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Semen cryopreservation is a crucial technique for preserving male fertility, playing a vital role in assisted reproductive procedures by storing frozen semen samples for artificial insemination (AI) and intra-cytoplasmic sperm injection (ICSI) to enhance reproductive success rates. This study aims to identify the most effective cryopreservation methods and assess their impact on semen quality, particularly sperm DNA fragmentation. A total of 30 semen samples were categorized into fertile and infertile groups. DNA fragmentation analysis was conducted using the Sperm Chromatin Structure Assay (SCSA). Each sample was divided into three portions and frozen using different cryoprotectants: (egg-yolk + glycerol), (sucrose + glycerol), and (glycerol alone). After one month of storage, the samples were analyzed to determine the most effective medium. The findings revealed a decline in sperm motility post-freezing compared to fresh samples, along with a slight increase in morphological abnormalities. Additionally, there was a rise in sperm DNA fragmentation and an increase in apoptotic marker (Caspase-3) levels after the freezing process. The study concluded that cryopreservation and thawing caused some degree of sperm cell damage, with infertile samples being more adversely affected than fertile ones.

Cryopreservation reduces ram sperm viability and fertilizing ability by inducing oxidative stress, membrane damage, and DNA fragmentation. This study evaluated whether adding chitosan-stabilized nano green tea extract (Nano-GTE) to a Tris-soybean lecithin extender improves post-thaw semen quality and fertility of Ossimi rams. Ejaculates (pooled from five mature rams) were extended with Tris-soybean lecithin containing 0 (control), 50, 100, 150, or 200 µg/mL Nano-GTE, equilibrated at 5 °C for 4 h, frozen in 0.25 mL straws, stored in liquid nitrogen and thawed at 37 °C for 30 s. Post-thaw progressive motility, viability and membrane integrity were significantly higher in the 100 µg/mL group (PrM: 46.3 ± 0.21% vs. control 41.5 ± 0.22%; Viability: 47.1 ± 0.27% vs. control 42.8 ± 0.24%; Membrane integrity: 47.6 ± 0.26% vs. control 43.9 ± 0.27%; P  < 0.001). DNA fragmentation (Comet assay) was lowest in the 100–150 µg/mL groups (DNA damage: 5.0 ± 0.21% at 100 µg/mL vs. control 8.3 ± 0.21%; P  < 0.001). Antioxidant capacity (TAC) in the post-thaw medium increased at 100 µg/mL, while markers of membrane damage (MDA, AST, ALT, LDH) were reduced compared with control ( P  < 0.05). Fertility (pregnancy rate after cervical AI) was higher with semen from the 100 and 150 µg/mL groups, whereas the 200 µg/mL dose impaired some parameters. Together, these results indicate that supplementing a Tris–soybean lecithin extender with Nano-GTE at 100 µg/mL markedly improves the post-thaw quality and fertility of ram sperm; higher doses (≥ 200 µg/mL) may be detrimental.

The aim of this study was to evaluate the cryoprotective capacity of Tris-Citrate-Fructose (TCF) extenders supplemented with soy lecithin (asolectin) at concentrations of 0.05% (Asol 0.05%) and 0.5% (Asol 0.5%), in comparison with the commonly used egg yolk-based extender (TCF-EY). Ten ejaculates, each obtained from a different dog, were included in the experiment. Both fresh and cryopreserved semen were assessed for total motility (TM) and progressive motility (PM) using computer-assisted sperm analysis (CASA). Plasma membrane integrity (LIVE), DNA fragmentation index (DFI), mitochondrial activity (MT−/PI+, MT+/PI+, MT+/PI−), and oxidative stress (CellROX+/DRD+, CellROX−/DRD−, CellROX+/DRD−) were measured by flow cytometry. Significant differences (p<0.05) were found in TM (%) between the TCF-EY and Asol 0.5% groups (51.16±5.80 vs. 22.33±9.62). However, no significant differences were observed in LIVE sperm. The DFI remained below 5% in all examined groups. Compared with the control TCF-EY extender, both soy-supplemented groups showed a significant reduction in the population of viable sperm without oxidative stress. The use of asolectin-based extenders also significantly reduced the percentage of viable sperm with active mitochondria, compared to the TCF-EY extender. There were no significant differences between the tested two soy lecithin concentrations in any of the evaluated parameters.. In conclusion, the TCF-EY extender demonstrated superior efficacy in preserving semen quality after cryopreservation. Further research is needed to explore alternative phospholipid sources, including different types and concentrations of soy lecithin, and their effects on sperm fertilizing capacity.

Boar semen cryopreservation is crucial for the porcine industry. The two‐step (S2) is currently used for cryopreservation of boar semen. In this method, a cryoprotectant is added at 4°C to reduce its toxicity. Another method is the one‐step (S1), which is easier, cheaper and reduces the need for equipment, but may increase the toxicity of cryoprotectant. This study compared S1 and S2 equilibration protocols, evaluating post‐thaw total motility, viability, motion parameters, membrane integrity, antioxidant indicators, apoptosis and related gene expression. Results showed S2 had higher motility (p < 0.05), while S1 had greater average path velocity (p < 0.05), better plasma membrane integrity (p < 0.05), lower malondialdehyde (MDA) (p < 0.01), higher total antioxidant capacity (T‐AOC) and catalase (CAT) (p < 0.01), lower superoxide dismutase (SOD) (p < 0.05) and reduced apoptosis (p < 0.05). S2 had higher Caspase‐9, Bax and SOD‐2 expression (p < 0.05). S1 shows superiority in membrane protection, antioxidant capacity and anti‐apoptosis, despite slightly lower motility. It is a promising method for large‐scale use with further refinement to enhance post‐thaw motility. This study compared the effects of one‐step and two‐step balanced treatment methods for cryopreservation of boar semen and evaluated their impact on sperm quality after thawing. The optimized balanced treatment method improved sperm integrity and antioxidant capacity, and reduced apoptosis and oxidative damage. These findings provide practical guidance for improving the efficiency of boar semen cryopreservation and reproductive performance.

Cryopreservation of semen was a crucial technology in livestock breeding; however, it compromised sperm quality due to oxidative stress, lipid peroxidation, and structural damage. This study aimed to determine the effect of adding α-tocopherol and glutathione antioxidants in Caudal Epididymal Plasma (CEP) diluent on the quality of frozen Bali bull semen. Four groups of CEP semen diluents were prepared with different antioxidant supplementation: P1 (CEP), P2 (CEP + 2 mM α-tocopherol), P3 (CEP + 0.75 mM glutathione), and P4 (CEP + 2 mM α-tocopherol + 0.75 mM glutathione), while Tris Egg Yolk extender was used as a comparator (P5). Semen collected from two Bali bulls was evaluated before and after the freezing and thawing processes using parameters such as motility, viability, membrane integrity, lipid peroxidation (MDA), and DNA integrity. The results showed that the P2 group supplemented with 2 mM α-tocopherol had the highest post-thawed motility, viability, and membrane integrity compared to other groups (P<0.05). The addition of α-tocopherol and glutathione proved to reduce MDA concentrations and improve the quality of frozen semen. This study concluded that α-tocopherol and glutathione improved the quality of Bali bull semen after cryopreservation, with α-tocopherol showing more significant results.

Background/Objectives: Semen cryopreservation has been widely used in recent decades, mainly in patients with male factor infertility (MFI) and patients with cancer (to preserve their fertility potential before undergoing gonadotoxic treatments, such as chemotherapy or radiotherapy). Methods: we evaluated the temporal trend and the existence of factors determining the usage of cryopreserved semen (in order, for instance, to conceive using assisted reproduction techniques (ARTs)). We evaluated 7044 patients who cryopreserved since 1991, evaluating clinical information such as the diagnosis, age, the method with which the collection took place and the number of pick-ups for use in ART. Results: The mean age of the patients was 33.12 ± 8.41 years. The most common cancers were testicular (1758/3262) and hematological cancers (942/3262). Patients who underwent cryopreservation for MFI picked up their samples much more than neoplastic patients (42.15% vs. 8.55%). Patients 35–40 years old picked up their samples more frequently when compared with other age groups. Moreover, we found several data regarding the sub-types of cancers and the methods of semen collection. Conclusions: According to our results, the indication for semen cryopreservation, age and the method of collection might represent a simple way to predict the future use of semen for ART.

In this study, we explore the effects of Poria cocos mushroom polysaccharides (PCPs) on the quality and DNA methylation of the cryopreserved spermatozoa of Shanghai white pigs. A total of 24 ejaculates (three ejaculate samples per boar) from eight Shanghai white pigs were manually collected. The pooled semen was diluted with a based extender supplemented with different concentrations of PCPs (0, 300, 600, 900, 1200, and 1500 μg/mL). Once thawed, the quality of the spermatozoa and their antioxidant function were assessed. In the meantime, the effect of spermatozoa DNA methylation was also analyzed. The results show that compared with the control group, 600 μg/mL of PCPs significantly improves the spermatozoa viability (p < 0.05). The motility and plasma membrane integrity of the frozen–thawed spermatozoa are significantly higher after treatment with 600, 900, and 1200 μg/mL of PCPs compared with the control group (p < 0.05). In comparison with the control group, the percentages of acrosome integrity and mitochondrial activity are significantly enhanced after the application of 600 and 900 μg/mL PCPs (p < 0.05). The reactive oxygen species (ROS), the malondialdehyde (MDA) levels, and the glutathione peroxidase (GSH-Px) activity, in comparison with the control group, are significantly decreased in all groups with PCPs (all p < 0.05). The enzymatic activity of superoxide dismutase (SOD) in spermatozoa is significantly higher in the treatment with 600 μg/mL of PCPs than in the other groups (p < 0.05). As compared with the control group, a significant increase in the catalase (CAT) level is found in the groups with PCPs at 300, 600, 900, and 1200 μg/mL (all p < 0.05). In comparison with the control group, the 5-methylcytosine (5-mC) levels are significantly decreased in all groups with PCPs (all p < 0.05). As a result of these findings, a certain amount of PCPs (600–900 μg/mL) added to the cryodiluent can significantly improve the quality of Shanghai white pig spermatozoa and can also reduce the methylation of spermatozoa DNA caused by cryopreservation. This treatment strategy may establish a foundation for the cryopreservation of semen from pigs.

Silk sericin plays a protective role in a variety of mammalian cells during cryopreservation. This study aimed to determine the effects of sericin supplementation to a freezing extender on sperm quality and lipid peroxidation in dairy bull sperm cryopreservation. Each semen sample of five Holstein-Friesian crossbred bulls was divided into four aliquots and diluted in a tris-egg yolk extender supplemented with different concentrations of sericin [0%, 0.25%, 0.5%, and 1.0% (wt/vol)]. Sperm motility (CASA), viability, acrosome integrity, mitochondrial membrane potential (fluorescent staining) and lipid peroxidation (malondialdehyde – MDA test) were analysed. The results show that the 0.25% and 0.5% sericin groups had the highest total sperm motility (P < 0.05). Sperm viability, acrosome integrity, and mitochondrial function were higher in the group supplemented with 0.25% sericin compared to the control and 1.0% (P < 0.05). Sericin supplementation with 0.25% and 0.5% significantly decreased MDA concentrations compared with the control (P < 0.05). In conclusion, supplementation of the semen freezing extender with sericin at the concentration of 0.25% significantly improved the post-thaw semen quality and reduced lipid peroxidation in Holstein-Friesian crossbred bulls.

Native breed conservation is an important component of poultry biodiversity. The aim of this work is to describe different steps that lead to donor selection for the implementation of the Italian Semen Cryobank of Autochthonous Chicken and Turkey Breeds. The variability within and between breeds was evaluated, and the stored semen reproductive capacity was in vivo tested using artificial insemination. Semen from Bionda Piemontese, Bianca di Saluzzo and Pepoi roosters was collected and processed. Concentration, volume, sperm membrane integrity, total motile sperm, progressive motile sperm and kinetic parameters were analyzed; sperm parameters accounting for bird variability were used to select male donors. Fresh semen quality parameters measured in donor ejaculates showed significant differences between breeds; no differences were found after cryopreservation. Variability in the fertilizing ability of cryopreserved semen was found within a breed (5–16%) and between birds within a breed (BP = 3–7%; BS = 7–31%; PP = 6–22%); only sperm quality parameters measured in fresh ejaculates, not frozen/thawed, may be associated with in vivo fertility results. In conclusion, sperm concentration and progressive motility were successfully used as selection parameters to identify chicken male donors with improved sperm quality for sperm cryobanking. However, new reliable sperm markers to predict cryopreserved semen’s fertilizing ability are required.

In the present study, the synergistic protective effect of co-supplementation of glutathione (GSH) with selenium nanoparticles (SeNPs) on the cryopreservation efficiency of bull semen was analyzed. After collection, the ejaculates of Holstein bulls were subsequently diluted with a Tris extender buffer supplemented with different concentrations of SeNPs (0, 1, 2, and 4 μg/ml), followed by semen equilibration at 4°C and assessment of sperm viability and motility. Subsequently, the ejaculates of Holstein bulls were pooled, split into four equal groups, and diluted with a Tris extender buffer supplemented with basic extender (negative control group, NC group), 2 μg/ml SeNPs (SeNPs group), 4 mM GSH (GSH group), and 4 mM GSH plus 2 μg/ml SeNPs (GSH + SeNPs group). After cryopreservation, motility, viability, mitochondrial activity, plasma membrane integrity, acrosome integrity, concentration of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), and ability of frozen-thawed sperm cells to support embryonic development were evaluated. No side effect of SeNPs concentrations applied in the current study on the motility and viability of equilibrated bull spermatozoa was found. Meanwhile, supplementation of SeNPs significantly promoted the motility and viability of equilibrated bull spermatozoa. Furthermore, the co-supplementation of GSH with SeNPs effectively protected bull spermatozoa from cryoinjury as expressed by promoting semen motility, viability, mitochondrial activity, plasma membrane integrity, and acrosome integrity. Finally, the enhanced antioxidant capacity and embryonic development potential in the frozen-thawed bull spermatozoa cryopreserved by co-supplementation of GSH with SeNPs further confirmed the synergistic protective effect of co-supplementation of GSH with SeNPs on the cryopreservation of bull semen.