Sperm Freezing Research Articles

Predictive Indicators of Cryotolerance and Fertility in Bovine Sperm: Evaluating Fresh Semen Quality to Improve AI Outcomes With Frozen-Thawed Sperm.

The success of artificial insemination (AI) with frozen-thawed semen in cattle is influenced by both female factors and sperm quality. In terms of sperm quality, prior studies indicate that the ability of frozen-thawed bovine sperm to fertilise an oocyte is dependent on their quality and resilience to cryopreservation. Cryopreservation induces oxidative stress, leading to ultrastructural damage in the sperm. This study aimed to determine whether the quality of fresh semen can identify bulls with good and poor sperm freezability. This difference between fresh and frozen semen from the same bull allows us to predict fertility. Motility and kinetic parameters were assessed using computer-assisted sperm analysis (CASA), while six functional variables were evaluated through flow cytometry, both before and after the freeze-thaw process on the sperm from 13 bulls. Invivo fertility was measured using 90-day non-return rates. The principal component analysis (PCA) of eight sperm variables post-thaw identified one principal component explaining 81.19% of the total variance and classified the bulls into two groups: Poor freezability bulls (progressive motility: 48.12% ± 8.41%; viability: 77.51% ± 7.61%) and good freezability bulls (progressive motility: 58.64% ± 6.64%; viability: 88.12% ± 2.52%). Bulls with higher freezability showed better sperm viability and motility, as well as lower levels of ROS, superoxides and intracellular calcium before cryopreservation that were significantly correlated with higher non-return rates (NRR). The results underscore the importance of assessing the quality and functionality of fresh semen to predict the fertility potential of cryopreserved sperm. This approach can aid in selecting ejaculates with the best potential for successful artificial insemination, ultimately improving reproductive performance in dairy cattle.

Investigation of the efficacy of different cryoprotectants in the freezing of testicular tissue and epididymal sperm: Spermatological parameters, tissue viability and PARP-1 gene expression

The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.

The effect of myo-inositol antioxidant activity on human sperm parameters and DNA damage in ultra-rapid and conventional freezing methods

Male fertility preservation is still challenged by cell damage induced during sperm cryopreservation and impaired sperm structure and function. Sperm ultra-rapid freezing, despite a higher protective effect compared to conventional freezing method, is still associated with suboptimal sperm cryosurvival and needs to be modified to increase its efficiency in sperm protection. Sperm freezing media supplemented with antioxidants can improve sperm parameters following freezing-warming process. In this study, we aimed to investigate the effect of employing ultra-rapid freezing and myo-inositol on sperm cryosurvival. Thirty semen samples with normal sperm parameters were collected and each one was divided into four portions to cryopreserve by conventional freezing, ultra-rapid freezing, conventional freezing + myo-inositol 2 mg/ml, and ultra-rapid freezing + myo-inositol 2 mg/ml. Sperm samples warmed after at least 24 h of freezing and sperm cryosurvival were analyzed by evaluation of sperm motility, viability, morphology and DNA fragmentation index (DFI). Freezing method had a significant influence on post-thaw sperm DFI and morphology (p < 0.05) and the interaction between freezing method and antioxidant supplementation significantly affected sperm morphology (p < 0.05). The highest percentage of sperm normal morphology and minimal DFI was achieved using ultra-rapid freezing supplemented by myo-inositol antioxidant compared to other groups (P < 0.05). The highest sperm DNA damage after freezing-warming was observed following the conventional freezing method. In conclusion, sperm freezing method was identified as factor strongly influencing sperm DFI and morphology after thawing/warming. Sperm samples can be rapidly frozen using the modified freezing media supplemented by myo-inositol without impacting sperm DNA and morphology.

Preserving Fertility in People With Rheumatic Diseases.

Rheumatology patients historically were told they "can't" or "shouldn't" become pregnant. Improved rheumatic diagnostics and treatments have led to decreased morbidity and mortality and increased quality of life resulting in an opportunity to focus on fertility and its preservation. In the same vein as rheumatic disease care, assisted reproductive technology (ART), which includes freezing of egg and sperm as well as in vitro fertilization, has made considerable strides in the recent past. ART is safe for those with rheumatic diseases and has comparable outcomes to the general public, but may take additional effort due to optimal timing, rheumatic medications, and other nuances. In a specialty that treats chronic inflammatory diagnoses using teratogens and gonadotoxins, it is important to address family building desires with patients so their goals can be met.Rheumatologists have little knowledge of ART and how it impacts or applies to their patients; however, patients want their rheumatologist to be the source of knowledge for this information (Arthritis Rheumatol. 2022;74:suppl 9). Many barriers to ART exist and will be explored, with the financial burden being paramount (Glob J Health Sci. 6;1:181-191). Future efforts to examine the future fertility of rheumatology patients in an era of biologics and "treat-to-target" are needed to better understand who would most benefit from this costly and not without risk medical treatment. Given the changing landscape of financial support for ART due to insurance mandates, rheumatologists should not modify counseling based on the anticipated ability of patients to afford care. Preservation should also be broached with patients without partners and those from the LGBTQAI+ community. In addition to expanding the education of rheumatologists regarding this topic and its incorporation into clinical care, advocacy for ART access and insurance coverage is a much-needed future direction.

Seminal plasma microbiomes, sperm parameters, and cryopreservation in a healthy fertile population.

Recent advances in microbiome research have revealed the presence of diverse microbial communities in human tissues previously thought to be sterile. The present study delves into the emerging field of seminal plasma microbiomics, examining the relationship between semen microbes and semen parameters and post-freezing tolerance. The study involved a cohort of healthy fertility males and microbial genome analysis using 16S rRNA to characterize the microbial diversity of seminal plasma. Microbial diversity analysis identified unique amplicon sequence variants (ASVs) and genera dominant in seminal plasma. Spearman's correlation coefficient was used to assess the relationship between flora and semen parameters. A paired t-test was used to compare the changes in microbiome expression in seminal plasma before and after cryo-resuscitation. The relevant results show that the top five phyla in terms of abundance of seminal plasma microbiome were Firmicutes, Bacteroidota, Proteobacteria, Actinobacteriota, and Campylobacterota. Spearman correlation analysis highlighted the association between specific microbial species and semen parameters, between Porphyromonas_asaccharolytica and sperm concentration. Microbial changed significantly after cryo-resuscitation, affecting taxonomic units such as Campylobacter and Muribaculaceae, and KEGG enrichment analyses, suggesting that metabolic pathways are associated with sperm freezing. Eubacterium_coprostanoligenes and Eptoniphilus_duerdenii exhibited a potential impact, while Orynebacterium_tuberculostearicum demonstrated a positive correlation with the recovery rate of progressive motile sperm. The semen of normal fertile individuals contains a microflora component that is closely related to semen quality, including the sperm's ability to withstand freezing.

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&lt;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&lt;.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.

Stallion Sperm Freezing with Different Extenders: Role of Antioxidant Activity and Nitric Oxide Production.

Sensitivity to freezing remains a critical issue in stallion semen cryopreservation procedures. To explore this topic in-depth, semen was collected from ten stallions, diluted with three different extenders, transported to the laboratory, and then centrifuged and frozen with four different extenders. We conducted analyses of sperm kinetics, mitochondrial membrane potential (MMP), and hydrogen peroxide content both before and after freezing. Additionally, we assessed antioxidant activity using the ABTS and FRAP methods and measured nitric oxide stable metabolites (NOx) in the blank extenders, seminal plasma, and extenders conditioned by spermatozoa before and after freezing. We found significant variability in the antioxidant activity and NOx content of the blank extenders and the seminal plasma. In the seminal plasma, ABTS-based antioxidant activity and NOx values were correlated with some sperm kinematic parameters and MMP in refrigerated semen, while no correlation was observed in frozen sperm parameters. Sperm function varied significantly between stallions but not between extenders, either before or after freezing. However, significant differences in antioxidant activities and NOx values were found among extenders conditioned following freezing. These results provide new insights into the factors contributing to the variability in individual stallions' tolerance to sperm freezing.

Comparison of Clinical Outcomes in the Slow-Developing Blastocysts With or Without Preimplantation Genetic Testing-Aneuploidy on Day 6 in the Frozen–Thawed Cycle

ObjectivesThis study investigated the potential of the slow-developing blastocysts using preimplantation genetic testing-aneuploidy (PGT-A) in patients undergoing frozen–thawed embryo transfer, stratified by age. MethodsA retrospective analysis was performed including a total of 743 cycles, the first frozen embryo transfer (FET) cycle with single embryo transfer, who underwent treatment between January 2020 and July 2023 in a single fertility centre, Gangnam CHA Fertility Center. A total of 743 cycles, in which we performed intracellular sperm injection and freeze-all strategy, from 743 patients were included. The patient group was divided into 4 groups as follows: group 1 (G1), 208 FET on day 5; group 2 (G2), 177 FET with PGT-A on day 5; group 3 (G3), 220 FET on day 6; group 4 (G4), 138 FET with PGT-A on day 6. We also divided into 2 groups—under 35 years of age and over 35 years of age—and performed the analysis separately for each group. ResultsIn the under 35 years of age group, there were no significant differences in clinical pregnancy and miscarriage rates in G1 and G2 (67.2% vs. 63.8%, not statistically significantly different). Also, G4 had a higher clinical pregnancy rate than G3, but it was not significant (51.8% vs. 54.7%, not statistically significantly different). In the 35 years or older group, G2 had higher pregnancy rates than G1 and lower miscarriage rates (clinical pregnancy rate: 43.3% vs. 67.7%, P = 0.001, miscarriage rate: 22.5% vs. 3.4%, P = 0.001). In addition, G4 had a higher pregnancy rate than G3 and a lower miscarriage rate (clinical pregnancy rate: 31.8% vs. 46.9%, P = 0.003, miscarriage rate: 22.9% vs. 2.2%, P = 0.023). ConclusionsIn the under-35-year-old group, PGT-A on day 5 and day 6 showed a high pregnancy rate and a low miscarriage rate. Therefore, using PGT-A seems advantageous for patients of an advanced maternal age.

Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus)

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

Assessment of bisphenol accumulation from disposable devices used sequentially in IVF routine procedures

Research question: To investigate the release of bisphenols (BPs) from disposable devices (DDs) used in ART procedures and to determine whether they accumulate when several DDs are used sequentially under routine conditions.Design: A comprehensive assessment of 19 individual DDs (31 measurements) and 9 combinations of DDs replicating the main steps in an ART procedure was undertaken. The extraction of BPs followed routine use conditions (temperature and duration). The concentration of 10 BPs was determined using on-line solid phase extraction/liquid chromatography/mass spectrometry methodology.Results: Bisphenol S (BPS) was quantified consistently from 100 mm culture dishes (32 ± 20 pg) and from high security sperm straws (3 ± 1 pg). Also, BPS and Bisphenol A (BPA) were quantified consistently from spermicide-free condoms (95 ± 78 and 83 ± 49 pg, respectively). No other BPs were detected in individually tested DD.When DDs were used together, BPA and BPS were both detected consistently in the association of 13 DDs mimickin;g the sperm collection in condom and its preparation (46 ± 16 and 43 ± 32 pg, respectively). BPS was quantified consistently in the association of 14 DDs mimicking the sperm collection, its preparation and freezing (10 ± 4 pg), and in the association of 17 DDs mimicking the oocyte pick-up (37 ± 22 pg).Conclusions: BPA and BPS are released in small quantities from some DDs used in routine conditions during ART procedures, but do not clearly accumulate when these consumables are used together.

Proteomic Analysis of Differences in the Freezability of Porcine Sperm Identifies α-Amylase As a Key Protein.

To investigate the mechanisms underlying the differences in the freezability of boar semen, Yorkshire boars with freezing-tolerant semen (YT, n = 3), Yorkshire boars with freezing-sensitive semen (YS, n = 3), Landrace boars with freezing-tolerant semen (LT, n = 3), and Landrace boars with freezing-sensitive semen (LS, n = 3) were selected for this study. Their sperm was subjected to protein extraction, followed by data-independent acquisition proteomics and functional bioinformatics analysis. A total of 3042 proteins were identified, of which 2810 were quantified. Some key KEGG pathways were enriched, such as starch and sucrose metabolism, carbohydrate digestion and absorption, mineral absorption, the HIF-1 signaling pathway, and the necroptosis pathways. Through PRM verification, we found that several proteins, such as α-amylase and epididymal sperm-binding protein 1, can be used as molecular markers of the freezing resistance of boar semen. Furthermore, we found that the addition of α-amylase to cryoprotective extender could significantly improve the post-thaw motility and quality of boar semen. In summary, this study revealed some molecular markers and potential molecular pathways contributing to the high or low freezability of boar sperm, identifying α-amylase as a key protein. This study is valuable for optimizing boar semen cryopreservation technology.

O-312 Home semen freezing for fertility patients at the time of global travel restrictions due to COVID-19 pandemic

Abstract Study question Can an alternative process for freezing semen be utilized for patients in need of fertility care during the coronavirus disease 2019 (COVID-19) pandemic travel restrictions? Summary answer At-home sperm freezing was developed during COVID-19 to offer fertility care for travel restricted patients, which provided similar IVF outcomes compared to laboratory frozen sperm. What is known already Cases of COVID-19 were first detected in December 2019 spreading rapidly across the world leading to declaration of a pandemic on March 11, 2020 and resulting in international border closures and global travel restrictions lasting into 2023. Sperm freezing by trained laboratory personnel is well established and safe clinical practice. While home semen collection kits existed to extend sperm survival for hours allowing sufficient time for the sample to reach facility to be frozen in the laboratory, there were no home semen freezing methods for longer time constraints available for our patients. Study design, size, duration A specialized home semen freezing process has been developed and validated in-house on research samples. Retrospective analysis was performed to assess home sperm freezing efficiency in patients undergoing fertility treatment. IVF outcomes were compared between home sperm freezing to the samples frozen in our laboratory (control) from 2020 to 2023. Participants/materials, setting, methods In total 508 semen samples frozen at home have been received at the clinic, with 495 (98%) of samples arriving within set time of 18 days in optimal frozen condition. Delivery of 12 samples were delayed causing sample thawing during transportation that required a new at home freezing. Only IVF with ova donors were included to compare outcomes of 448 IVF with semen frozen at home to 421 IVF with semen frozen in the laboratory. Main results and the role of chance Only 17 (3%) of home semen samples arrived from patients within Canada, while 491 (97%) were from patients of 27 different countries of several regions: Europe 346 (68%), Australia 73 (15%), Middle East 36 (7%), USA 26 (5%), Asia 7 (1.5%), and Latin America 3 (0.5%). Men from the study group were similar to control in terms of age (36 ± 5.2 versus 36.8 ± 5.8). Post thaw test was performed on all home freezing samples as a quality control to ensure that sufficient sample was available for IVF: mean sperm concentration was 25.3 M/ml ± 14.8 and motility of 21% ± 10. IVF outcomes between samples frozen at home or in the lab were compared; fertilization (80.3% ± 14.6 v. 79.6 ± 19.3), blastulation (66.5% ± 20.7 v. 66.6% ± 19.3%), clinical pregnancy (39.1% v. 38.8%), miscarriage (14.6% v.6.7%) or life birth (30.9% v. 31.6%), respectively, were not statistically different. PGT-A was performed on 7224 blastocysts with results controlled by the age of ova donors and were not statistically different for embryos derived from sperm frozen at home and in the laboratory with euploidy (65.3% v. 64.6%), mosaic (14.8% v.13.8%) and aneuploidy (19.9% v. 21.6%), respectively. Limitations, reasons for caution Pre-freezing semen analysis was only available for laboratory frozen samples. Post-thaw results were not compared between home and laboratory frozen sample as different freezing protocols with two dilution factor (1:1 v. 3:1) and different sample volume per frozen unit (0.1-1.2 ml in vial v. 0.5 ml in straw) were used. Wider implications of the findings In cases where it is logistically difficult or impossible for sperm providers to travel to the clinic for IVF treatment, home semen freezing performed by the patient is a viable and safe method. Trial registration number not applicable

Evaluation of the effectiveness of the use of exosomes in the regulation of the mitochondrial membrane potential of frozen/thawed spermatozoa.

Numerous studies confirm the involvement of extracellular vesicles (EVs) in the regulation of physiological processes of mammalian sperm cells. It has been proven that they take part in the processes of capacitation, acrosonmal reaction, and anti-oxidation. Despite growing interest in the biomedical potential (including the search for new reproductive biomarkers) of EVs, the role of extracellular seminal vesicles in maintaining semen quality during cryopreservation has not yet been established. Therefore, the objective of this experiment was to evaluate the effectiveness of the use in the regulation of the mitochondrial membrane potential of bovine sperm and to explain the mechanisms of EV action during cell cryopreservation. Exosomes were isolated from bull semen plasma, measured, and used for extender supplementation. Semen samples were collected from Simmental bulls, diluted, and pre-evaluated. Then they were divided into equal fractions that did not contain EVs or were supplemented with 0.75; 1.5 and 2.25 mg/ml of EVs. The test samples were frozen/thawed and the mitochondrial membrane potential, DNA integrity, and viability were evaluated. EVs have been established to have a positive effect on cryopreserved sperm structures. The most favourable level of EVs was 1.5 mg / ml, which can be successfully to improve cell cryostability during freezing/thawing. In this study, exosomes isolated from the sperm plasma and supplemented with a concentrated dose in the extender for sperm freezing were shown to significantly improve cryostability of cells by supporting the potentials of the mitochondrial membrane and protecting the cytoplasmic membrane of spermatozoa.

The effects of vitamin E supplementation on sperm parameters, chromatin integrity, and gene expression before and after freezing in aged mice.

Some age-related testicular changes, such as Sertoli cell vacuolization and blood-testis barrier breakdown, reduce total sperm production and male fertility. Therefore, this study investigated the effect of vitamin E on restoring testicular function in aged mice. Sperm cryo-resistance was also assessed. Twenty-eight 48-week-old male Naval Medical Research Institute mice were divided into four groups for a daily gavage of vitamin E: the control group received distilled water, while the three treatment groups were administered 100, 200, and 400 mg/kg, respectively, for 4 weeks. Subsequently, semen analyses, DNA fragmentation index (DFI), and protamine deficiency tests were conducted. Testicular histology, tissue antioxidant enzyme activity, and gene expression levels were also assessed. The two higher dosages of vitamin E were associated with a higher sperm count, greater progressive motility, and improved sperm morphology (p<0.05). These benefits were also evident after sperm freezing (p<0.05). Although chromatin abnormalities increased following vitrification, the treatment groups showed better outcomes (p<0.05). The tubular diameter, epithelium height, and luminal diameters remained unchanged with age. The tissue antioxidant capacity was greater in the groups receiving the high doses of vitamin E. Additionally, significant increases in inhibitor of DNA binding protein-4 (Id4) and GDNF family receptor alpha-1 (Gfra1) expression were observed in the higher vitamin E dosage groups, and promyelocytic leukemia zinc finger protein (Plzf) expression was notably present in the 400 mg/kg treatment group compared to the control group (p<0.05). Antioxidant supplementation might enhance reproductive outcomes in aging males. The observed effects included improved sperm cryo-resistance, which is advantageous for future applications such as sperm freezing or fertility preservation.

Effects of MnTBAP on Porcine Semen Cryopreservation and Capacitation.

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 μM of MnTBAP based on a Tris-egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 μM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 μM and 150 μM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 μM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 μM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 μM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze-thawing, improving the fertilization capacity, and leading to increased embryo development.

Correction of metabolic and structural disorders in bull spermatozoids during cryopreservation using molecular hydrogen

The influence of molecular hydrogen on the fertility parameters and energy metabolism of spermatozoids of black-and-white Holstein bulls was studied. Sperm was diluted with sterile BioXcell medium (France), diluted in hydrogen water. We studied native sperm diluted with BioXcell and BioXcell with molecular hydrogen, as well as after deep freezing with and without pre-treatment with molecular hydrogen. The addition of molecular hydrogen to the medium for diluting sperm and subsequent freezing changed the functional status of cells after thawing, leading to an increase in their motility, an increase in the number of motile and fast sperm, and a decrease in the number of slow cells relative to the analyzed indicators of regenerative cells after cryopreservation that were not exposed to molecular hydrogen. Sperm motility after thawing was 12% higher (p ≤ 0.05), the average movement speed was 9% (p≤0.05), and the ATP content was two times higher than in sperm samples not treated with molecular hydrogen. The results of the experiment showed that the addition of molecular hydrogen to the semen extender improves the biological indicators of the quality of bull sperm, its fertility, and increases the energy metabolism of spermatozoids.

Sperm melatonin receptors, seminal plasma melatonin and semen freezability in goats

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.

Advancements in Understanding and Enhancing Antioxidant-Mediated Sperm Cryopreservation in Small Ruminants: Challenges and Perspectives.

Cryopreservation poses significant challenges to the preservation of sperm integrity and function, particularly in small ruminants where cryodamage is pronounced. This review explores the molecular mechanisms underlying sperm cryodamage and strategies for improving cryopreservation outcomes, with a focus on the role of antioxidants. Cryopreservation-induced alterations in proteins and RNA transcripts critical for sperm function, including motility, capacitation, fertilization, and embryo development, are discussed. Proteomic, transcriptomic, and epigenomic advancements have provided valuable insights into these mechanisms, offering potential biomarkers for predicting sperm freezability and enhancing cryopreservation strategies. Combining technologies such as mass spectrometry and flow cytometry allows for a comprehensive understanding of molecular and cellular changes induced by the freezing-thawing process. However, challenges remain in optimizing cryoprotectant formulations and antioxidant supplementation to improve post-thaw sperm fertility. Further research is needed to explore a wider range of novel cryoprotectants, antioxidants, and proteins for cryopreservation media, as well as to validate their efficacy in enhancing sperm viability and function. Additionally, investigations into the effects of cryopreservation on RNA transcripts and epigenetic factors in small ruminant species are warranted to advance our understanding of sperm preservation. Overall, this review highlights the importance of antioxidants in mitigating cryodamage and underscores the need for continued research to refine cryopreservation protocols and improve reproductive outcomes in small ruminants.

Reproductive Strategies for Declining Fertility: Fertility Preservation

There is a global trend of declining fertility among people of childbearing age and mankind is confronted with great challenges of fertility problems. As a result, fertility preservation technology has emerged. Fertility preservation involves interventions and procedures aimed at preserving the patients' chances of having children when their fertility may have been impaired by their medical conditions or the treatments thereof, for example, chemotherapy and/or radiotherapy for cancer. The changes in patients' fertility can be temporary or permanent damage. Fertility preservation can help people diagnosed with cancer or other non-malignant diseases. More and more fertility preservation methods are being used to preserve the fertility of cancer patients and protect their reproductive organs from gonadotoxicity. Fertility preservation may be appropriate for young patients with early-stage cancers and good prognosis before they undergo treatments (chemotherapy and/or radiotherapy) that can negatively affect their fertility. It is also appropriate for patients with chronic conditions or those who have encountered environmental exposures that affect their gonadal function. Fertility preservation methods include oocyte cryopreservation, embryo cryopreservation, and ovarian tissue cryopreservation (OTC) for women and sperm freezing and testicular tissue freezing for men. The survival rates of children and adolescents diagnosed with malignant tumors have been steadily increasing as a result of advances in cancer treatments. Cryopreservation of oocytes and sperm is recognized as a well-established and successful strategy for fertility preservation in pubertal patients. OTC is the sole option for prepubertal girls. On the other hand, cryopreservation of immature testicular tissue remains the only alternative for prepubertal boys, but the technology is still in the experimental stage. A review showed that the utilization rate of cryopreserved semen ranged from 2.6% to 21.5%. In the case of cryopreserved female reproductive materials, the utilization rate ranged from 3.1% to 8.7% for oocytes, approximately from 9% to 22.4% for embryos, and from 6.9% to 30.3% for ovarian tissue. When patients have needs for fertility treatment, cryopreserved vitrified oocytes are resuscitated and in vitro fertilization-embryo transfer (IVF-ET) was performed to help patients accomplish their reproductive objectives, with the live birth rate (LBR) being 32%. On the other hand, when cryopreserved embryos are resuscitated and transferred, the LBR was 41%. OTC has the advantage of restoring natural fertility and presents a LBR of 33%, compared with the LBR of 19% among 266 IVF patients. In addition, OTC has the benefit of restoring the endocrine function. It has been observed that the shortest recovery time of the first menstruation after transplantation was 3.9 months, and the recovery rate of ovarian function reached 100%. To date, a growing number of cancer survivors and patients with other diseases are benefiting from fertility preservation measures. In the face of declining human fertility, fertility preservation provides a new approach to human reproduction. Fertility preservation should be applied in line with the ethical principles so as to fully protect the rights and interests of patients and their offsprings.

Effects of meteorology and lunar cycle on the post-thawing quality of avian sperm.

Various climatological and lunar cycle parameters have a direct impact on animal reproduction, and in the case of the avian species, spermatozoa are extremely sensitive to heat stress. These parameters could influence sperm freezability, which will ultimately affect post-thawing semen quality, being sperm motility in roosters a relevant indicator of this quality as it is highly related to fertility. Therefore, the objective of the present study is to determine which are the climatological and lunar cycle parameters that have a greater effect on sperm freezability in roosters. Sperm was obtained from 16 Utrerana breed roosters and a total of 27 replicates were performed. A pool was made with those ejaculates that met the minimum quality criteria for each replicate, and four freezing-thawing samples per replicate were analyzed. The straws were thawed, and sperm motility was evaluated, classifying the results obtained into four seminal quality groups according to the guidelines of the Food and Agriculture Organization of the United Nations (Group 1: Good, Group 2: Satisfactory, Group 3: Acceptable but undesirable and Group 4: Unsatisfactory). The following traits were recorded for each day of semen collection: maximum temperature, minimum temperature, maximum barometric pressure, minimum barometric pressure, maximum gust, wind direction, mean wind speed, sunshine hours, rainfall, moon phase, and percentage of illuminated lunar surface over the total area. A discriminant canonical analysis was performed to determine which of these parameters offered the most information when classifying an ejaculate in each quality group, with minimum temperature, the new moon as moon phase, minimum barometric pressure, and rainfall being the most significant variables. According to the results obtained, semen quality decreases when temperature and precipitation are lower, pressure is higher, and when there is a new moon phase. Therefore, these environmental conditions should be avoided for sperm collection and processing.