identical, all four assessment indexes (R2, RMSE, SI, MAPE) demonstrate that the four groups of polypropylene fibre concrete exhibit superior performance to the control group of ordinary concrete. This indicates that polypropylene fibre can enhance the mechanical properties and freezing resistance of the concrete matrix, delay the process of freezing and thawing damage to the matrix, and extend the lifespan of the matrix, yet cannot prevent the ultimate failure of the matrix. The application of intelligent algorithms to optimise the parameters of an artificial neural network model can enhance its capacity to generalise and predict the mechanical properties of concrete. In terms of the coefficient of determination (R2), the Beetle Antenna Search algorithm (0.9782) outperforms the Particle Swarm Optimization (PSO; 0.9676), the Genetic Algorithm (GA; 0.9645), and the backpropagation neural network (BPNN; 0.9460). The improved backpropagation neural network based on the Beetle Antenna Search algorithm not only avoids the trap of local optimality but also improves the model accuracy while further accelerating the convergence speed. This approach can address the complexity, non-linearity, and modelling difficulties encountered during the freezing process of concrete. Moreover, it offers relatively accurate prediction outcomes at a reduced cost in comparison to traditional experimental methodologies.

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

Association of Endogenous Seminal L-Carnitine Levels with Post-Thaw Semen Parameters in Humans

Cryopreservation of semen is a useful tool for male fertility preservation. Some evidence for a beneficial effect of L-carnitine supplementation of freezing media on cryopreserved semen samples has been reported. Here, we examined the association of endogenous levels of seminal L-carnitine with post-thaw semen parameters. We also investigated the effect of freezing medium supplemented with L-carnitine on sperm characteristics, related to endogenous seminal L-carnitine levels. Semen analyses were performed on 125 fresh samples, and after standard cryopreservation and with L-carnitine as a supplement. Participants were categorized into two groups based on the median levels of endogenous seminal L-carnitine: low L-carnitine (≤38.8 µg/ml) and high L-carnitine (&gt;38.8 µg/ml). After standard cryopreservation, semen samples with high L-carnitine levels showed higher rapid progressive, progressive and total sperm motility and a reduced seminal static oxidation–reduction potential (ORP) level than samples with low L-carnitine levels. Only in post-thaw samples with low L-carnitine levels, there was an increase in the amount of sperm neck midpiece defects, compared to the fresh samples. Cryopreservation with L-carnitine had the most beneficial effect on rapid progressive sperm motility in samples with high endogenous L-carnitine levels. In conclusion, L-carnitine has a beneficial impact on sperm characteristics in post-thaw samples both as an endogenous component in seminal plasma and as a supplement in the freezing medium, by improving sperm motility and reducing seminal oxidative stress.

Optimization of Zwitterionic Polymers for Cell Cryopreservation.

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3C)_DSion(100%)/1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.

Effects of taurine, cysteine and melatonin as antioxidant supplements to the freezing medium of Prochilodus brevis sperm

Cryopreservation consist of a set of methods to preserve cells and tissues by drastically reducing the temperature. Among some undesired effects, cryopreservation might generate reactive oxygen species that lead to an increase of oxidative stress, causing damage to cells. This study aimed to test taurine, cysteine, and melatonin on the freezing of Prochilodus brevis sperm and assess its effects on post-thawed sperm quality. Sperm was collected and seven pools were formed (n = 7). They were diluted (1:9) in standard medium (5% glucose, 10% dimethyl sulfoxide and 5% egg yolk) supplemented or not (control) with taurine (0.3, 1.0, 3.16 or 10.0 mM), cysteine (0.3, 1.0, 3.16 or 10.0 mM) or melatonin (0.6, 1.12, 2.0 or 3.56 mM). Post-thawed sperm was evaluated for kinetic (total motility, velocities, and percentage of rapid cells), morphology and membrane and DNA integrity. Differences were found when melatonin was used as an antioxidant. For the variables rapid sperm and sperm velocities, 3.56 mM melatonin presented higher results than the control (melatonin 0 mM). Melatonin 2 mM was similar to 3.56 mM on rapid sperm, average path velocity (VAP) and curvilinear velocity (VCL). No difference was found between concentration 0 mM (control) and taurine treatments. As for cysteine, 0.3 mM presented the best results for rapid sperm than 10 mM, and higher VCL and VAP than 1 mM. Melatonin 3.56 mM presented higher results on kinetic parameters (rapid motility, VCL, VSL and VAP) than other tested antioxidants. Therefore, melatonin 3.56 mM is recommended to be added to the sperm freezing medium of P. brevis.

Evaluation of the effect of mitoquinone on functional parameters, DNA structure, and genes expression related to the apoptotic and antioxidants of human sperm after freezing-thawing.

Sperm freezing is considered as an effective way in assisted reproductive technology (ART) programs, it has detrimental effects on sperm function, due to the production of reactive oxygen species (ROS). This study aimed to investigate the potential of Mitoquinone (MitoQ) in inhibiting the production of mitochondrial ROS during sperm freezing. A total of 20 human normozoosperm samples were collected for this study. The samples were divided into four groups, each containing different concentrations of MitoQ (0, 0.2, 2, and 20 nM), and then subjected to the freezing process. After thawing, the sperm suspensions were evaluated for parameters including motility, morphology, acrosome integrity, adenosine triphosphate (ATP) level, intracellular ROS, viability, chromatin packaging, DNA denaturation, DNA fragmentation, as well as the expression of antioxidants (GPX, SOD) and apoptotic (Bax, Bcl2) genes. The results showed that total and progressive mobility of sperms significantly increased in the 2 nM group, while significantly decreased in the 20 nM group (p ≤ 0.05). Sperm morphology did not significantly improve across all the tested concentrations (p ≥ 0.05). Intracellular ROS levels showed a significant decrease and increase in the concentrations of 2 and 20 nM, respectively (p ≤ 0.05). Furthermore, a significant increase was observed in viability, ATP, acrosome integrity, chromatin packaging, and non-denatured and non-fragmented DNA after treatment with 2 nM of MitoQ, compared with the control group (p ≤ 0.05). Regarding gene expressions, the relative expressions of oxidative stress genes were increased in the 2 nM group and decreased in the 20 nM group (p ≤ 0.05), while no significant difference was observed in the expressions of apoptotic genes compared with the control group (p ≥ 0.05). All the comparisons were made with respect to the control group. Adding the optimal concentration of MitoQ (2 nM) to the sperm freezing medium not only improves sperm functional parameters and reduces DNA damages, but also stimulates the expression of antioxidant genes, leading to even greater benefits for sperm cryopreservation.

The antioxidant effects of butylated hydroxytoluene on cryopreserved goat sperm from a proteomic perspective.

At present, there are few reports about the proteomics changes provoked by butylated hydroxytoluene (BHT) supplementation on cryopreserved semen in mammals. Thus, we aimed to evaluate the effects of different concentrations of BHT on goat sperm and to investigate the proteomics changes of adding BHT to cryopreserved goat (Capra hircus) sperm. Firstly, semen samples were collected from four goats, and frozen in the basic extenders containing different concentrations of BHT (0.5 mM, 1.0 mM, 2.0mM) and a control without BHT, respectively. After thawing, the protective effects of dose-dependent replenished BHT to the freezing medium on post-thaw sperm motility, integrities of plasma membrane and acrosome, reactive oxygen species levels were confirmed, with 0.5 mM BHT being the best (B group) as compared to the control (without BHT, C group). Afterwards, TMT-based quantitative proteomic technique was performed to profile proteome of the goat sperm between C group and B group. Parallel reaction monitoring was used to confirm reliability of the data. Overall, 2,476 proteins were identified and quantified via this approach. Comparing the C and B groups directly (C vs. B), there were 17 differentially abundant proteins (DAPs) po-tentially associated with sperm characteristics and functions were identified, wherein three were upregulated and 14 were downregulated, respectively. GO annotation analysis demonstrated the potential involvement of the identified DAPs in metabolic process, multi-organism process, reproduction, reproductive process, and cellular process. KEGG enrichment analysis further indicated their potential roles in renin-angiotensin system and glutathione metabolism pathways. Together, this novel study clearly shows that BHT can effectively improve quality parameters and fertility potential of post-thawed goat sperm at the optimal concentration, and its cryoprotection may be realized through regulation of sperm metabolism and antioxidative capability from the perspective of sperm proteomic modification.

Impact of Curcumin Supplementation on Sperm Characteristics and Gene Expression in Fish Sperm Freezing Medium

This study aimed to determine whether curcumin addition in freezing media affected the ability of trout semen to resist cryodamage. Fish semen was collected and cryopreserved in a curcumin-supplemented freezing medium at 20 μM. Sperm motility parameters, and nuclear factor 2 gene expressions (NRF-2 and Keap-1) were assessed following freezing and thawing. Adding 20 μM curcumin to the sperm extender increased the rate and duration of motility, as well as the percentage of live sperm at 277 mOsm with a 1:3 dilution. However, the sperm quality decreased at 135 and 365 mOsm with 1:2, 1:3, and 1:4 dilutions. Following cryopreservation, curcumin-supplemented frozen-thawed semen exhibited significantly elevated levels of NRF-2 and Keap-1 gene expressions. The findings demonstrated that adding curcumin to freezing media protected fish and motility parameters against damage induced by the freeze-thawing process.

Coenzyme Q10 improves the quality and invitro fertility of post-thawed buffalo (Bubalus bubalis) semen via its antioxidative effect.

This study aimed to determine the effects of Coenzyme Q10 (CoQ10) in the freezing medium on functional and oxidative stress parameters and invitro fertilization (IVF) rate of buffalo sperm. Collected samples were relocated to the laboratory for initial evaluation, gentle dilution in extenders, cooling (4°C, 2 h), equilibration (4°C, 4 h), packaging (straws, 0.5 mL), programmable freezing, and thawing (37°C, 30 s). Statistical analysis depicted that adding CoQ10 (100 μM) in a freezing medium caused a significant augmentation in total motility (%), average path, and straight-line velocities (μm/sec) of buffalo sperm than control. Adding CoQ10 (100 μM) improved sperm progressive motility, rapid velocity, and functional parameters (%) compared to the control and 10 μM of CoQ10. Moreover, CoQ10 in a freezing medium caused a significant augmentation in seminal plasma catalase (U/mL) and glutathione reductase (GSH; nmol/109 ) at 100 μM than control and other treatments. CoQ10 inclusion (100 μM) ameliorates seminal plasma superoxide dismutase (U/mL), glutathione-S-transferase (GST; nmol/mL/min) fructose (μg/mL), and ATP (nmol/million) than control. Furthermore, CoQ10 at 100 μM improved seminal plasma glutathione peroxidase (μM) levels than control, 10 μM, and 20 μM. Lastly, hydrogen peroxide (H2 O2; nM) production was significantly lower at 100 μM than at control and 10 μM. CoQ10 (100 μM) caused a significant augmentation in the un-capacitated pattern followed by a reduction in the capacitated pattern, and apoptosis-like changes (%) than control, and other treatments, whereas viability was increased than control and other treatments. CoQ10 (100 μM) significantly improved the IVF rate in comparison with control, CoQ10 at 10 μM, and 20 μM groups. In conclusion, the addition of CoQ10 (100 μM) in the freezing medium can improve the quality and invitro fertility of post-thawed buffalo semen via its antioxidative effect. Further studies are needed to evaluate the effect of CoQ10 on the invivo fertility of buffalo bull semen.