Search Results (190)

Liquid storage is an important tool used to prolong fresh semen shelf-life while protecting spermatozoa from damage, conserving their overall functionality, and ensuring better fertility than frozen semen from sheep. The increased production of reactive oxygen species (ROS) during sperm storage leads to a decline in sperm quality, particularly with regard to sperm nuclear DNA damage and mitochondrial membrane potential (MMP). This study evaluated the effect of storing Sarda ram semen at 15 °C for 7 h on its redox status, motility, morphology, acrosome integrity, ATP content, mitochondrial potential membrane, and in vivo fertility after artificial insemination. Two different extenders were compared: a lab-made skimmed milk (SM)-based extender and a commercial extender (OviXcell^®, IMV-Technologies, France). Lower ROS levels in the SM (p < 0.001) indicated that its oxidative status was better maintained compared to the commercial extender (CE). Antioxidant defenses (total antioxidant capacity, TEAC; superoxide dismutase, SOD; total thiols) were higher in the SM (p < 0.01) than in the CE. SM also had higher MMP (p < 0.05), acrosome integrity (p < 0.05), ATP content (p < 0.01), and in vivo fertilizing capacity (p < 0.05) compared to the CE, which indicated higher semen quality. In conclusion, the SM extender, while maintaining a better oxidative/antioxidant balance, ensured higher semen quality after 7 h of storage at 15 °C in vitro compared to the CE. Full article

Semen cryopreservation is associated with sperm vulnerability to oxidative stress and ice crystal-induced damage, adversely affecting in vitro fertilization (IVF) success. This study aimed to investigate the effects of freezing diluent supplemented with antioxidant limonin (Lim), myo-inositol (MYO), and the ice crystal formation inhibitor L-proline (LP) through sperm motility, morphological integrity, and antioxidant capacity. The Lim (150 mM), MYO (90 mM), and LP (100 mM) significantly ameliorated the quality of post-thaw sperm in Debao boar, and combined treatment of these agents significantly enhanced sperm motility, structural integrity, and antioxidant capacity compared with individual agents (p < 0.05). Notably, the combined use of these agents reduced glycerol concentration in the freezing diluent from 3% to 2%. Meanwhile, the integrity of the sperm plasma membrane, acrosome membrane, and mitochondrial membrane potential was significantly improved (p < 0.05), and the result of IVF revealed the total cell count of the blastocysts was also greater in the 2% glycerol group (p < 0.05). In conclusion, the newly developed freezing diluent for semen, by adding Lim (150 mM), MYO (90 mM), and LP (100 mM), can enhance the quality of frozen–thawed Debao boar sperm and reduce the concentration of glycerol from 3% to 2% as high concentrations of glycerol can impair the quality of thawed sperm and affect in vitro fertilization outcomes. In conclusion, the improved dilution solution formulated demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing. Full article

The cryopreservation of rabbit semen is a valuable strategy for genetic resource preservation and efficient artificial insemination, but outcomes remain inconsistent, partly due to variations in sperm concentration per dose. This study aimed to evaluate the in vivo effects of different sperm concentrations (15, 25, 35, 55, and 75 million per straw) on fertility, prolificacy, and offspring growth in nulliparous and multiparous does. A total of 384 rabbit females were inseminated using frozen–thawed semen, and their reproductive performance was compared with fresh semen. Fertility and kindling rates varied with sperm concentration and parity: nulliparous does showed the highest fertility at 15 million sperm/straw (84.4%), while multiparous does reached peak values at 25–55 million/straw (78.1–81.3%). Litter size and live-born kits were consistently higher in multiparous than in nulliparous does. Offspring body weight at 19 and 60 days was influenced by both sperm concentration and maternal parity, with better growth generally observed in multiparous groups. Weaning success remained high across all groups. Our results indicate that sperm concentrations ranging from 15 to 35 × 10⁶/straw are the most suitable for cryopreservation, as they maintain high fertility, prolificacy, and offspring growth, comparable to fresh semen. These results confirm that optimizing sperm concentration during cryopreservation improves reproductive efficiency and that tailoring insemination strategies to the physiological status of the female enhances outcomes. The results provide useful recommendations for improving cryopreservation techniques in rabbit breeding programs. Full article

Infertility is a significant global health concern, and incorporating antioxidants into sperm preparation media is one strategy to enhance sperm quality and decrease infertility rates. This study aimed to investigate the phytochemical compounds of red cotton stamen extracts and their effects as antioxidants in improving the quality of bull frozen semen. Among the extracts, RCU contained the highest levels of total phenolics, total tannins, and total monomeric anthocyanins along with the strongest ABTS free radical scavenging activity and protein denaturation inhibition. Exposing sperm to FeSO₄-induced oxidative stress resulted in significantly reduced motility, viability, and normal morphology. However, treatment with RCD, RCU, and RCM improved these parameters. Additionally, the FeSO₄-induced group showed elevated levels of reactive oxygen species (ROS) and advanced glycation end products (AGEs) compared to the normal control, whereas all red cotton stamen extracts effectively reduced these levels. In conclusion, red cotton stamen extracts, rich in phenolic bioactive compounds, demonstrated strong free radical scavenging capacity and improved sperm motility, viability, and morphology by neutralizing free radicals and enhancing antioxidant defenses. These findings suggest that the red cotton stamen extracts, particularly RCD and RCU, offer benefits for sperm preservation. Full article

The significant decline in amphibians worldwide is demanding the development of reliable techniques to save species and their genetic diversity. Considerable efforts are currently in progress to develop assisted reproductive technologies (ARTs), focusing mainly on sperm cryopreservation and in vitro fertilization (IVF). In Xenopus, a simple and efficient transgenesis method based on the intracytoplasmic injection (ICSI) of cryoconserved sperm was developed several decades ago, allowing for quick generation of large numbers of transgenic animals, for biological research. Such a methodology could be critical for the recovery of species and their genetic diversity, contributing to amphibian conservation. However, this approach raised the question of whether the sperm preservation method used with ICSI is compatible with long-term storage. To address this question, animals were generated by ICSI using a twenty-year-old cryopreserved sperm preparation. Their development, behavior, and reproduction ability were compared with those of animals obtained using a recently frozen sperm preparation and those of animals obtained via IVF using fresh semen. Although lower than with IVF, we showed that fertilization rates using ICSI after 20 years of cryopreservation are similar to those of a recent preparation, with viable offspring leading to normal F2 generation. This pioneering achievement is proof of concept for long-term sperm cryopreservation using simple and readily available technologies for the conservation of endangered amphibians. Full article

The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA^® semen analysis system. We found that semen cryopreservation significantly reduced sperm motility and was associated with an increased percentage of acrosome-reacted spermatozoa. It should be noted, however, that, in this case, there was no negative control, and the results may reflect acrosomal damage rather than the elicited responses. Further validation of the methods with equine sperm and the inclusion of a control are recommended. Full article

The physiological functions of mammalian sperm, such as motility, hyperactivation, and capacitation, require substantial energy. This study investigates the effects of two classic cryopreservation extenders—TCG (tris-citrate-glucose) and LEY (lactose-egg yolk)—on the energy metabolism of frozen–thawed boar semen. By comparing the quality indicators, key metabolite levels, and the activities of critical enzymes involved in glycolysis and the tricarboxylic acid cycle, we aim to understand how these different semen extenders influence the spermatozoa vitality of frozen–thawed boar semen. Following thawing, the LEY-cryopreserved sperm demonstrated significantly elevated motility parameters (viability, VCL, VSL, and VAP) and enhanced plasma membrane and acrosomal integrity compared with the TCG group (p < 0.05), though both cryopreserved groups exhibited significantly reduced performance relative to fresh semen controls. Cryopreservation markedly reduced intracellular adenosine triphosphate (ATP), pyruvate, and acetyl coenzyme A (A-CoA) levels (fresh > LEY > TCG; p < 0.05). The LEY-preserved spermatozoa retained higher activities of glycolysis-related enzymes (phosphofructokinase, PFK; pyruvate kinase, PK) compared with the TCG group, which, in turn, showed elevated lactate dehydrogenase (LDH) activity. Critically, TCG-suppressed pyruvate dehydrogenase (PDH) activity (p < 0.05) coincided with diminished A-CoA, indicating impaired mitochondrial oxidative phosphorylation. These results demonstrate LEY’s superior preservation of motility and membrane stability but highlight cryodamage-induced energy metabolism dysregulation, particularly TCG’s disruption of the glycolysis–TCA cycle coordination essential for spermatozoa function. In conclusion, the choice of semen extender has a significant impact on the energy metabolism and overall quality of frozen–thawed semen, highlighting the importance of optimizing cryopreservation protocols for improved spermatozoa viability and functionality. Full article

The study investigated the sperm motility and morphometry of pre-freeze and post-thaw boar epididymal semen cooled at increasing holding times at 18 °C. A total of 50 testes of heterogeneous boars were collected (5 testes/day) from the local abattoir and transported to the laboratory at 5 °C within 30 min after slaughter. Semen was retrieved from the caudal part of the epididymis using the slicing float-up method, diluted with Beltsville Thawing Solution extender, pooled in a 50 mL centrifuge tube/5 testes/day, and cooled at 18 °C. Following each holding time (0, 3, 6, 9, 12, 24, and 48 h), the cooled semen sample was re-suspended with Fraction A extender and stored at 5 °C for an additional 45 min. A cooled resuspended semen sample was then diluted with Fraction B extender, loaded into 0.25 mL straws, and frozen using liquid nitrogen vapour. Thawing was accomplished by immersing the semen straws in warm (37 °C) water for 1 min and the samples were evaluated for sperm motility and morphometry traits using the computer-assisted sperm analyzer system. The data were analyzed using variance analysis. Descriptive statistics were used to assess sperm morphometry, establishing the minimum and maximum values. Boar epididymal sperm survived for up to 48 h when held at 18 °C. Furthermore, the highest post-thawed sperm motility rates were observed in semen frozen after 3 h of holding time, with a sperm total motility of 85.9%, a progressive motility of 60.3%, and a rapid motility of 33.2%, as compared to other holding times (p < 0.05). The acceptable ranges for pre-freeze and post-thawed sperm morphology were head length (8.4–9.1 µm), width (4.4–4.8 µm), area (29.9–38.2 µm²), perimeter (20.1–23.7 µm), midpiece width (1.1–2.8 µm), and sperm shape, were consistent regardless of the holding time. A holding time of 3 h enhances the cryoresistance of sperm cooled at 18 °C. Therefore, these findings suggest that boar epididymal sperm can be effectively conserved and can maintain fertilization capability when cooled for 3 h at 18 °C before freezing. Full article

Semen quality plays a crucial role in bovine in vivo embryo production. This study aimed to compare the effects of 0 °C-refrigerated semen and liquid nitrogen-frozen semen on embryo quality in Simmental cattle. Semen collected from five bulls was equally divided into two groups: one diluted with a 0 °C refrigeration solution and stored at 0 °C, and the other diluted with a cryopreservation solution and stored in liquid nitrogen for 24 h. We evaluated sperm motility, progressive motility (assessed via a computer-assisted sperm analyzer), acrosome integrity, and plasma membrane integrity in both groups. Fifty superovulated Simmental cows were artificially inseminated with semen from both groups. Embryos were non-surgically flushed on day seven, followed by BrdU proliferation staining and TUNEL apoptosis staining. Proliferation and apoptosis levels were quantified using marker genes. Results showed that 0 °C-refrigerated semen exhibited significantly higher sperm motility, progressive motility, acrosome integrity, and plasma membrane integrity compared to liquid nitrogen-frozen semen (p < 0.05). While total embryo numbers showed no significant difference between groups (p ≥ 0.05), embryos from 0 °C-refrigerated semen contained significantly more proliferative cells (p < 0.05) and fewer apoptotic cells (p < 0.05) than those from frozen semen. These findings demonstrate that 0 °C-refrigerated semen outperforms liquid nitrogen-frozen semen in both sperm quality parameters and resultant embryo quality. Full article

Cryopreserved pig semen tends to produce excessive reactive oxygen species (ROS) during the thawing process, which leads to a decline in semen quality during in vitro storage. Mogroside V (MV) has been proven to be an effective antioxidant, and previous research has shown that MV can delay oocyte aging and improve the in vitro maturation efficiency of pig oocytes. However, the role of MV in the cryopreservation capacity of animal sperm remains unclear. To evaluate the effect of MV on sperm motility after thawing, different concentrations of MV (0, 25, 50, 75, 100 μmol/L) were added to the thawing medium. By comparing the sperm motility and kinematic parameters in the thawing medium with different MV concentrations and incubation times (0, 1, 2, and 4 h), we ultimately selected sperm thawed immediately in the medium supplemented with 75 μmol/L MV for subsequent experiments. Compared with the control group, the sperm thawing medium containing MV improved sperm quality during the freeze–thaw process. Immediate evaluation after thawing at 37 °C showed that supplementation with 75 μmol/L MV produced an optimal effect on the maintenance of motility, plasma membrane integrity, the acrosome integrity, the ROS levels, and the T-AOC activity. In conclusion, MV supplementation improves the quality of frozen–thawed sperm by enhancing sperm function and preventing oxidative stress. Full article

Ferroptosis is implicated in cryodamage to sheep sperm, potentially due to glutathione peroxidase 4 (GPX4) degradation during freezing; however, the pathway underlying GPX4 degradation remains unclear. In this study, a comparison of cryoprotective effects between the autophagy inhibitor chloroquine (CQ) and the ubiquitination inhibitor MG132 revealed that 5 μM CQ treatment significantly enhanced the motility (p < 0.01) and sperm plasma membrane integrity rate (p < 0.01) of frozen–thawed sperm; no protective effects were observed in any MG132 treatment group. Mechanistic analysis indicated that CQ treatment substantially restored GPX4 protein expression (p < 0.01), and concurrently reduced lipid peroxidation (p < 0.01) and free iron ion accumulation (p < 0.01), in frozen–thawed sperm. These findings suggest that GPX4 degradation during cryopreservation occurs via the autophagy pathway. This study established a ferroptosis–GPX4–autophagy axis during sheep sperm cryopreservation and identified autophagy-mediated GPX4 loss as a potential target for enhancing sperm cryoprotection. Full article

Sperm cryopreservation is helpful for maintaining the genetic diversity of fish species. This study was aimed at developing efficient methods to cryopreserve the sperm of three fish species, including koi carp (Cyprinus carpio L.), Ya fish (Schizothorax prenanti), and Glyptosternum maculatum. Firstly, based on the analysis of sperm viability, the cryomedium, dilution ratio, volume, and cooling procedure were assessed and optimized in koi carp. The results showed that the highest sperm viability was up to 63.23 ± 1.36% after a 14-day cryopreservation using the optimal method, briefly, sperm frozen with a volume of 50 μL (Vol.sperm:Vol.cryomedium = 1:9) of cryomedium containing 10% DMSO and 3% sucrose in D17 through ultrarapid cooling. Secondly, both the mitochondrial membrane potential and the DNA fragmentation index of sperm were examined and found to be significantly damaged after the cryopreservation. Intriguingly, the fertilization rate of sperm after 14-day cryopreservation is up to 63.03 ± 1.36% and the elongation of cryopreservation time (210 days) just slightly affected the fertilization rate (55.09 ± 4.70%) in koi carp. Thirdly, the optimal cryopreservation method was applied to cryopreserve Glyptosternum maculatum sperm; the cell viability was 45.39 ± 4.70%. And then this method, after a minor modification (3% sucrose of cryomedium replaced with 3% SMP) was adopted to cryopreserve Ya fish sperm, the cell viability was up to 70.45 ± 2.23%. Lastly, the ultrastructure and morphology of sperm was observed by SEM, and it was found that the cryopreservation prominently caused sperm head swelling and tail shortening in three fish species. In conclusion, this study established effective methods for cryopreserving sperm in three fish species and elaborated the injuries on sperm caused by cryopreservation. And the findings facilitate developing more protocols with practical value to cryopreserve sperm in different fish species. Full article

This study evaluated the impact of different processing techniques on microbial load and sperm quality in frozen–thawed equine semen to identify alternatives to reduce the preventive use of antibiotics. Semen was obtained and processed under rigorous hygiene measures from ten stallions, using four protocols: Simple Centrifugation with antibiotics (S+) and Simple Centrifugation (S−), Filtration (F−) and Single-Layer Colloidal Centrifugation (C−) in an antibiotic-free extender. Microbial load in different culture media, sperm viability and motility were assessed. Microbial load results were consistent across protocols, except in Columbia 5% Sheep Blood Agar media, where S− exhibited higher microbial load than S+ (p < 0.05). However, F− and C− showed similar microbial loads to S+. No significant differences were observed in progressive motility, average path velocity, straight-line velocity or wobble parameters between protocols. Total motility and viability were significantly higher in S+ compared to other treatments (p < 0.05). Thus, regardless of antibiotics, the proposed methods achieved results similar to the traditional antibiotic-inclusive protocol in terms of microbial load and the most relevant semen quality parameters. These findings suggest that the use of F− and C−, combined with optimized hygiene measures, offers an effective alternative to reduce the prophylactic use of antibiotics in semen extenders. Full article

This study aimed to analyze the effect of curcumin on the antioxidant properties and fertility of freeze–thawed bovine spermatozoa and bovine oocytes. In this study, curcumin concentrations of 0, 5, 10, 25, and 50 µM were added bovine sperm cryopreservation solution and oocyte IVM medium to assess sperm quality, antioxidant properties, oocyte maturation, IVF rate, and embryonic development. The results demonstrated that adding curcumin to the cryopreservation solution significantly improved the viability, motility, and acrosome integrity of bull sperm after freezing and thawing (p < 0.05). The addition of 25 µM curcumin resulted in the best sperm quality. Analysis of antioxidant capacity showed that 25 µM curcumin significantly increased the activities of MMP and antioxidant enzymes, such as CAT, SOD, and GSH-PX, and lowered the levels of MDA and ROS (p < 0.05). Adding curcumin to the in vitro maturation medium notably enhanced the maturation rate and decreased DNA fragmented nuclei of bovine oocytes (p < 0.05), with optimal outcomes observed at 25 and 50 µM curcumin. Totals of 25 and 50 µM curcumin markedly elevated GSH and MMP (p < 0.05), reduced ROS and malondialdehyde concentrations (p < 0.05), and significantly enhanced fertilization rates and blastocyst formation (p < 0.05). In conclusion, incorporating curcumin into both the bovine semen cryopreservation solution and the oocyte IVM medium significantly improved the quality of frozen–thawed sperm, antioxidant activity, oocyte maturation, IVF rate, and embryonic development. Full article

This study aimed to investigate the effects of adding different levels of punicalagin or oleuropein to TRIS diluent on the quality of frozen–thawed semen from Najdi rams. Semen was diluted using TRIS-based diluter with 15% egg yolk (control group); supplemented with 0.1, 0.5, or 1 mg/100 mL punicalagin (in Experiment 1); or supplemented with 1, 2.5, or 5 mg/100 mL oleuropein (in Experiment 2). The collected semen was evaluated and cryopreserved, with the motility and concentration of sperm assessed using a CASA system. The results showed that the total motile spermatozoa (TMS), percentage of progressive motile spermatozoa (PMS), curvilinear velocity (VCL), rectilinear velocity, average path velocity (VAP), linearity coefficient, straightness index, minor defects, and sperm vitality were higher in the 0.1 mg/100 mL punicalagin group (p < 0.05) than in other groups. HOST% post-thawing was significantly higher in all punicalagin groups compared to the control group (p < 0.001). The percentages of PMS, TMS, VCL, minor defects, and sperm vitality were higher in the 1 mg/100 mL oleuropein group (p < 0.05) than in other groups. Oleuropein supplementation at 5 mg/100 mL decreased VAP in cooled sperms, while all levels increased VAP post-thawing. HOST-positive sperms% post-thawing was higher in all oleuropein-treated groups than the control group (p < 0.001). Moreover, oleuropein nonsignificantly increased the acrosome integrity in cooled sperms, while higher studied concentrations of oleuropein (2.5 and 5 mg/100 mL) decreased the acrosome integrity in frozen sperms. In conclusion, adding punicalagin (0.1 mg/100 mL) or oleuropein (1 mg/100 mL) to TRIS diluent improved the quality of frozen–thawed semen from rams. Full article